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PROCEEDINGS OF THE 3RD ENURS AND ESRF-DAY MEETING OF SYNCHROTRON RADIATION USERS FROM PORTUGAL, APRIL 8TH, 2014.

Current Synchrotron-based Research in Portugal 2014

Editors Geoffrey Robert Mitchell Paulo Jorge da Silva Bártolo (also University of Manchester) Maria João Romão (Universidade Nova de Lisboa) Ana Patrícia Oliveira Tojeira Daniela Filipa de Sousa Rocha Dino Miguel Fernandes Freitas Elodie Maria Pinto Margarida Rosa da Costa Lima Cabrita Franco Sara Isabel da Silva Biscaia Tânia Filipa Quitério Viana Telma Margarida Dias Ferreira Centre for Rapid and Sustainable Product Development, Polytechnic Institute of Leiria, Portugal


Š 2014 Polytechnic Institute of Leiria Published by: Centre for Rapid and Sustainable Product Development, PolytechnicInstitute of Leiria Centro Empresarial da Marinha Grande Rua de Portugal – Zona Industrial 2430-028 Marinha Grande, Portugal Email: cdr-sp@ipleiria.pt cdrsp.ipleiria.pt ISBN: 978-972-8793-67-8 (Print version) ISBN: 978-972-8793-68-5 (ebook version)


Foreword The Centre for Rapid and Sustainable Product Development of the Institute Polytechnic of Leiria was proud to host the 3rd ENURS meeting and to welcome distinguished guests from the European Synchrotron Research Facility in Grenoble France. 2014 is the UNESCO Year of Crystallography. 2014 marks the centenary of the birth of crystallography. A century later, the International Year of Crystallography 2014 highlights the continuing importance of crystallography underpinning many sciences and its role in addressing post-2015 development issues such as food security, safe drinking water, health care, sustainable energy and environmental remediation; as well as commemorating auspicious crystallography accolades. This Year also commemorates the 50th anniversary of another Nobel Prize, awarded to Dorothy Hodgkin for her work on vitamin B12 and penicillin. Portugal was the first country to join the ESRF as a Scientific Associate Member on 10th January 2003. Portugal's Secretary of State for Science and Technology, Manuel Fernandes Thomaz, said on that occasion. "We would like to boost Portugal's participation to the ESRF, since the scientific projects at the synchrotron have a lot of applications that are relevant to society". This small book records the almost 50 scientific contributions presented at the 3rd ENURS Meeting in Marinha Grande from researchers across Portugal. Reviewing these contributions it is certainly the case that the scientific projects represent a strong vibrant community whose work has a strong relevance to society. Here within CDRSP we have employed synchrotron radiation facilities to evaluate the complex molecular organisation present in polymer scaffolds prepared by additive manufacturing. These objects are for eventual use as scaffolds for tissue regeneration which will provide an enhanced quality of life for many citizens as well as stimulate economic growth in these emerging technologies. We have set out to record all the relevant information in one place for those interested in the use of synchrotron radiation facilities in their research programmes. I would like to thank all those who contributed to the success of the 3rd ENURS meeting and to congratulate those awarded the best poster prizes.

Professor Geoffrey Mitchell Conference Chair 3rd ENURS and ESRF Day

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Introduction of the National Delegate to the ESRF Portugal is a Scientific Associate Member of the ESRF since November 1997 and in 2013 this membership was renewed for another 5 years. Portuguese researchers have been users in fields as diverse as Structural Biology, Materials Science, Chemistry, Physics and Cultural Heritage. With a participation share of 1%, the Portuguese community of around 100 users has been actively involved in experiments at the ESRF, with an excellent outcome and a steady increase in the number and impact of publications. There is also an increasing participation in experiments at other Synchrotron Radiation (SR) labs in Europe. The national community of SR users has grown over the years but there are still many ways to expand and profit even more. The latest developments, in particular the upgrade phases, offer new possibilities and new areas of application, that may also attract new potential users. The main purpose of the Meeting of Portuguese SR Users (ENURS) has been to provide the users with an insight and update on the great opportunities offered, in particular at the ESRF, as well as to report the research that has been carried out. In addition to scientific discussions, one important aim is to continue strengthening the Portuguese users community, in particular at the ESRF. After the 1st ENURS Meeting (January 2012, FCT-UNL) in 2013 it was decided to combine the 2nd ENURS with an ESRF-day, which proved to be very successful. With its 3rd edition the Portuguese users community will be able once more to share experiences, discuss science and be aware of the ever-increasing opportunities offered by the ESRF Research Institute!

Maria João Romão National Delegate to the ESRF Council

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Contents Foreword 3 Introduction of the National Delegate to the ESRF 5 Synchrotron Facilities in Europe 9 European Synchrotron Radiation Facility 13 Portuguese Participation in the ESRF 19 Fundação para a Ciência e Tecnologia 23 Centro para o Desenvolvimento Rápido e Sustentado de Produto 25 ENURS and ESRF-DAY 27 Invited Speakers 29 Program 31 Current Synchrotron-Based Research in Portugal 33

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Synchrotron Facilities in Europe In Europe there are 23 light sources either in operation, commissioning or construction. A list of some examples is showed below.

ALBA ALBA is a 3rd generation Synchrotron light facility located in Barcelona (Spain). It first welcomed users in May 7th, 2012 in one of its 7 current beamlines. ALBA Synchrotron Light Source Cerdanyola del Vallès (Barcelona), Spain www.cells.es

ANKA The ANKA synchrotron radiation facility opened in March 2003. It currently operates with 17 beamlines. ANKA - Synchrotron Radiation Facility, Karlsruhe Institute of Technology (KIT) Eggenstein-Leopoldshafen, Germany www.anka.iss.kit.edu

BESSY II The BESSY II synchrotron was open to the public in September 1998. The total number of beamlines or end stations is 43. BESSY II - Berlin Electron Storage Ring Berlin, Germany www.helmholtz-berlin.de

CLIO The CLIO Free-Electron Laser (FEL) produces a high peak power and tunable wavelength laser beam and it will operate with 10 beamlines for the fields of surface spectroscopy, molecular physics, near-field infrared microscopy, and quantum wells. Centre Laser Infrarouge d'Orsay (CLIO) Orsay, France www.clio.lcp.u-psud.fr 9


DAFNE Four beamlines have become operational since the facility came online in 2001, which receive light from the intense photon emission of DAFNE, via a 0.51 GeV storage ring allowing experiments with soft Xrays, ultraviolet light, infrared, and extreme ultraviolet radiation, and they are open to external users. DAFNE-Light - INFN-LNF Synchrotron Radiation Facility Frascati, Italy web.infn.it/Dafne_Light

DIAMOND It welcomed its first scientific users in January 2007, and currently has 22 beamlines in operation. Diamond Light Source Ltd was established in 2002 and employs approximately 500 scientists, engineers, technicians and support staff from over 40 countries worldwide. Diamond Light Source Didcot (Oxfordshire), UK www.diamond.ac.uk

XFEL The European X-ray Free-Electron Laser is a 3.4-kilometer-long research facility currently under construction in the Hamburg area, Germany. Starting in 2015, it will generate extremely intense X-ray flashes. European X-Ray Free-Electron Laser Facility Hamburg (Germany) www.xfel.eu

ELETTRA Elettra, the 2/2.4 GeV third generation Italian light source operates with 25 beamlines and one storage ring free electron laser that enable the most important x-ray based techniques in the areas of spectroscopy, spectro-microscopy, diffraction, scattering and lithography. Elettra - Sincrotrone Trieste S.C.p.A. Basovizza, Trieste, Italy www.elettra.eu

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ESRF The European Synchrotron Radiation Facility (ESRF) is a joint facility supported and shared by 20 countries. The ESRF operates the most powerful synchrotron radiation source in Europe, at 6 GeV. European Synchrotron Radiation Facility Grenoble (France) www.esrf.eu

ISA The synchrotron machine ASTRID2 represents developments in the ISA's synchrotron programme. It operates at 580 MeV. It is expected that ASTRID2 will become fully operational in 2014, and replace the existing ASTRID synchrotron machine. ASTRID/ASTRID2 Aarhus (Denmark) www.isa.au.dk

SLS The Swiss Light Source (SLS) at the Paul Scherrer Institute is a 3rd generation synchrotron light source. With an energy of 2.4 GeV, it provides photon beams of high brightness for research in materials science, biology and chemistry. Swiss Light Source Villigen (Switzerland) www.psi.ch/sls

SOLARIS The storage ring is a heart of Solaris Synchrotron Radiation Centre. It is a replica of the 1.5 GeV storage ring built at MAX IV There are two beamlines in construction and other two at the stage of conception and design. Polish synchrotron radiation facility Solaris Krak贸w, Poland www.synchrotron.uj.edu.pl

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PETRA III at DESY With a circumference of 2.3 km, PETRA III is one of the most brilliant storage ring light source in the world. Since the end of 2012, all 14 beamlines are available for users. DESY Photon Science Hamburg, Germany www.photon-science.desy.de

DELTA DELTA is an electron storage ring with a ďŹ nal energy of 1.5 GeV, located at the TU Dortmund and maintained by the Center for Synchrotron Radiation. Dortmund Electron Storage Ring Facility Dortmund, Germany www.delta.tu-dortmund.de

SOLEIL SOLEIL is a third-generation (2.75 GeV) machine. The facility inauguration was in December 18, 2006, and became operational in 2007, having planned for 24 beamlines, although it has the capacity for 43 in total. Synchrotron SOLEIL Saint-Aubin- Essonne (France) www.synchrotron-soleil.fr

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European Synchrotron Radiation Facility “The ESRF is an international research institute for cutting-edge science with photons: Discovery of the structure and dynamics of our complex world, down to the single atom.” The European Synchrotron Radiation Facility is one of the world's largest synchrotron science centres with a ring-shaped machine with 844 metres in circumference. It is the brightest source of X-rays up to date. The ESRF welcomed its first users in 1994 offering 15 operational beamlines. In recent years, more than 6000 scientists from 12 member countries and from around the world travel to the ESRF to perform about 1500 experiments. From the data collected in such experiments, about 2000 scientific articles are published each year, reaching a total of more than 20,000 articles published since the first experiments in 1994. The ESRF Upgrade Programme 2009-2018 maintains our world-leading role through a continuous quest for higher performance figures, meeting the needs of returning users, and attracting scientists from new disciplines. A wide range of scientific techniques are availed in the facilities, such as X-ray microscopy, magnetic scattering, absorption spectroscopy and microfluorescence and new techniques are being added constantly. The opportunities for beamtime occur twice a year through the submission of a proposal and further acceptance after external review. Use of the facility is free for academic research or published industrial research. Industry users can pay if they want to do proprietary research. This year alone, the ESRF had more applications for beamtime than at any other time in its 20 year history. Almost 1200 proposals have been received for experiments between August this year and April next year. This translates to a number of proposals two and a half times higher than the available experiment time. During scheduled operation, the machine runs continuously, 24 hours a day, six days a week with one day each week used for maintenance, tests and the handover between experiments. The ESRF employs 600 people from more than 40 different countries. The operation of the ESRF has an annual budget of around 80 million euros. ESRF shares its site with the European Molecular Biology Laboratory (EMBL) and the Institute Laue-Langevin (ILL). Grenoble also hosts a renowned research university and major laboratories of the French agencies CEA and CNRS focusing on nanotechnologies and energy research. The ESRF Experiments Division has built more than 40 specialised beamlines that are installed by the ESRF for use by the international scientific community. These beamlines are designed for research in areas as diverse as engineering, physics, chemistry, crystallography, earth science, biology and medicine, surface and materials science. In addition, several Collaborating Research Groups (CRGs) made up of institutes from countries participating in the ESRF, build and operate beamlines at the ESRF with independent funding.

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The ESRF beamline groups in the ESRF Experiments Division and their support services are organised in the following research areas:         

Structural biology Structure of materials Electronic structure and magnetism Dynamics and extreme conditions Structure of soft matter X-ray imaging Collaborating research groups Theory Scientific infrastructure

Structural Biology The Structural Biology group operates a world leading suite of synchrotron radiation beamlines dedicated to the study of biological macromolecules:    

four highly intense, tunable beamlines (ID14-4, ID23-1, ID29 and ID30B) two fixed-wavelength beamlines (ID30A-1 and ID30A-2) two microfocus beamlines dedicated to protein crystallography (ID23-2 and ID30A-3) a protein solution scattering beamline (BM29)

Structure of Materials The Structure of Materials Group provides facilities for a range of X-ray scattering, imaging and spectroscopy experiments, relevant to the fields of energy research, catalysis, engineering, metallurgy, electronics, semiconductors, MEMs, nanoscience and other elements of advanced technology, along with aspects of fundamental structural physics and chemistry. Beamline     

ID01 Microdiffraction imaging ID03 Surface diffraction ID11 Materials science, time-resolved diffraction ID15A/B High-energy diffraction/scattering ID22 Powder diffraction Associated facilities

 Surface Characterisation Laboratory - Several UHV facilities including a scanning-

tunneling microscope and transfer chambers  Electrochemistry Laboratory  Material Science Support Laboratory - Joint ESRF/ILL facility for the support

of materials engineering applications.

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Electronic Structure and Magnetism The Electronic Structure and Magnetism (ESM) group supports studies of the structural arrangement of atoms and the configuration of the electronic states in material. Beamline facilities are specialized for local and long-range structural and spectroscopic X-ray methods. The introduction to the group's chapter in the annual ESRF Highlights gives an overview of some recent accomplishments. The principle areas of study are the magnetic, chemical and electronic properties of materials. Of particular importance is in understanding the interplay of electronic (charge, spin, orbital) degrees of freedom and their relation to the structure. This dependence is closely related to the functionality of the material, be it catalytic, chemical, magnetic or electrical. The fields of research include catalysis, chemistry, correlated materials, dynamics, electronic structure, geochemistry, geophysics, magnetism, optical activity, physics, reaction kinetics, superconductivity etc. Beamlines        

ID12 Circular polarisation ID20 Magnetic scattering (Closed May 2011) BM23 X-ray absorption spectroscopy (upgrade beamline replacing BM29) ID24 Energy dispersive X-ray absorption spectroscopy beamline ID26 X-Ray Absorption and Emission Spectroscopy (XAS-XES) ID32 Previously ID08: Dragon — soft X-ray beamline for polarization-dependant studies

Dynamics and extreme conditions The scientific programs of the Dynamics and Extreme Conditions Group cover all classical domains of natural sciences with a significant impact in applied fields such as Earth, environmental and planetary science, catalysis, soft matter, material synthesis, and nanoscience. The introduction to the group's chapter in the annual ESRF Highlights gives an overview of some recent accomplishments. High pressure structural studies employ monochromatic (angle-dispersive) diffraction using diamond anvil cells (DAC) to pressures beyond 200 GPa and in temperature from 5K to 5000K. The DAC techniques are complemented by the Paris-Edinburgh press (on ID27), and the large volume press (currently being commissioned on ID06). Structural dynamics such as collective excitations in disordered materials and phonons in crystalline materials are investigated by inelastic X-ray or by nuclear inelastic scattering. Furthermore, nuclear resonance scattering allows the study of diffusion and rotational motions directly in the time domain. Electronic and magnetic properties are studied utilising nuclear resonant scattering (including their corresponding dynamics in the nanosecond to microsecond time regime), X-ray absorption (XAS) and X-ray emission spectroscopies (XES). These spectroscopies are employed for chemical characterisation, including in situ studies of low-Z elements and of valence shell and collective excitations in inorganic compounds.

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    

Beamlines ID09A High pressure I ID18 Nuclear resonance ID26 High-brilliance X-ray spectroscopy (beamline has moved to Electronic Structures and Magnetism Group) ID27 High pressure II ID28 Inelastic scattering II Associated facilities

 ID06 Internal techniques and instruments test beamline: large volume press Standard

support facilities Structure of soft matter The scientific portfolios of the beamlines in the Structure of Soft Matter Group cover the broad field of soft matter and related areas in biology and nanosciences. The introduction to the group's chapter in the annual ESRF Highlights gives an overview of some recent accomplishments. Soft matter studies address a large variety of questions concerning the microstructure, dynamics, self-assembly, structural kinetics and rheology of complex and nanostructured materials (polymers, colloids, surfactants, liquid crystals, proteins, etc.) in bulk, at interfaces as well as in confined geometry. Soft matter activities involve in situ investigations of material processing, site-selective chemistry and tailoring of supermolecular complexes, structural dynamics of molecular assemblies, structure and dynamics in thin films and membranes, diffraction from fibres, small unit cell systems and biological entities with hierarchical structures.    

Beamlines ID02 High brilliance beamline ID09B Time-resolved diffraction ID10 Soft interfaces and coherent scattering beamline ID13 Microfocus beamline

Associated facilities  Partnership for Soft Condensed Matter (PSCM)f low-Z elements and of valence shell and collective excitations in inorganic compounds. X-ray imaging The last few years have seen major developments in synchrotron-radiation-based X-ray imaging techniques (absorption, phase contrast, diffraction, fluorescence). These developments are based on the coupling of beam features with new detectors and computers. As a result, users have many new options, including:  three-dimensional imaging  higher spatial resolution  the possibility to exploit the coherence of the beam  setups permitting in situ and real-time observation as well as combinations of these options. 16


The X-ray Imaging Group supports not only traditional physical, medical, materials science and engineering subjects, but also new areas for X-ray imaging, such as environmental, archaeological, palaeontological and biological studies. Collaborating research groups Several bending-magnet beamlines at the ESRF are operated independently by Collaborating Research Groups (CRGs). For administrative purposes, CRGs are third-party contractors to the ESRF, and each maintains a research program independent of the ESRF. Further information on CRGs is available from the CRG Liaison Office. Users have two routes for accessing these beamlines:  One-third of the beamtime on these beamlines is managed through the ESRF public user program.  The remaining two-thirds is allocated by each CRG through an independent proposal process. Allocation procedures may differ slightly among CRGs.  Both routes are handled through the ESRF User Portal. The CRG Liaison Office can advise on proposal submission for CRGs. BM1 SNBL (Swiss-Norwegian Beamlines) - X-ray absorption and diffraction BM2 D2AM (French CRG Beamline) - Materials science BM8 GILDA (General Purpose Italian Beamline for X-ray Diffraction and Absorption) Bm20 ROBL (Rossendorf Beamline) - Radiochemistry and ion beam physics BM25 SpLine (Spanish CRG beamline) - X-ray absorption and diffraction BM26 DUBBLE (Dutch-Belgian Beamlines) - Small-angle X-ray scattering and interface diffraction; Protein crystallography and extended X-ray absorption fine structure (EXAFS) BM28 XMaS (UK CRG) - Magnetic scattering BM30A FIP (French Beamline for Investigation of Proteins) - Protein crystallography BM30B FAME (French Absorption Spectroscopy Beamline in Material and Environmental Sciences) - Extended X-ray absorption fine structure (EXAFS) BM32 IF (French Interface Beamline) - Interfaces Theory The research carried out in the Theory Group concerns mostly solid state theory, with a particular emphasis on magnetism. In particular, we are currently working on the following topics:-ray imaging, such as environmental, archaeological, palaeontological and biological studies.  Magnetic and structural properties of transition metal alloys under high pressure.

This work is the subject of the doctoral thesis of Yaroslav Kvashnin. It is carried out in close collaboration with the group of Sakura Pascarelli at Id24.  Multiferroics. These materials are the focus on an intense research activity worldwide,

including work done on various ESRF beamlines. The activity of Andres Cano in this field concerns the theory of excitations in multiferroics (electro-magnons), as well as questions related with inhomogenous polarization states in multiferroics.

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 Magnetic and electrical properties of graphene. This is one of the hottest fields in

condensed matter physics. Our activities in the field concern the theory of new phenomena due to strain-induced pseudo-magnetic fields (Andres Cano), the magnetism of transition metal impurities deposited on graphene (Richard Bouzerar, Patrick Bruno), a problem which is investigated experimentally by the Electronic Structure and Magnetism Group, as well as new quantum Hall phenomena in graphene (Patrick Bruno).  Theory of quantum spin systems and molecular magnets. Here our activity addresses the

quantum dynamics of spin systems in the quantum regime (Patrick Bruno). This work applies directly to molecular magnets, but has implications for various apparently unrelated problems, such as ultra-cold quantum gases or superconducting mesoscopic devices. Scientific infrastructure The Scientific Infrastructure Group provides specialized support to users and develops new techniques in the areas of:  chemistry and micromanipulation,  sample environments, and  surface science.

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Portuguese Participation in the ESRF Portugal is a Scientific Associate Member of the ESRF since November 1997. The renegotiation of such agreement occurred in January 2008 for five more years. As associate member, Portugal contributes annually with about 1% to the cost with operation of the facilities. The membership of Portugal to the ESRF allowed the Portuguese scientific community to explore with more regularity the use of synchrotron radiation through experiments in different scientific and technological areas. Since 2005, there is an average of 40 Portuguese researchers using ESRF beamtime in the development of their experiments. The beamtime available for Scientific Associates for experimentation is directly related to the percentage of the contribution to the organization, which has for base the presentation of projects previously evaluated by committees composed by scientists from the Members. This evaluation takes into account the relevance and scientific worth. The total percentage of beamtime assigned to the national community has been varied between 0.85% and 1%.

1% Portugal 1% 2%

1%

2%

4% 4%

28%

4% 6%

14%

26% 15%

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European Synchrotron Users Organisation ESUO is an organization with the aim of coordinating the synchrotron radiation user activities within Europe to guarantee the realization and access to the best beamlines in Europe. It was established in January 18th 2010. The aim of ESUO is: 1. To facilitate direct access to European national accelerator based radiation sources throughout programmes of the European Union. (For brevity all such light sources will be referred to below as SR sources). 2. To enable all European scientists to access appropriate SR facilities solely on the basis of scientific merit. 3. To promote an integrated approach throughout Europe to the use of European SR facilities. 4. To disseminate information about scientific opportunities and funding mechanisms for access to SR facilities. 5. ESUO is an organisation composed of national user delegates chosen by national SR users groups (1 voting delegate per country) plus the representatives of the European facility users' organisations as none-voting members. 6. ESUO and its members are independent of the SR facilities and of national funding agencies 7. ESUO will be represented by an Executive Committee composed of: - A Chair/spokesperson - A vice Chair - A maximum of 5 ordinary members The composition will reflect the breadth of SR science. 8. The founding Executive committee will continue in their roles for up to 4 years (2010-14) subject to ratification from the ESUO delegates after 2 years (2012). 9. After 4 years (i.e. in 2014) up to 4 of the 7 members can be re-elected for a further 2 years and ESUO delegates will be invited to elect at least 3 new members for the 4 year period 2014-8) Thereafter elections to vacant places will occur every 2 years for the 4 year vacancies arising. 10. ESUO should meet annually to: (a) Receive a report from the Executive Committee. (b) Ratify any proposals made by the Executive Committee. (c) Receive reports from national delegates. (d) Discuss new initiative. Website: www.esuo.org 21


Fundação para a Ciência e Tecnologia The support to the internationalization of the National Scientific and Technological System lies in the framework of the current FCT structures, with the Department of European, Bilateral, and Multilateral Relations. That includes the support to the participation of the scientific community in Multilateral Programmes and Scientific Networks in International Organizations, as well as, in joint projects or undertakings, part of scientific and technological cooperation agreements or bilateral agreements between Portugal and other countries and institutions. FCT supports and follows the participation of the scientific and technological community in the international organizations of which Portugal is a member. The resulting benefits for the scientific community include the access of national teams to international scientific infrastructures, increased industry competitiveness, access to training programmes and calls for scientific or management positions in international organizations.

Human resources advanced training Since November 2002, an agreement between Portugal and the Partnership for Structural Biology (PSB), enables portuguese participation in a project leading to the construction of a beamline dedicated to methodologies and high-throughput techniques. This project funds four scientific and technician positions and includes a number of collaboration projects development between PSB and Portugal. Resulting from this agreement, several research students could follow a trainee program in ESRF during the period between 2003 and 2008. In 2010, the referred agreement was renewed for more five years. Council Maria João Romão mjr@fct.unl.pt (351) 21 294 83 10 Industrial connection to CERN, ESO and ESRF Emir Sirage emir.sirage@fct.pt (351) 21 391 15 30 Administrative and Finance Committee Maria José de Almeida m.j.almeida@fct.pt (351) 21 391 15 29

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CDRSP Centro para o Desenvolvimento RĂĄpido e Sustentado do Produto Instituto PolitĂŠcnico de Leiria Centro Empresarial da Marinha Grande Rua de Portugal - Zona Industrial 2430-028 Marinha Grande Portugal T. +351 244 569 441 | F. +351 244 569 444 www.cdrsp.ipleiria.pt

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Centro para o Desenvolvimento Rápido e Sustentado de Produto The mission of the Centre for Rapid and Sustainable Product Development (CDRSP) is to contribute to the development of science and technology through the development of innovative and efficient products, materials and processing, adding value to the industry and creating public awareness about the importance of rapid and sustainable product development. To accomplish such goal, CDRSP undertakes scientific and technological researches, develops activities in training, consultancy and knowledge transfer in strategic product development areas. CDRSP management and organization is performed by a Board of Directors supported by a Scientific Council which encompasses all CDRSP senior researchers holding PhD degrees. An Advisory Board comprises reputed national and international people from academia and industry. CDRSP activities are multidisciplinary, involving Mechanical, Electronic, Chemical, Civil, Materials and Biomedical Engineers, Mathematicians, Physicists and Managers. KEY RESEARCH THEMES: Emerging Technologies – production technologies to exploit the potential of emerging technologies (in particular bio- and multi-scale technologies); leveraging simulation and modelling techniques to address manufacturing challenges; flexible, rapidly responsive production systems for customized manufacturing. Advanced Materials – materials with novel functionalities enhancing new manufacturing technologies and processes and the opportunities to manufacture entirely new materials-based technologies and products (smart materials, sustainable polymers and functionally graded structures). Sustainable manufacturing and manufacturing of green technologies. Research areas:  Advanced materials for rapid manufacturing and biofabrication;  Biofabrication;  Biomimetics;  Composites materials;  Computer assisted tissue engineering;  Concurrent engineering;  Eco-design;  Haptic CAD nD modelling (multidimensional modelling);  Micro-nanofabrication;  Material's Recycling and Reuse;  Reaction injection moulding;  Rapid manufacturing;  Reverse engineering;  Smart materials;  Sustainable product development

Main Industrial Sector:  Automotive Industry  Construction and Architecture  Energy  Manufacturing industry (especially for plastics and moulds industry)  Medical industry

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ENURS AND ESRF­DAY 3RD Meeting of Synchrotron Radiation Users from Portugal The Meeting brought together Portuguese synchrotron radiation users and potential users to share their experiences and successes with each other, to develop new collaborations and to build an even stronger synchrotron user community in Portugal. It also provided an opportunity for the community to hear from scientists at the European Synchrotron Radiation Facility (ESRF) about the enormous research opportunities offered by this European research infrastructure. This meeting added value to both current users of synchrotron radiation and to those who would like to learn more about the opportunities available for adding value to their own research programs. The national community of ESRF users has grown over the years but there are still many ways to profit even more from the unlimited possibilities offered by the ESRF research portfolio. This applies above all to scientists new to synchrotron radiation. The Meeting provided opportunities for networking and informal discussions involving the national community and the ESRF scientists, to increase awareness on the advantages of using this infrastructure, thus strengthening our user's community. The field of research and academic degrees of the participants of the 3rd ENURS and ESRFday was diverse.

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Best Poster Award Viviana Correira REQUIMTE - Universidade Nova de Lisboa Title: “Implementation of the Microarray Technology for studying molecular interactions of the bacterial Cellulosome�

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Invited Speakers

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Programme Reception 9.30 am Welcome/Opening Session 10.30 am

Gema Martinez-Criado “Exploring single nanowires with a multimodal hard x-ray nanoprobe”

Tobias Schulli “Nanomaterials investigated with hard X-rays: from diffraction microscopy to high resolution powder crystallography” Lunch + Poster Session

Christoph Mueller-Dieckmann “Structural biology facilities at the ESRF”

Harald Reichert “Future developments at the ESRF including Phase II” Coffee-break

Joanne McCarthy “Access to the ESRF and how to prepare a good proposal”

F. M. Braz Fernandes European Synchrotron Users Organization Closing Session 6.30 pm

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Current Synchrotron足Based Research in Portugal

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Current Synchrotron-based Research in Portugal 2014 Structure of Materials

Thermal evolution of Pd/Al nanomultilayers using two distinct heating cycles A.J. Cavaleiroa, A.S. Ramosa, F.M. Braz Fernandesb, C. Baehtzc & M.T. Vieiraa a

b

CEMUC®, Department of Mechanical Engineering, University of Coimbra, Portugal CENIMAT/I3N, Department of Materials Science, University Nova de Lisboa, Portugal c Helmholtz Zentrum Dresden Rossendorf HZDR, Dresden, Germany Email of corresponding author: andre.cavaleiro@dem.uc.pt

Keywords: Palladium, Aluminium, multilayers, phase transformations, joining The energy released by highly exothermic Pd/Al multilayer (ML) thin films can be used for joining applications, namely for providing extra energy in diffusion bonding processes and consequently diminish joining time, temperature or pressure [1]. Pd/Al MLs were deposited onto Ti alloys by dual cathode magnetron sputtering. Different substrates’ rotation speeds were selected in order to achieve different modulation periods (5, 15 and 35 nm). The thermal evolution of the Pd/Al nanomultilayers was studied in-situ by XRD using synchrotron radiation. The experiments were conducted at the ESRF Materials Research station of the Rossendorf beam line (BM20). Two thermal cycles were defined: i) slow heating through 50ºC steps from 100 to 900ºC with isothermal Ө-2Ө scans at each temperature step (Fig. 1a); ii) single step heating with linear array of detector’s pixels fixed enabling relatively fast (~1.3s) acquisitions (Fig.1b). The use of synchrotron radiation allowed the phase evolution sequence of Pd/Al nanomultilayers to be unequivocally resolved, while highlighting the influence of the heating rate. It is possible to avoid undesired intermediate phases by heating in a single step at a moderate heating rate. References 1. A.J. Cavaleiro, A.S. Ramos, F.M. Braz Fernandes, N. Schell, M.T. Vieira, 2014, J Mater Eng Perform. In press.

Figure 1. XRD diffractograms of Pd/Al ML (Λ = 35 nm) as function of temperature. a) Slow heating, b) Single step heating

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Current Synchrotron-based Research in Portugal 2014 Structure of Materials

Programmable Sample Environment to provide controlled shear and temperature profile to mimic polymer processing Geoffrey Mitchella a

Centre for Rapid and Sustainable Product Development Institute Polytechnic of Leiria. Marinha Grande, Portugal Email of corresponding author: geoffrey.mitchell@ipleiria.pt

Keywords: Polymer Processing, Shear flow, Small angle X-ray Scattering, Crystallisation, Orientation The final properties of a plastic part are strongly influenced by the processing procedure. The polymer is shaped in the liquid state using flow fields and the cooled to room temperature to form a solid to retain the processed shape. Time-resolving smallangle and wide-angle x-ray scattering provide quantitative data on the changes in molecular organisation and morphology which take place during shear flow. This contribution describes a shear flow cell designed and developed to enable x-ray scattering data to be collected during shear flow and for polymers subjected to controlled thermal profiles. The objective is to evaluate structural changes relevant to industrial polymer processing. We describe the key features of the system which has been used extensively at the ESFR and provide examples of the results obtained.

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Current Synchrotron-based Research in Portugal 2014 Structure of Materials

Tensiometer to enable time-resolving x-ray scattering data on polymers during mechanical testing Geoffrey Mitchella, Ana Tojeiraa & Miguel Gaspar a a

Centre for Rapid and Sustainable Product Development Institute Polytechnic of Leiria. Marinha Grande, Portugal Email of corresponding author: geoffrey.mitchell@ipleiria.pt

Keywords: Polymer testing, mechanical properties, Small angle X-ray Scattering, Orientation Mechanical testing of polymer based materials is a critical component of a materials development programme. The use of time-resolving small-angle and wide-angle x-ray scattering during mechanical testing provides an insight in to the properties of the material and enables structure-property relationships to be developed. This contribution describes a tensiometer for the measurement of the mechanical properties of polymers which has been designed and developed to enable x-ray scattering data to be collected during testing. We describe the key features of the system which has been used extensively at the ALBA and Daresbury Synchrotron Facilities and we provide examples of the results obtained.

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Current Synchrotron-based Research in Portugal 2014 Structure of Materials

Controlling the morphology of polymers on multiple scales Geoffrey Mitchella & Ana Tojeiraa a

Centre for Rapid and Sustainable Product Development Institute Polytechnic of Leiria. Marinha Grande, Portugal Email of corresponding author: geoffrey.mitchell@ipleiria.pt

Keywords: Polymer morphology, properties, Small angle X-ray Scattering, Orientation Crystalline polymers spontaneously form hierarchical structures although the precise manner in which these scales of structure are interconnected especially terms of the formation and evolution of the complete structure remains unclear. We have set out to control these scales of structure by introducing additional components which self-assemble in to nano-scale units which then direct the crystallisation of the polymer matrix. In other words, we first assemble a low concentration top-level structure which is designed to template or direct the subsequent crystallisation of the matrix polymer. This top level structure takes on the role of controlling the structure. We have set out to both establish the design principles of such structures and to develop experimental procedures which allow us to follow the formation of such complex hierarchical polymer structures. In particular we focus of the relationships between these different levels of structure and time sequence of events required for the structure to evolve in the targeted manner. In this programme, we have exploited time-resolving small-angle x-ray scattering and electron microscopy together with neutron scattering to probe and quantify the different scales of structure and their evolution. We highlight new neutron scattering instrumentation which we believe have great potential in the growing field of hierarchical structures in polymers. The addition of small quantities of nanoparticles to conventional and sustainable thermoplastics leads to property enhancements with considerable potential in a number of areas. Most engineered nano-particles are highly stable and these exist as nano-particles prior to compounding with the polymer resin, they remain as nano-particles during the active use as well as in the subsequent waste and recycling streams. In this work we explore the potential for constructing nano-particles within the polymer matrix during processing from organic compounds selected to provide nanoparticles which can effectively control the subsequent crystallization process. Typically these nano-particles are rod-like in shape. We consider the key requirements for organic compounds which can be exploited in this manner and the range of control which is possible. The research leading to these results has received support from the European Community's Seventh Framework Programme [FP7/2007-2013] under grant agreements n° 218331 NaPolyNet and nº 226716.

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Current Synchrotron-based Research in Portugal 2014 Structure of Materials

The development of preferred orientation in PEO Electrospun Fibres Geoffrey Mitchella, Saeed Mohanb & Mohamed Al-Azabc a

Centre for Rapid and Sustainable Product Development Institute Polytechnic of Leiria. Marinha Grande, Portugal b Department of Chemistry, University of Reading, Whiteknights, Reading UK c Department of Chemistry, Faculty of Science, United Arab Emirates University, UAE Email of corresponding author: geoffrey.mitchell@ipleiria.pt

Keywords: Polymer morphology, electrospinning, orientation Electrospinning is a procedure for the preparation of nanoscale fibres through the application of a high voltage to a droplet of polymer solution. The charging of the droplet generates a force which draws the fibre towards the grounded electrode and overcomes the surface tension to generate fine fibres. We have used small-angle x-ray scattering procedures to study the morphology and especially the preferred orientation these fibres. We explore the influence of a rotating collector on the internal structure of polyethylene oxide fibres electrospun from a solution in water. We find that above a threshold collector speed, the mean fibre diameter reduces as the speed increases and the fibres are further extended. Small-angle and wide-angle X-ray scattering techniques show a preferred orientation of the lamellar crystals normal to the fibre axis which increases with collector speed to a maximum.

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Current Synchrotron-based Research in Portugal 2014 Structure of Materials

SAXS/WAXS study on electrically conducting nanocomposite Imran Khana, Artur Mateusa & Geoffrey Mitchella a

Centre for Rapid and Sustainable Product Development, Institute Polytechnique Leiria, Portugal Email of corresponding author: imran.khan@ipleiria.pt

Keywords: Thermosetting polyurethane, carbon nanotubes, carbon black, electrical conductivity, percolation, SAXS, WAXS We present a recent work using thermosetting polyurethane (TSPU) with multi-wall carbon nanotubes (MWCNT) and carbon black (CB), as fillers to obtain electrically conducting nanocomposites for different applications like ESD, EMI etc. A percolation threshold of 0.5%wt and 3%wt is observed in case of TSPU/MWCNT and TSPU/CB nanocomposites, respectively. The dispersion of fillers (MWCNT, CB) in thermosetting polyurethane (TSPU) is a critical work that further facilitates networking. This intense dispersion process is achieved, first by dispersing MWCNT into dimethylformamide (DMF) with ultra-sonication and then mixing with polyol followed by heating to get fully dispersed mixture that is further mixed with isocyanate to get desired nanocomposite. A 3-D network of MWCNT within the TSPU matrix becomes the source of electronic conduction that can be explained with the help of percolation theory. Synchrotron radiations source (SAXS, WAXS) is one of the best structural/morphological tools that were used to understand the dispersion and networking of fillers (MWCNT, CB) in TSPU matrix. We are able to interpret the formation of the percolated network, on the basis of morphology study, that cause to enhance electrical conductivity of nanocomposite. Our method provide a simple and effective rout for carbon nanotube dispersion in thermosetting polyurethane matrix with dramatically increased maximum electrical conductivity 2.06*10-1S/m (5%wt) and 5.41*10-2 S/m (10%) for MWCNT and CB respectively.

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Current Synchrotron-based Research in Portugal 2014 Structure of Materials

Morphology changes during in-situ mechanical testing of scaffold fibres using SAXS Ana Tojeiraa, Tânia Vianaa, Sara Biscaiaa, Christina Kamma-Lorgerb & Geoffrey Mitchella a

Centre for Rapid and Sustainable Product Development, Polytechnic Institute of Leiria, Marinha Grande, Portugal b NCD Beamline, ALBA Synchrotron Light Source, Barcelona, Spain Email of corresponding author: ana.tojeira@ipleiria.pt

Keywords: Bone Tissue Engineering, Scaffold fibres, Mechanical properties, Morphology, Small-angle X-ray scattering Complex and highly precise technologies have been currently used to respond to healthcare demands, such as treatment for bone defects, with customised products available within shorter periods of time. Additive Manufacturing (AM) has been a reliable approach to obtain tri-dimensional constructs with controlled architectures (best fitting to the outline of the defect) and functions (temporary mechanical support to the growing tissue and contact cues to the proliferating cells). Properties of tissue engineered-scaffolds do not depend exclusively on the biomaterial itself but also in its processing conditions. Hence, scaffold constructs have been extensively optimised in order to mimic the chemical composition and physical architecture (at different scales) of natural extracellular matrix to promote cell adhesion, proliferation, differentiation and new tissue formation. In this study, poly(ε-caprolactone) (PCL) based fibres were produced by Fused-Deposition Modelling (via Bioextruder) and were submitted to mechanical deformation. Time-resolving x-ray scattering experiments were performed using a monochromatic x-ray source – the beamline NCD at the ALBA Synchrotron Light Source in Barcelona, Spain. From such experiments, highly anisotropic characteristics of the SAXS patterns indicated a high level of preferred orientation of the crystals in consequence of the extrusion process. By applying deformation of the scaffold fibres, SAXS patterns recorded, on a continuous basis, the alteration of the morphology correlated with deformation. These data were used in conjunction with the results of mechanical properties tests to identify the most appropriate processing parameters to use in preparing bone scaffolds for further testing.

References S.J. Hollister, 2009. Adv Mater, 21, 3330-3342. C. Cui, S.M. Jorgensen, D.R. Eaker, E.L. Ritman, 2008. Coherent X-ray Scattering for Discriminating Biocompatible Materials in Tissue Scaffold. In S.R. Stock (ed), Developments in X-Ray Tomography, VI, Bellingham, Wash, California (USA). A. Tojeira, S. Biscaia, Viana, T., P.J. Bártolo, G.R. Mitchell, 2014. Structure Development during Additive Manufacturing. In P.J. Bártolo et al, High Value Manufacturing, pages, CRC Press – Taylor and Francis Group,221-226 London (UK).

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Current Synchrotron-based Research in Portugal 2014 Structure of Materials

In situ studies of shape memory alloys F.M. Braz Fernandesa, K.K. Mahesha, J.P. Oliveiraa, R.J.C. Silvaa & N. Schellb a

CENIMAT - I3N, Materials Science Department, FCT/UNL, Monte de Caparica, Portugal b Institute of Materials Research, Helmholtz-ZentrumGeesthacht, Geesthacht, Germany Email of corresponding author: fbf@fct.unl.pt

Keywords: Shape Memory Alloys, XRD, Synchrotron Radiation

u.]

The Shape Memory Effect (SME) and Superelasticity (SE), involving a phase transformation, requires the use of techniques where the structural changes may be determined from different perspectives. A great number of techniques is used, going from the structural morphology by optical and electron microscopy (SEM, TEM), to the crystallographic parameters determination by diffraction, either of X-rays (XRD), neutrons (ND) or electrons (selected area electron diffraction, SAED, in association with TEM). AFM has also been used to characterize the surface topography, mainly in the case of thin films. Another group of techniques uses the dependence of thermal, volume, or electrical characteristics of the material as a function of the structure. The fact that SME and SE are related with a mechanical / structural interaction, makes the mechanical testing an interesting tool for the determination of the stress conditions capable of inducing the austenite ↔ martensite transformation (SIM), besides the classical mechanical characterization of each phase in their different domains of existence. In situ XRD has been used to follow the structural evolution during the production of crystallized thin films or during thermal cycling. The singularities of the SME and the SE make the simultaneous combination of several characterization techniques a very interesting approach: (i) in situ study of the deformation of SMA by diffraction (of neutrons or X-rays), (ii) XRD in situ analysis of the structural evolution during thermal treatment / cycling.

Inte nsit y [a.

Resistivity

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66.0

2nd Cycle

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BM HAZ FZ HAZ

50.0 -100

-80

-60

-40

-20

0

20

40

60

BM

2 Theta[°]

(a)

(b)

(c)

Figure 1. In situ studies: (a) thermal cycling, (b) laser welding (c) thermal cycling/ electrical resistivity measurement.

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80

100


Current Synchrotron-based Research in Portugal 2014 Structure of Materials

Study of Endodontic Files Using Synchrotron Radiation F. M. B. Fernandesa, J. P. Oliveiraa, A. Machadoa & N. Schellb a

CENIMAT - I3N, Materials Science Department, FCT/UNL, Monte de Caparica, Portugal b Institute of Materials Research, Helmholtz-ZentrumGeesthacht, Geesthacht, Germany Email of corresponding author: fbf@fct.unl.pt

Keywords: Shape Memory Alloys, X-Ray Diffraction, Stress-Induced Transformation In clinical conditions, Ni-Ti endodontic rotary files work under flexion/rotation. Published data on fracture focus on two approaches to this problem: mechanical bench assays and numerical calculations based on FEA. No data is reported on literature about a high spatial resolution structural analysis of endodntic files (neither straight, nor bent). The present research work aims at studying two different brand files: ProFile and M-Two. Previous work has been carried out as a Ph.D. thesis [2]. Remarkably, although different files resist differently to fracture, all brand new instruments seemed to fracture near the mid-point of flexure arch. This is comparable to what is seen clinically on intra-oral radiographs: files broken inside root canals typically separate at the mid-point of root canal curvature arch. Those findings strongly suggest an accumulation of residual stresses in Ni-Ti that might account for fracture of clinically reused instruments [3].

(a)

(b)

Figure 1. 3D plots of XRD patterns along (a) unbent (mostly austenite) and (b) bent (austenite + stress-induced martensite) Mtwo file (X-axis: 2θ; Y-axis: spot nr, points spaced 0.25 mm, from tip to handle).

References 1. M.H. Lee, A. Versluis, B.M. Kim, C.J. Lee, B. Hur, H.C. Kim. 2011. Int. Endod. J., 37, 1152-1157. 2. S.V. Correia. 2009. Service Study of Superelastic Ni-Ti Endodontic Files. Ph.D. Thesis (written in Portuguese). University of Lisbon, Lisbon, 3. S.V. Correia, M.T. Nogueira, R.J.C. Silva, L. Pires Lopes, F.M. Braz Fernandes. 2009. Phase Transformations in Ni-Ti Endodontic Files and Fatigue Resistance. In Proceedings ESOMAT 09, European Symposium on Martensitic Transformation and Shape Memory, Prague, Czech Republic, 07-11/09/2009. Paper ID-07004.

SM |9


Current Synchrotron-based Research in Portugal 2014 Structure of Materials

XESA – a programming system for the analysis of 2-D and 1-D X-ray Scattering Data from Polymers and other Soft Matter

Geoffrey Mitchella and Ana Tojeiraa a

Centre for Rapid and Sustainable Product Development Institute Polytechnic of Leiria. Marinha Grande, Portugal Email of corresponding author: geoffrey.mitchell@ipleiria.pt

Keywords: Small angle X-ray Scattering, Wide-angle X-ray scattering, preferred orientation, scripting Small-angle and Wide-angle X-ray Scattering techniques provide powerful methodologies for the analysis of the structure and morphology of polymers. When utilised together with the powerful x-ray sources available at a synchrotron such as the ESRF a large quantity of data can be produced. This can prove invaluable in the study of transitions as a function of temperature or the development of preferred orientation during deformation and flow. Typically a large number of time-resolved frames are obtained and these need processing to extract the quantitative parameters available. The nature of the models which are applied can vary enormously from system to system and we have developed over many years a flexible environment which facilities this analysis of large quantities of data. XESA – X-ray Experimental data handling for Structural Analysis [1] provides a command driven interactive or non-interactive system. The interactive system can be used to develop the script or code for the subsequent analysis, while the non-interactive mode can be for processing large sets of data. The core of XESA provides a programming environment with 1d and 2d Workspaces which can be mathematically manipulated together with a system of variables, subroutines, conditional branching and loops. A particular feature of XESA is that it contains routines for performing analysis of 2-d patterns arising from samples with a preferred anisotropy. Spherical Harmonics can be calculated and manipulated following the procedures described by Mitchell [2]. Peaks can be fitted with a variety of peak shapes using using a powerful non-linear least squares procedure. XESA provides facilities for correcting experimental data for the effects of absorption, polarisation and multiple scattering. Data can be scaled to absolute units and transformed to real space functions. Monte Carlo type minimisation can be performed using any function. XESA forms part of a larger programme suite which includes MESA – A molecular editor for structural analysis which provides facilities for constructing molecular models and calculating scattering and other functions from those models. Currently the XESA system runs in a linux operating system. 1. G.R. Mitchell. 2014. Scattering Methods for the Measurement of Orientation, Springer. 2. G.R. Mitchell. 2014. XESA. Centre for Rapid and Sustainable Product Development, Marinha Grande, (Portugal).

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Current Synchrotron-based Research in Portugal 2014 Environmental & Cultural Materials

Selenium in wastes from the old mine of São Domingos in southern Portugal (IPB): already attached to oxygen or retained in relics of former chalcogenide minerals? Maria O. Figueiredoa,b, Teresa P. da Silvaa, João P. Veigab, Maria J. Batistaa, Eduardo Salas-Colerac & Daniel P. de Oliveiraa a

Unity of Mineral Resources and Geophysics, National Laboratory of Energy and Geology (LNEG), Apartado 7586, 2610-999 Amadora b CENIMAT/I3N, Dept. Materials Science, Faculty of Sciences and Technology, New University of Lisbon, 2829-516 Caparica, Portugal c SpLine, Spanish CRG Beamline, European Synchrotron Radiation Facility (ESRF), BP 220-38043 Grenoble Cedex, France Email of corresponding author: ondina.figueiredo@lneg.pt

Keywords: mine wastes; São Domingos pyrite mine; selenium; XANES; Se K-edge Selenium is a naturally occurring element that may be released by various anthropogenic processes - mining, industry, agriculture. Despite being an essential nutrient for humans, animals and microorganisms, it configures a serious concern because it becomes toxic at concentrations slightly above the nutritional levels [1]. In the environment, this element displays a variety of oxidation states ranging from selenide (Se=) to Se6+, including Se4+ and elemental selenium (Se0). Recently [2], selenium contents above 900 ppm were assigned in mine wastes from the old sulphur factory at the São Domingos exhausted pyrite mine [3], exploited since Roman times until fourty years ago. In the perspective of sustainably remediating this mining site, an X-ray absorption spectroscopy study using synchrotron radiation, combined with X-ray diffraction, was carried out to clarify the speciation state of selenium and the nature of possible Se-carrier phase(s) in the debris left by this exhausted IPB mine. The results of this Se K-edge XANES study are described, showing that selenium does not significantly replace sulphur in the dominant sulphate phases of the mine waste materials, being rather linked to oxygen as a selenite anion (SeO3)= and remaining as a minor substituting selenide in residual sulphide phases contained in the mine debris. References 1. M. Lenz, P.N.L. Lens. 2009. Sci. Total Environ. 407, 3620-3633. 2. M.J. Batista, J.X. Matos, M.O. Figueiredo, D. de Oliveira, T.P. Silva, H. Santana, L. Quental, 2011, VIII Iberian Congr. Geochem., Castelo-Branco (Portugal). Extended Abstract, 6. http://repositorio.lneg.pt/bitstream/10400.9/1450/1/34970.pdf. 3. A.M. Álvarez-Valero, R. Pérez-López, J. Matos, M.A. Capitán, J.M. Nieto, R. Saéz, J. Delgado, M. Caraballo, 2008. Environ. Geol. 55, 1797-1809.

ECM |1


Current Synchrotron-based Research in Portugal 2014 Chemistry

Towards a sustainable recovery of valuable metals from mining residues: a XANES approach to tungsten speciation in debris from the Panasqueira mine Maria O. Figueiredoa,b, Teresa P. da Silvaa, Daniel P. de Oliveiraa, JoĂŁo P. Veigab, Eduardo Salas-Colerac & Maria J. Batistaa a

Unity of Mineral Resources and Geophysics, National Laboratory of Energy and Geology (LNEG), Apartado 7586, 2610-999 Amadora b CENIMAT / I3N, Dept. Materials Science, Faculty of Sciences and Technology, New University of Lisbon, 2829-516 Caparica, Portugal c SpLine, Spanish CRG Beamline, European Synchrotron Radiation Facility (ESRF), BP 220-38043 Grenoble Cedex, France Email of corresponding author: ondina.figueiredo@lneg.pt

Keywords: mining residues; Panasqueira mine; tungsten; XANES; W L-edges Tungsten displays unique physical-plus-chemical properties that hinder its replacement in relevant specialized industrial applications and render it a metal of high strategic importance, nowadays considered a critical commodity in the EU. Believed to be mainly transported by high-temperature magmatic fluids [1], tungsten has long been mined at Panasqueira (BeiraBaixa district), making Portugal the main European producer. With a history surpassing a century, this mine has given rise to a huge tonnage of debris (attaining 0.3% WO3 at Barroca Grande slimes [2]). Considering Horizon 2020 objectives and recent EU efforts to implement a sustainable retrieval of critical mineral resources, it is a mandatory purpose to enhance the recovery of tungsten from those mine tailings and, simultaneously, to identify mineral phases carrying other scarce metals (e.g. rhenium, that according to recent analyses [3] occurs in those debris at a level exceeding ten times its mean concentration in the Earth´s crust). The association W-Re was focused in studies on WO3 powders for electrochromic and catalytic applications and the occurrence of W5+ ions with a localized 5d1 electronic configuration was then assigned in (1-x)WO3-y.xReO2 synthetic combinations [4]. Accordingly, to pursue the above quoted objectives it is appropriate to look for the eventual occurrence of similar phases in the Panasqueira mine tailings by combining X-ray diffraction (XRD) with X-ray absorption spectroscopy at W L1- and L3-edges (XANES) using synchrotron radiation. The results of a preliminary experiment [5] carried out at the ESRF using the instrumental set-up of beam-line BM 25-A (SpLine) are described and the aims of a future experiment are briefly addressed. References 1. A. Bernard, R. B. Symons, W. I. Rose, 1990. Applied Geochem. 5, 317-326. 2. A. M. Botelho de Sousa, 1994. Estudos, Notas e Trabalhos, I.G.M., 36, 77-90. 3. ProMine project (Nano-particle products from new mineral resources in Europe, FP7-NMP-2008-LARGE-2) 4. A. Kuzmin et al. 1998. J. Appl. Phys,. 84, 5515. 5. MinReMol project (EXPL/AAG-REC/0978/2012), supported by FEDER Funds through FCT-MCTES.

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Current Synchrotron-based Research in Portugal 2014 Chemistry

Houston, we have a problem! Synchrotron radiation needed for structural characterization of new bio-inspired materials S. Quaresmaa, V. Andréa,b & M. T. Duartea a

Centro de Química Estrutural, Instituto Superior Técnico, Universidade de Lisboa, Av. Rovisco Pais, 1049-001 Lisbon, Portugal b CICECO, Universidade de Aveiro, 3010-193 Aveiro, Portugal. Email of corresponding author: silvia.quaresma@tecnico.ulisboa.pt

Keywords: BioMOFs, Mechanochemistry, Synchrotron radiation, Adenine, Gabapentin Polymeric systems in addition to mesoporous silicas and zeolites have been commonly used for the controlled release of drugs. However, some of these systems might present strong drawbacks: low drug-storage capacity, too rapid delivery and high toxicity. The use of Metal Organic Networks (MOFs) as new drug carriers has been proposed as a way to tackle these problems, requiring a biological-friendly composition with acceptable toxicity levels.[1-4] We are exploring a new pathway for the development of improved precursors to new bioinspired materials (BioMOFs), using safe metals and having active pharmaceutical ingredients (API) as linkers. This type of compounds is traditionally synthesized by solvothermal methods, but in this project we are also deeply engaged in using “green” techniques such as mechanochemistry and microwave. Gabapentin, a neuroleptic drug, was one of the first compounds that we used to synthesize new BioMOFs by mechanochemistry. New gabapentin coordination networks with several lanthanide chlorides (LnCl3, Ln= La3+, Ce3+, Nd3+, Er3+ and Y3+) were successfully synthesized and characterized.[5] More recently, we are exploring coordination networks with adenine and several safe metals such as Zn, Cu and others. Novel compounds have been synthesized and identified by X-ray powder diffraction, but in this case, despite the many efforts made to crystallize them, no single crystals suitable for single crystal X-ray diffraction analysis were obtained. It would be very important for this project if we could have the opportunity to collected data at ESRF for the structural characterization of these novel compounds. References 1. P. Horcajada, et al, 2006. Angew. Chem. Int. Ed., 45(36), 5974-5978. 2. S. Keskin, et al, 2011. Ind. Eng. Chem. Res., 50(4), 1799-1812. 3. A. C. McKinlay, et al, 2010. Angew. Chem. Int. Ed., 49, 6260 – 6266. 4. P. Horcajada, et al., 2012, Chem. Rev., 112(2), 1232-1268. 5. S. Quaresma, et al, 2013, Cryst. Growth Des., 13, 5007-5017.

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Current Synchrotron-based Research in Portugal 2014 Chemistry

In-situ small-angle x-ray scattering study of reaction times and morphology development in reaction injection moulding Geoffrey Mitchella & Artur Mateusa

a

Centre for Rapid and Sustainable Product Development Institute Polytechnic of Leiria. Marinha Grande, Portugal Email of corresponding author: geoffrey.mitchell@ipleiria.pt

Keywords: Polymer morphology, RIM, reaction kinetics Reaction injection moulding involves the mixing of two fluid reactants, in this work, a polyol and isocyanate and the injection of that mixture in to shaped mould. Reaction between the two components forms a solid product. Time-resolved small-angle x-ray scattering coupled with a specially designed laboratory scale RIM system are used to follow the development of structure during the RIM cycle. The small-angle x-ray scattering data coupled with data obtained from differential scanning calorimetry show that the characteristics of the morphology are fixed on a time scale much shorter than the reaction of the components. These results underpin the importance of including the consequences of phase separation and the morphology which develops in any predictive simulation of the RIM process.

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Current Synchrotron-based Research in Portugal 2014 Structural Biology

Superoxide Reductase – Structural characterization from two Archaea organisms Cristiana M. Sousaa, A. F. Pintob, J. V. Rodriguesb, M. Teixeirab, P.M. Matiasb, C.V. Romãob & T.M. Bandeirasa a

Instituto de Biologia Experimental e Tecnológica, Apartado 12, 2701-901 Oeiras, Portugal Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, Apartado 127, 2781-901, Oeiras, Portugal

b

Email of corresponding author: csousa@itqb.unl.pt

Keywords: Superoxide reductase; 1Fe-SOR; Archaeoglobus fulgidus; Ignicoccus hospitalis

stress

oxidation;

superoxide

anion;

Superoxide radical O2.- is a reactive oxygen species, known to be involved in a variety of cell toxicity mechanisms. While aerobes contain several antioxidant defence systems, such as superoxide dismutases and catalases, anaerobes and microaerophiles may depend only on the superoxide reductases (SOR) to keep oxygen toxic species below poisonous thresholds. SOR structures are organized in a seven-stranded β-barrel with an immunoglobulin-like fold, and the catalytic site is composed by one iron centre coordinated in a square-pyramidal geometry to four histidines and one cysteine. In the SOR resting state (ferric form) a glutamate residue is present as sixth ligand, completing an octahedral geometry around the iron atom. Our research focuses on the molecular mechanism of 1Fe-SORs, and aims to understand the role of the conserved glutamate and lysine residues, proposed to have an imperative role in the catalytic cycle. Herein we present the crystal structures of two wild-type SORs. References A.F. Pinto, J.V. Rodrigues, M. Teixeira. 2010. Biophys Acta., 1804(2), 285-97. T.M. Bandeiras, C.V. Romão, J.V. Rodrigues, M. Teixeira, P.M. Matias. 2010. Acta Crystallogr Sect F Struct Biol Cryst Commun, 66(Pt 3), 316-319. F.G. Pinho, C.V. Romão, A.F. Pinto, L.M. Saraiva, H. Huber, P.M. Matias, M. Teixeira, T.M. Bandeiras. 2010. Acta Crystallogr Sect F Struct Biol Cryst Commun, 66(Pt 5), 605-607.

SB |1


Current Synchrotron-based Research in Portugal 2014 Structural Biology

Structure and characterization of the family 46 carbohydrate-binding module (CBM46) of the endo-β-1,4glucanase B (CelB) from Bacillus halodurans Immacolata Vendittoa, Helena Santosa, Arun Goyalb, Luís Ferreiraa, Kazuo Sakkac, Harry Gilbertd, Carlos M.G.A. Fontesa & Shabir Najmudina a

CIISA-Faculdade de Medicina Veterinária, Avenida da Universidade Técnica, 1300-477 Lisbon, Portugal b Department of Biotechnology, Indian Institute of Technology Guwahati, Guwahati, Assam, India c Faculty of Bioresources, Mie University, Tsu 514-8507, Japan d Institute for Cell and Molecular Biosciences, University of Newcastle-upon-Tyne, Newcastle-upon-Tyne NE2 4HH, United Kingdom

Email of corresponding authors: adivenditto@fmv.utl.pt, shabir@fmv.utl.pt Keywords: family 46 carbohydrate binding module, endo-β-1,4-glucanase CelB, plant cell wall degradation, Bacillus halodurans

Carbohydrate Binding Modules (CBM), which improve the efficacy of associated Carbohydrate Active enZYmes (CAZYmes), are organized in families based on primary sequence homology (www.cazy.org). Currently, there are more than 65 different CBM families. Encoded at locus BH0603 (GenBank accession no. BA000004) in the genome of the alkaliphilic bacterium Bacillus halodurans [1] is a putative modular endo-β-1,4-glucanase (CelB) composed of N-terminal glycoside hydrolase family 5 catalytic module (GH5), followed by an immunoglobulin-like module (Ig) and a C-terminal family 46 CBM (CBM46). CBM46 was shown to bind insoluble forms of cellulose [2]. It forms a significant part of the CelB polypeptide. Truncation studies revealed that removal of CBM46 results in an enzyme with very limited overall action on cellulosic substrates [2]. Thus, these data suggest that CBM46 members might display unique properties within the CBM world and structural information on CBM46 will lead to a better understanding of its targetting function towards different substrates. We have solved the structure of CBM46 to a resolution of 2.3 Å using a SeMet derivative by SAD experiment using AUTOSOL from the PHENIX suite. Subsequently, the structure of the native form was solved to a resolution of 2.4 Å [3]. CBM46 has a classic β-jelly roll fold and is a dimer in the crystal. It has a very hydrophobic cleft lined with aromatics amino acid residues, which are thought to be important in the targeting role. We have mutated these and tested their role in substrate binding by affinity gel electrophoresis and isotherm titration calorimetry experiments. References 1. H. Takami, K. Nakasone, Y. Takaki, G. Maeno, P.R. Sasaki, N. Masui, F. Fuji, C. Hirama, Y. Nakamura, N. Ogasawara, S. Kuhara, K. Horikoshi. 2000. Nucleic Acids Res., 28, 4317–4331. 2. B.M. Wamalwa, M. Sakka, P.M. Shiundu, K. Ohmiya, T. Kimura, K Sakka. 2006. Appl Environ Microb, 72, 6851–6853. 3. I. Venditto, H. Santos, L.M.A. Ferreira, K. Sakka, C.M.G.A. Fontes, S. Najmudin. 2014. Acta Crysta F, reference: BO5134.

SB |2


Current Synchrotron-based Research in Portugal 2014 Structural Biology

Structural and functional characterization of novel Carbohydrate Binding Module families in Ruminococcus flavefaciens cellulosome Immacolata Vendittoa, Vânia Fernandesa, Maja Gro Rydahlb, Arun Goyalc, Maria S.J. Centenoa, Luís M.A. Ferreiraa, Harry J. Gilbertd, William G.T. Willatsb, Carlos M.G.A. Fontesa & Shabir Najmudina a

CIISA-Faculdade de Medicina Veterinária, Avenida da Universidade Técnica, 1300-477 Lisbon, Portugal Department of Plant Biology and Biotechnology, University of Copenhagen, 1871 Frederiksberg, Denmark c Department of Biotechnology, Indian Institute of Technology Guwahati, Guwahati, Assam, India d Institute for Cell and Molecular Biosciences, University of Newcastle-upon-Tyne, Newcastle-upon-Tyne NE2 4HH, United Kingdom b

Email of corresponding authors: adivenditto@fmv.utl.pt, shabir@fmv.utl.pt Keywords: carbohydrate binding module, plant cell wall degradation, Microarray, Ruminococcus flavefaciens Cellulosomes are multienzyme complexes that catalyze the efficient degradation of plant cell wall polysaccharides. A range of anaerobic bacteria and fungi were shown to produce cellulosome systems and recently the genome of a ruminal cellulolytic bacterium, Ruminococcus flavefaciens was sequenced providing an opportunity to discover novel cellulosomal enzymes. The genome sequence of R. flavefaciens revealed that it encodes more than 200 dockerin containing proteins, a significant proportion of them with unknown function (1). Since cellulosomes play a key role in plant cell wall deconstruction, it is believed that they comprise an extremely interesting source for the discovery of new Carbohydrate-Active Enzymes (CAZymes) and Carbohydrate Binding Modules (CBMs). We used Affinity gel electrophoresis (AGE) and Microarray technology to identify novel ligand specificities in cellulosomal modules of unknown function. Our data reveal that there are 11 novel CBMs families. The structures of two of these, Rf1 and Rf6, have been solved. Rf1 was solved to a resolution of 1.5 Å by a SeMet-MAD experiment in the trigonal space group R32 (H32). Rf1 comprises 2 anti-parallel β-sheets forming a β-sandwich with a long helix, giving a new fold. It is a dimer burying a surface of 1600 Å2, with the two monomers binding to each other as a 3-pronged clamp covering the potential substrate binding cleft. Rf6 was solved by SeMet-SAD experiments in the orthorhombic space group I212121 and cubic space group I213 to a resolution of 2.0 Å and 1.5 Å, respectively. The resolution of the orthorhombic form was improved to 1.75 Å using the native Rf6. The Rf6 structure also comprises a novel βsandwich fold made of two anti-parallel β-sheets. It has a flat binding surface which is dominated by 2 pairs of aromatics, Tyr 563-Trp 564 & Trp 606-Trp 607, and Tyr 597. These are the likely key residues for substrate recognition. References M.T. Rincon, T. Čepeljnik, J.C. Martin, R. Lamed, Y. Barak, E.A. Bayer, H.J. Flint. 2005. J. Bacteriol.187 (22), 7569.

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Current Synchrotron-based Research in Portugal 2014 Structural Biology

Structural insights into O2 inactivation in the [NiFeSe] Hydrogenase from Desulfovibrio vulgaris Hildenborough Pedro M. Matiasa,b, Marta C. Marquesa, Ricardo Coelhoa & Inês A. C. Pereiraa a

Instituto de Tecnologia Química e Biológica Universidade Nova de Lisboa, Apartado 127, 2780 Oeiras (Portugal) b Instituto de Biologia Experimental e Tecnológica, Av. da República, EAN, 2780 Oeiras (Portugal) Email of corresponding author: matias@itqb.unl.pt

Keywords: Biological Hydrogen Production, Hydrogenases, 3D structure At a critical stage of the fossil fuel crisis and global warming, hydrogen is drawing increased attention as an energy carrier with great potential to become an environment-friendly fuel in the future. Using hydrogenases, biological hydrogen production from renewable sources can replace the expensive Pt-based catalysts presently used to transform fossil fuels. Hydrogenases can also be used in bioelectrical devices for the direct conversion of hydrogen into electricity. The most studied hydrogenases are of the [NiFe] type, and while most [NiFe] hydrogenases are inactivated by oxygen, the few known O2-tolerant enzymes are hydrogen-uptake enzymes, unsuitable for hydrogen production, due to strong product inhibition. In contrast, the [NiFeSe] hydrogenases, where a selenocysteine is bound to the nickel, are very attractive alternatives because of their high hydrogen production activity and fast reactivation after O2 exposure. High-resolution crystallographic 3D structures of the soluble form of the [NiFeSe] hydrogenase from D. vulgaris Hildenborough in three different redox states (oxidized asisolated, H2-reduced and air re-oxidized) revealed the structural changes that take place at the active site during enzyme reduction and re-oxidation. These results provide new insights into the pathways of O2 inactivation in [NiFeSe] hydrogenases, different from those found in [NiFe] hydrogenases. This knowledge is essential to understand their properties and for the correct design of devices using the [NiFeSe] enzymes for H2 production.

References H.S. Lee, W.F. Vermaas, B.E. Rittmann, 2010. Trends Biotechnol., 28, 262-271. B. Friedrich, J. Fritsch, O. Lenz, 2011. Curr. Opin. Biotechnol., 22, 358-364. A. Parkin, G. Goldet, C. Cavazza, J.C. Fontecilla-Camps, F.A. Armstrong, 2008. J. Am. Chem. Soc., 130, 1341013416. C.S.A. Baltazar, M.C. Marques, C. M. Soares, A.M. De Lacey, I.A.C. Pereira, P.M. Matias, 2011. Eur. J. Inorg. Chem., 2011, 948-962.

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Current Synchrotron-based Research in Portugal 2014 Structural Biology

Stabilization and Crystallization Studies on a Molecular Chaperone of Escherichia coli PaoD A.R. Otrelo-Cardosoa, V. Schwuchowb, D. Rodriguesa, E.J. Cabritaa, M.J. Romãoa, S. Leimkühlerb & T. Santos-Silvaa a

REQUIMTE–CQFB, Departamento de Química, Faculdade de Ciências e Tecnologia, Universidade Nova de Lisboa, 2829-516 Caparica, Portugal b Institute of Biochemistry and Biology, Department of Molecular Enzymology, University of Potsdam, Karl-Liebknecht Str. 24-25, 14476 Potsdam, Germany Email of corresponding author: a.cardoso@campus.fct.unl.pt

Keywords: Chaperone, Molybdenum cofactor, Molybdoenzyme, Ionic liquid. Molybdenum is found in the active site of a diverse group of enzymes that are present in almost all forms of life. For molybdoenzymes of the xanthine oxidase family, the final step of the protein synthesis is the insertion of the matured cofactor (Moco) [1]. The insertion of Moco into the target apo-enzymes requires specific cofactor storage/binding proteins such as PaoD which is a chaperone for Periplasmic Aldehyde Oxidoreductase from Escherichia coli, PaoABC [2,3]. The main objective of this work is to stabilize and characterize PaoD. As described in the literature [4], either the anionic or cationic counterpart of the ionic liquids (IL) can stabilize proteins through protein-ion interactions. Since PaoD is highly unstable, ionic liquids were used to avoid protein precipitation and promote the crystallization process. In fact, the thawing of PaoD in the presence of the IL [C4mim]Cl or [C2OHmim]PF6 lead to a reduction of protein precipitation and the identification of two crystallization conditions using ammonium sulphate or PEG as precipitating agents. These crystals were used in a diffraction experiment and two data sets, with a maximum resolution of 3.4 and 2.6 Å, were collected using a 0.97 Å wavelength radiation on the ID23-1 beamline of the ESRF. Structure determination is in process, with selenomethionine expression and heavy atoms derivatives. STD – NMR and DLS experiments have also been performed in order to investigate the effect of the ionic liquids in the stabilization process. STD shows an interaction between the acidic ring protons of the cation and, most likely, negatively charged residues at the protein surface. DLS assays show a reduction of the overall size of the protein aggregates in presence of ionic liquids. These interactions may prevent protein precipitation, increasing the stability and aid in the crystallization process [5]. References 1. M.J. Romão. 2009. Dalton Trans, 4053-4068. 2. M. Neumann, S. Leimkühler. 2011. Biochem Res Int, 2011, 1-13. 3. M. Neumann, G. Mittelstädt, C. Iobbi-Nivol, M. Saggu, F. Lendzian, P. Hildebrandt, S. Leimkühler. 2009. FEBS J, 276, 2762-2774. 4. M. Kowacz, A. Mukhopadhyay, A.L. Carvalho, J.M.S.S. Esperança, M.J. Romão, L.P.N. Rebelo. 2012. CrystEngComm, 14, 4912-4921. 5. A.R. Otrelo-Cardoso,V. Schwuchow, D. Rodrigues, E.J. Cabrita, S. Leimkühler, M.J. Romão, T. Santos-Silva. 2014. PLoS ONE, 9, e87295.

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Current Synchrotron-based Research in Portugal 2014 Structural Biology

Structure of a type III cohesin-dockerin complex from R. flavefaciens revealed by X-Ray crystallography Pedro Bulea, Carlos Fontesa & Shabir Najmudina a

Faculdade de Medicina Veterinária da Universidade de Lisboa, 1300-477 Lisboa

Email of corresponding authors: shabir@fmv.ulisboa.pt, adpedrobule@fmv.ulisboa.pt

Keywords: Ruminococcus cristallography,

flavefaciens,

celllulosome,

cohesin-dockerin,

X-ray

Cellulosomes are massive cell bound multienzyme complexes tethered by macromolecular scaffolds that centralize anaerobic bacteria’s efforts to hydrolyze plant cell wall polysaccharides, a major untapped source of carbon and energy. Integration of cellulosomal components occurs via highly ordered protein: protein interactions between cohesins, located in the scaffold, and dockerins, usually found in the enzymes. The proposed cellulosomic architecture for the Ruminococcus flavefaciens strain FD-1 consists in a scaffoldin (ScaB) that acts as the backbone to which other components will attach, possessing 9 cohesins and a dockerin with an X-module that binds to the cohesin on ScaE that in turn covalently attaches itself to the cell wall. ScaA dockerin binds to ScaB cohesins allowing more carbohydrate active modules to be assembled. ScaC acts as an adaptor that binds to ScaA and ScaB cohesins increasing the repertoire of proteins that can integrate the complex. In previous studies, we screened for novel cohesin: dockerin complexes which led to the identification of a total of 58 probable coh-doc pairs. Four were selected for subsequent structural and biochemical characterisation based on the quality of their expression and diversity in their specificities. One of these is the C12D22, which comprises the cohesin from the adaptor ScaC and the dockerin from a CBM containing protein. This complex has been purified, crystallized and data collected to a resolution of 2.7 Å (hexagonal, P65) and 2.1 Å (orthorhombic, P212121) from two different crystalline forms, respectively. References C.M.G.A Fontes, H.J Gilbert. 2010. Annu. Rev. Biochem., 79, 655-81. M.E.B. Miller, D.A. Antonopoulos, A. Hernandez, J. Thimmapuram, B. Henrissat, P.M. Coutinho, I. Borovok, S. Jindou, R. Lamed, H.J Flint, E.A. Bayer, B.A. White. 2009. Plos One, 4 (8) e6650. M.T. Rincon, B. Dassa, H.J. Flint, A.J. Travis, S. Jindou, I. Borovok, R. Lamed, E.A. Bayer, B. Henrissat, P.M. Coutinho, A.D. Antonopoulos, M.E.B. Miller, B.A. White, 2010. PLos One, 5 (8), e12476.

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Current Synchrotron-based Research in Portugal 2014 Structural Biology

Use of Gold Nanoparticles as Additives in Protein Crystallization Diana Ribeiroa, Alina Kulakovaa, Pedro Quaresmab, Eulália Pereirab, Cecília Bonifácioa, Maria J. Romãoa, Ricardo Francoa & Ana L. Carvalhoa a

REQUIMTE/CQFB, Departamento de Quím Universidade Nova de Lisboa, 2829-516 Caparica, Portugal b Universidade do Porto, 4169-007 Porto, Portugal Email of corresponding author: almc@fct.unl.pt

Keywords: Gold nanoparticles (AuNPs), crystal growth improvement, nucleation, X-ray diffraction Several obstacles can be present when attempting to grow protein crystals and subsequently determine its structure and strategies that facilitate the production of suitable crystals for Xray diffraction techniques, such as nucleating agents, are highly sought for. Since gold nanoparticles (AuNPs) exhibit unique properties that have made them a very attractive material for application in biological assays and the potentially interesting interactions between AuNPs and biological macromolecules [1, 2], we investigated AuNPs-induced protein crystal growth [3, 4]. Differently functionalized AuNPs were tested as additives in co-crystallization studies with model proteins (hen egg white lysozyme (HEWL), ribonuclease A (RNase A) and proteinase K) as well as with case studies where there were problems in obtaining well-diffracting crystals. Trials were performed considering different crystallization drawbacks, from total absence of crystals to improvement of crystal morphology, size, twinning and number of crystals per drop. Improvement of some of these factors was observed in the cases of HEWL, RNase A, phenylalanine hydroxylase (PAH), myoglobin, native aldehyde oxidase (AOH) and human albumin. In these proteins, the presence of the AuNPs promoted an increase in the size and/or better crystal morphology. From the systematic trials and subsequent observations, it can be concluded that the introduction of AuNPs should definitely be considered in crystal optimization trials to improve previously determined crystallization conditions [3].

References 1. J. Kim, J.W. Grate, P. Wang. 2008. Trends Biotechnol., 26, 639−46. 2. F. Kurniawan. 2008. New Analytical Applications of Gold Nanoparticles, 2−50, University of Regensburg, Germany. 3. D. Ribeiro, A. Kulakova, P. Quaresma, E. Pereira, C. Bonifácio, M.J. Romão, R. Franco, A.L Carvalho. 2014. Cryst. Growth Des. 14, 222–227. 4. F. Hodzhaoglu, F. Kurniawan, V. Mirsky, C. Nanev. 2008. Cryst. Res. Technol., 593, 588−593.

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Current Synchrotron-based Research in Portugal 2014 Structural Biology

Crystal Structure of the first Type I ScaB Cohesin from Bacteroides cellulosolvens Cellulosome Kate Camerona, Carlos Fontesa & Shabir Najmudina a

Faculdade de Medicina Veterinária da Universidade de Lisboa, 1300-477 Lisboa, Portugal Email of corresponding authors: kate.cameron44@gmail.com, shabir@fmv.utl.pt

Keywords: Cohesins, Dockerins, Cellulosomes, Plant cell wall degradation, Protein:Protein interactions, Bacteroides cellulosolvens Cellulosomes, secreted by anaerobic micro-organisms, are highly elaborate, extracellular, multi-enzyme complexes, responsible for the efficient degradation of plant cell wall polysaccharides. Cohesin and dockerin recognition pairs are crucial for the architecture of the cellulosome [1]. Integration of cellulosomal components was first shown to occur in the Clostridium spp. via highly ordered protein: protein interactions between cohesins located in a primary macromolecular scaffold and dockerins found in the enzymes (type I interaction). Primary scaffoldins also contain a C-terminal divergent type II dockerin that specifically recognizes type II cohesins located on the bacterium’s envelope, thereby providing a mechanism for the cell surface attachment of cellulosomes. The cellulosome of Bacteroides cellulosolvens also has a similar two-component scaffoldin arrangement, except that the types of cohesins carried by the primary and anchoring scaffoldins are reversed [2]. The cellulosome of B. cellulosolvens comprises a primary scaffoldin, named ScaA, that contains eleven type II cohesins with a C-terminal type I dockerin and an anchoring scaffoldin, named ScaB, that bears ten type I cohesins and an S-layer homology module that tethers the whole cellulosome to the bacterial cell surface. In order to understand the specificity determinants of these type I interactions, we have co-expressed, purified and crystallised the ScaA dockerin complexed with the seventh cohesin of the ScaB. However, data we obtained from the crystals indicated that just a single ScaBCoh7 module was present, with Matthew’s coefficient of 2.4 Å3 Da-1 and solvent content of 49%. The data extends to a resolution of 1.84 Å in the orthorhombic space group P212121. The structure was solved by molecular replacement. The C-terminal His6-tag binds to the dockerin-binding cleft of the symmetryrelated Coh7 molecules, thereby occluding the ScaA-dockerin from binding. This cohesin has a similar fold to the known cohesins from the Clostridium spp. with a sequence identity ranging from 30-40%, further work is being carried out where the His6-tag will be introduced at the N-terminus of the ScaBCoh7 or on the ScaADoc. References 1. C.M.G.A. Fontes, H.J. Gilbert, 2010. Annu. Rev. Biochem., 79, 655-81. 2. Q. Xu, E.A. Bayer, M. Goldman, R. Kenig, Y. Shoham, R. Lamed. 2004. J. Bacteriol., 186, 968-77.

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Current Synchrotron-based Research in Portugal 2014 Structural Biology

Structural Studies on Human and Mouse Aldehyde Oxidases C. Coelhoa, M. Mahrob, A. Fotib, E. Garattinic, S. Leimkühlerb, T. Santos-Silvaa & M. J. Romãoa a

REQUIMTE/CQFB, Faculdade de Ciências e Tecnologia, Universidade Nova de Lisboa, 2829-516 Caparica, Portugal;bDepartment of Molecular Enzymology, Institute of Biochemistry and Biology, University of Potsdam, Karl-Liebknecht-Str. 24-25, 14476 Potsdam, Germany;cLaboratory of Molecular Biology, Istituto di Ricerche Farmacologiche “Mario Negri”, via La Masa 19, 20156 Milan, Italy. E-mail of corresponding author: c.coelho@fct.unl.pt Keywords: Aldehyde Oxidase (AOX); Molybdenum-Enzymes; Crystal Structure Aldehyde oxidases (AOX; E.C. 1.2.3.1) are molybdo-flavoenzymes with broad substrate specificity, oxidizing aldehydes and N-heterocycles. AOX belongs to the xanthine oxidase (XO) family of Mo-containing enzymes. The true physiological function of AOX is still unknown, although it is recognized to play a role in the metabolism of compounds with medicinal and toxicological relevance [1]. The protein importance has increased in recent years since it is substituting Cyt-P450 as the central drug metabolizing system in humans. We have solved the 3D structure of mouse AOX3 to 2.9 Å resolution at beamline ID14-1 at the ESRF [2]. This is the first structure of an aldehyde oxidase, providing important evidences on substrate and inhibitor specificities between AOX and XO (Fig.1). The complement of AOX proteins in mammals varies from one in humans (hAOX1) to four in rodents (mAOX1, mAOX3, mAOX4 and mAOX3L1) as a result of evolutionary genetic events [1]. Due to this unusual complement of AOX genes in different animal species, conclusions regarding protein metabolism in humans cannot be taken exclusively from the mouse model. To overcome this limitation we have also crystallized and solved the crystal structure of the human recombinant protein (hAOX1) to 2.7 Å resolution (unpublished data). There are important differences between the human and mouse proteins located particularly at the active site and in the substrate funnel. Structural comparisons between the two proteins will allow drawing relevant mechanistic and functional implications for the two classes of enzymes. The ensemble of these structures provides important insights into the role of aldehyde oxidases, contributing to elucidate the clinical metabolism implications of hAOX1 in human which has particular relevance for novel therapeutically drug design studies. This work would not be possible without access to a synchrotron facility due to the limited size of the crystals and diffraction. References 1. E. Garattini, M. Fratelli, M. Terao. 2008 Cell Mol Life Sci, 65, 1019-1048. 2. C. Coelho, M. Mahro, J. Trincao, A.T. Carvalho, M.J. Ramos, M. Terao, E. Garattini, S. Leimkuhler, M.J. Romão. 2012. J Biol Chem, 287, 40690-40702. Figure 1. (a) homodimeric mAOX3 crystal structure, (b) arrangement and distances between the different protein cofactors.

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Current Synchrotron-based Research in Portugal 2014 Structural Biology

The crystal structure of Erythrin Joana P. Carrilhoa, Pedro M. Matiasa, Miguel Teixeiraa & Célia V. Romãoa a

Instituto de Tecnologia Química e Biológica, Oeiras, Portugal Email of corresponding author: joanacarrilho@itqb.unl.pt

Keywords: Oxidative stress, Hyperthermophile, Erythrin, Hydrogen peroxide, Rubrerythrins The erythrins are the simplest example of the rubrerythrins family, composed only by the one four helix bundle domain containing a diiron center [1]. The identification of the first rubrerythrin is in the sulfate reducing bacterium Desulfovibrio vulgaris, they have been found in the three life domains. The rubrerythrins are a large family of enzymes present in aerobic prokaryotes (Bacteria and Archaea), as well as in anaerobic protozoa. Rubrerythrins, together with superoxide reductases, constitute a new family of enzymes that couples the reduction of reactive oxygen species to the oxidation of reducing equivalents from NAD(P)H [2]. This type of reactivity contrasts with the one of classic enzymes, which perform the detoxification by dismutation of superoxide ion and hydrogen peroxide. The erythrin from the hyperthermophile Acidianus ambivalens, characterized by the ability to grow by oxidation or reduction of sulfur with O2 or H2 [3], was overexpressed in E. coli, yielding a dimeric protein harboring a diiron center, confirmed by UV-Visible and EPR spectroscopies. By the absence of additional domains in this protein was possible the study in detail the molecular mechanism of hydrogen peroxide. Protein crystals were obtained, which diffracted to a resolution 1.5 Å and the structure was then solved by Molecular Replacement. A detailed description of this structure will be presented.

References 1. T. Wakagi. 2003. FEMS Microbiol Lett, 222(1), 33-37. 2. J. LeGall, et al. 1988. Biochemistry, 27(5), 1636-1642. 3. T.M. Bandeiras, et al. 2002. FEBS Lett, 531(2), 273-277.

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Current Synchrotron-based Research in Portugal 2014 Structural Biology

Structural Characterization of a VIVO(pic)2-Lysozyme Adduct – New Insights on Vanadium Complexes as Potential Therapeutics Marino F. A. Santosa, Isabel Correiab, Ana R. Oliveiraa, Eugenio Garribbac, João C. Pessoab & Teresa Santos-Silvaa a

REQUIMTE-CQFB, Departamento de Química, Faculdade de Ciências e Tecnologia, Universidade Nova de Lisboa, 2829-516 Caparica, Portugal b Centro de Química Estrutural, Instituto Superior Técnico, Universidade de Lisboa, Av. Rovisco Pais, 1049-001 Lisboa, Portugal c Dipartimento di Chimica e Farmacia, and Centro Interdisciplinare per lo Sviluppo della Ricerca Biotecnologica e per lo Studio della Biodiversità della Sardegna, Università di Sassari, Via Vienna 2, I-07100 Sassari, Italia Email of corresponding author: mf.santos@campus.fct.unl.pt

Keywords: Vanadium, X-ray crystallography, Vanadium(IV)-protein adduct, Lysozyme Nowadays, the role of vanadium and vanadium complexes in biological systems is carefully studied due to their possible physiological actions namely as insulin-enhancer, as anti-cancer and as anti-parasitic agents [1] The coordinated ligands are particularly important as these are able to improve the absorption and possibly the transport and uptake of vanadium to the cells, reducing the dose necessary for producing the same effect [1]. In this work, [2] we report the interaction of a vanadium picolinate complex with hen egg white lysozyme (HEWL). HEWL crystals were soaked with VIVOSO4:picolinic acid (molar ratio of 1:3) and diffracted up to 1.28 Å resolution. X-ray crystallography shows that VIVO(pic)2 complex is covalently bound to the COO− of the side chain of Asp52 of HEWL. A relatively long VIV=O bond distance (~1.82 Å) was obtained (theoretical value: ~1.60 Å). However, EPR and DFT results confirm the oxidation state of vanadium: VIV. We propose that the long VIV=O bond was a result of the exposure of the HEWL-VIVO(pic)2 crystals to the intense X-ray beam which induce a progressive reduction of VIV to VIII. This was confirmed by analysis of several sub-sets of the collected data. In conclusion, we present the first example of a VIVO-protein adduct characterized by X-ray crystallography. Similar results are expected to occur with human serum transferrin – responsible for vanadium in vivo transport – which could be important for the development of new anti-diabetic drugs. References 1. S. Mehtab et al., 2013. J. Inorg. Bio., 121, 187-195. 2. M. F. A. Santos et al., 2014. in prep.

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Current Synchrotron-based Research in Portugal 2014 Structural Biology

Structure and function of a membrane enzyme from the CDP-alcohol phosphatidyltransferase family Przemyslaw Noglya*, Ivan Gushchinb*, Alina Remeevab*, Ana M. Estevesa, Nuno Borgesa, Pikyee Maa, Cecília Pintoa, Andrii Ishchenkoc, Sergei Grudinind, Ekaterina Roundb, Isabel Moraese,f, Valentin Borshchevskiyc, Helena Santosa, Valentin Gordeliyb,c & Margarida Archera a

ITQB-UNL, Oeiras, Portugal; bIBS, CNRS, Grenoble, France, cICS-6, Juelich, Germany d Lab J Kuntzmann, Grenoble, France, eICL, London, UK, fDLS, Didcot, UK *Equal contribution, Email of corresponding author: archer@itqb.unl.pt

Keywords: membrane proteins, CDP-alcohol phosphatidyltransferases, phospholipid biosynthesis, X-ray structure Phospholipids play major roles in the structure and function of all cell membranes. Most integral from the large CDP-alcohol phosphatidyltransferase family are involved in phospholipid biosynthesis across the three domains of life. They share a conserved sequence pattern and catalyze the displacement of CMP from a CDP-alcohol by a second alcohol. We report here the first crystal structure of a bifunctional enzyme comprising a cytoplasmic nucleotidyltransferase domain (IPCT) fused with a membrane CDP-alcohol phosphotransferase domain (DIPPS) at 2.65 Å resolution (Fig. 1). The bifunctional protein dimerizes through the DIPPS domain, each comprising six transmembrane α-helices. The active site cavity is hydrophilic and widely open to the cytoplasm with a magnesium ion surrounded by four highly conserved aspartate residues from helices TM2 and TM3. We show that magnesium is essential for the enzymatic activity and is involved in catalysis. Substrates-docking was validated by mutagenesis studies and a structure-based catalytic mechanism is proposed.

Figure 1 Structure of IPCT/DIPPS bifunctional enzyme. The protein is dimerized via the transmembrane domain. Red arrows indicate the access to their active sites with a putative Mg2+ drawn as a cyan sphere. Membrane surface position in grey. Top view of DIPPS domain (perpendicular to the membrane plane).

References 1. P. Nogly, under revision.

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Current Synchrotron-based Research in Portugal 2014 Structural Biology

Structural and functional analysis of proteins involved in oxidative stress resistance reveals promising alternatives for the treatment of amoebiasis Micael C. Freitasa, Henrique G. Colaçob, Pedro M. Matiasa, João B. Vicente,b, c, & Tiago M. Bandeirasa a

Instituto de Biologia Experimental e Tecnológica, Apartado 12, 2781-901 Oeiras, Portugal b Institute for Medicines and Pharmaceutical Sciences, Faculty of Pharmacy, University of Lisbon, Portugal c Department of Biochemistry and Human Biology, Faculty of Pharmacy, University of Lisbon, Portugal Email of corresponding author: mfreitas@itqb.unl.pt

Keywords: Amoebiasis, Entamoeba (E.) histolytica, O-acetylserine sulfhydrylase, inhibitor. The protozoan Entamoeba (E.) histolytica is the etiologic agent of amoebiasis, a global leading cause of death by parasitic infection. The parasite invades and disrupts the host colon, causing severe tissue destruction and inflammation. Upon host infection, E. histolytica is confronted with reactive oxygen and nitrogen species. We have previously identified oxidative and nitrosative stress responsive genes using whole-genome expression profiling. Under the premise that these genes are related to E. histolytica pathogenic potential, we are characterizing from a structural-functional viewpoint a subset of such genes, overexpressing them in bacteria and studying the recombinant proteins by biochemical and biophysical techniques. The targets under study comprise proteins of unknown and known function, particularly enzymes involved in the sulfur-containing amino acid metabolism that lack structural homologues in the host and have been validated targets for new antiamoebic drugs. Focusing on the validated drug targets O-acetylserine sulfhydrylase (EhOASS) and methionine gamma-lyase (EhMGL), we have identified a small set of boronic acid-based compounds with promising inhibitory effect on the in vitro enzymatic activity. Structural studies are now ongoing to complement and evaluate protein/compounds interactions. EhOASS crystallization was previously published and was now re-established by us [1] and first results were already obtained for the apo-protein using synchrotron radiation. Future steps involve co-crystallization and crystal soaking with promising inhibitory compounds followed by structure resolution that will guide the design of more specific and higher-affinity molecules. References Funding: FCT grant PTDC/SAU-MIC/111447/2009 1. Chinthalapudi Krishna et al. 2007. Acta Cryst.F, F63, 512–515

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Current Synchrotron-based Research in Portugal 2014 Structural Biology

The structure of the first heterotrimeric periplasmic aldehyde oxidoreductase from Escherichia coli. Márcia A.S. Correiaa, Ana Rita Otrelo-Cardosoa, Viola Schwuchowb , Teresa Santos-Silvaa, Silke Leimkühlerb & Maria João Romãoa a b

REQUIMTE-CFQB, Departamento de Química, FCT-UNL, Caparica, Portugal.

Institut für Biologie und Biochemie, Universität Potsdam, Potsdam, Deutschland. Email of corresponding author: marcia.correia@fct.unl.pt

Keywords: Aldehyde oxidoreductase; Synchrotron radiation; X-ray crystallography Molybdoenzymes are involved in a large number of enzymatic reactions in the nitrogen, carbon and sulfur cycles Depending on the structure and orientation of the molybdenum centres, the molybdoenzymes can be divided in three families: the XO family, the sulfite oxidase (SO) family and the dimethylsulfoxide (DMSO) reductase family [1,2]. In general, enzymes of the XO family are dimmers that possess the same overall architecture [3], with two distinct [2Fe2S] clusters bound to the N-terminal domain, the FAD bound to a central domain and the Moco-binding domain at the C-terminus. In this work we crystallized and solved the X-ray structure of periplasmic aldehyde oxidase PaoABC from Escherichia coli. Using synchrotron radiation, a complete dataset was collected at PXIII (X06DA) beamline of the SLS (Paul Scherrer Institut, Switzerland). The crystals diffracted up to 1.8 Å resolution and belong to C2 space group with cell constants a=109.42, b=78.08, c=151.77 Å; ß=99.77º. PaoABC is the first example of an Escherichia coli protein containing a molybdopterin cytosine dinucleotide (MCD) and it is the only heterotrimer (aßγ) of the XO family known in the literature. This 135 kDa enzyme contains a large subunit (PaoC - 78.1 kDa), a medium subunit (PaoB - 33.9 kDa), and a small subunit (PaoA - 21.0 kDa). Based on the metal content and EPR studies previously performed, the enzyme was suggested to contain two [2Fe-2S], one FAD, and one Mo cofactor per monomer [2]. However, the crystal structure revealed the presence of an additional [4Fe-4S] cluster in subunit B, bound to four cysteine residues, in an additional 40 residues subdomain. Clear peaks in the anomalous difference maps confirmed the existence of this unexpected [4Fe-4S] cluster [4, 5]. PaoABC, together with 4hydroxybenzoyl-CoA reductase (4-HBCR), are the only known proteins to carry such a cluster. The crystal structure is in the last steps of refinement with an R and Rfree factor of 14,2% and 17,5% respectively. The active site of PaoABC has remarkable differences regarding other members of the family, namely a highly solvent-exposed active site and the absence of aromatic residues capable of π stacking interactions. References 1. S. Leimkühler, et al. 2011. Coordin Chem Rev, 255 (9-10), 1129-1144. 2. M. Neumann, et al. 2009. FEBS Journal, 276, 2762–74. 3. M.J. Romão. 2009. Dalton T, 21, 4053-4068. 4. R .Cardoso, et al. 2014. Int. J. Mol. Sci, 15(2), 2223-2236. 5. M.A.S. Correia, et al, in preparation.

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Current Synchrotron-based Research in Portugal 2014 Structural Biology

Low resolution structure of Dps1 and Dps2 from Deinococcus radiodurans obtained by SAXS S. P. Santosa, M.G. Cuypersb, A. Roundc, E. P. Mitchellc, T. Narayananc & C.V. Romãoa a

Instituto de Tecnologia Química e Biológica, Av. da República (EAN), 2784 - 505 Oeiras, Portugal b Institut Laue Langevin, Rue Jules Horowitz, 6, BP 156, 38042 Grenoble Cedex 9, France c European Synchrotron Radiation Facility, 6 rue Jules Horowitz, BP 220, 38043 Grenoble Email of corresponding author: cmromao@itqb.unl.pt

Keywords: Dps, SAXS, DNA, structure. The radiation resistant organism Deinococcus radiodurans has two genes that encode for two Dps (DNA-binding proteins from starved cells): DrDps1 and DrDps2. These proteins can protect DNA against oxidative damage by a dual action: DNA binding by an effective physical shield; or storing iron that avoids the formation of reactive oxygen species (1-2). The crystallographic structure was determined for both DrDps in two different states: apoform and in the presence of iron (3-4). Both proteins share the same overall structure with other members of the family, being hollow spheres comprising 12 identical subunits. The two DrDps proteins have unique long N-terminal extensions as compared to the other family members known to date. However and importantly, the crystal structures lack electron density for these N-terminal extensions to the first 29 amino acids residues for DrDps1 and to the first 41 amino acids residues for DrDps2, probably due to disorder and flexibility (3-4). Small Angle X-ray Scattering technique (SAXS, at BM29 and ID02, ESRF-France) was used to determine the position of the N-terminal on both proteins. These extensions have been proposed to be involved on Dps-DNA binding interactions, mediated by the positively charged residues present in them (1). The SAXS scattering curves and distribution functions obtained for both proteins are typical of a spherical structure with an extra tail, that could correspond to the presence of the N-terminals. In order to confirm this result, the construct of DrDps1 without the N-terminal region, DrDps1∆29, was analysed by SAXS. The distribution function obtained for the DrDps1∆29 is characteristic of a sphere structure, however the radius gyration (RG) and maximal particle size (Dmax) is smaller than the values obtained for the full-length proteins DrDps1 or DrDps2. This Suggests the presence of the N-terminal regions outside the dodecamer assembly, what was confirmed by ab initio modelling obtained from the distribution function generated by DAMMIN or GASBOR programs. References 1. P. Ceci, et al. 2005 J Biol Chem, 280, 34776-34785. 2. P. Ceci, et al. 2004 Nucleic Acids Res, 32, 5935-5944. 3. C.V. Romao, et al. 2006. J Biol Inorg Chem, 11, 891-902. 4. M.G. Cuypers, et al. 2007. J Mol Biol, 371, 787-799.

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Current Synchrotron-based Research in Portugal 2014 Structural Biology

Computational perspective towards understanding the structural mechanism of PaoABC with aliphatic and aromatic substrates: Molecular docking and binding free energy analysis Jayaraman Muthukumarana, Maria J. Romãoa & Teresa Santos-Silvaa a

REQUIMTE-CQFB, Departamento de Química, Centro de Química Fina e Biotecnologia, Faculdade de Ciências e Tecnologia, Universidade Nova de Lisboa, 2829-516 Caparica, Portugal. Email of corresponding author: tsss@fct.unl.pt

Keywords: PaoABC, Auto dock, Molecular docking, Benzaldehyde, Cinnamaldehyde PaoABC is a molybdenum enzyme that belongs to the Xanthine oxidase family and participates in detoxification of several aldehydes in the cell. In order to understand the structural mechanism, in silico interaction studies were performed with Auto Dock 4.2 using the atomic coordinates of Escherichia coli PaoABC and different aliphatic and aromatic substrates. Docking results (Fig. 1) showed that benzaldehyde and cinnamaldehyde have better binding affinity with Pao ABC rather than aliphatic substrates. Moreover, the amino acid residues Pro352 and Arg440 made significant interaction with the substrates whereas this was not observed in other cases. In order to confirm their role in mediating enzyme-substrate interaction, the in silico alanine scanning mutagenesis and docking analysis was also performed with all the substrates. The binding free energy of mutated forms of Pao ABC with most of the substrates was decreased in comparison with wild type. From all these results, we suggest that the order of binding affinity towards protein is cinnamaldehyde > benzaldehyde which is agreed with experimental results (Neumann et al 2009).

Figure 1: In silico interaction results of Pao ABC with two aromatics substrates, namely Benzaldehyde and Cinnamaldehyde. Hydrogen bonding interaction was presented in dotted lines. Zoom View: Three-dimensional substrate interaction diagram.

References M. Neumann et al. 2009. The FEBS journal, 276, 2762–2774.

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Current Synchrotron-based Research in Portugal 2014 Structural Biology

Crystal structure of chlorite reducing enzyme Chlorite Dismutase at 1.4 Å resolution Hugo D. Correiaa, Diana M. Freirea, Maria G. Rivasa, b, Cristina Costaa, Sabine V. Doorslaerc, Teresa Santos-Silvaa & Pablo J. Gonzaleza, b a

REQUIMTE/CQFB, Departamento de Química, Faculdade de Ciências e Tecnologia, Universidade Nova de Lisboa, 2829-516 Caparica, Portugal. b Physics Departament, Facultad de Bioquímica y Ciencias Biológicas, Universidad Nacional del Litoral, Ciudad Universitaria, Paraje El Pozo, S3000ZAA Santa Fe, Argentina c University of Antwerp, Physics Department, Universiteitsplein 1, B-2610 Wilrijk, Belgium Email of corresponding author: h.correia@campus.fct.unl.pt Keywords: Bioremediation, Water pollution, Chlorite, Chloride, Perchlorate, Chlorate, Heme enzyme, Crystal structure, Azide Chlorite Dismutase (Cld) is a heme protein that was purified from Magnetospirillum sp. strain Lusitani, a perchlorate reducing bacteria. It is a ~28 kDa enzyme that catalyses the reduction of chlorite to chloride while forming an O–O bond and releasing molecular oxygen.[1] Oxochlorides, such as perchlorate (ClO4-), chlorate (ClO3-) and chlorite (ClO2-), are harmful compounds and important water pollutants.[2] The chlorite anion in particular is chemically inert and highly soluble, making it the most dangerous of these species for public health. Chronic exposure to chlorite induces anemia and nervous system problems in children.[3] Thus, this enzyme can be used as a biocatalyst for bioremediation, since it catalyses the decomposition of chlorite at outstandingly high rates. Chlorite Dismutase was crystallized using several conditions; however, the tested crystals diffracted poorly. After crystal optimization the crystal structure of Cld was solved at 1.4 Å resolution in the presence of azide, a known inhibitor. The X-ray data shows five monomers of Cld present in the asymmetric unit related by NCS, forming a homopentamer. Each monomer harbours a heme b cofactor coordinated by a histidine residue, hydrogen-bonded to a glutamic acid residue. An azide molecule was found coordinating the iron atom in the active site, close to an arginine residue that is important for substrate positioning and cofactor stabilisation. The high resolution structure will contribute to the better understanding of the catalytic mechanism of Cld. Furthermore, it will provide clues for site-directed mutagenesis in order to develop a Cld-mutated specie with higher resistance to inactivation and, consequently, higher chlorite-decomposition efficiency. References 1. P. L. Hagedoorn, D. C. de Geus, W. R. Hagen, 2002. Eur J Biochem, 269, 4905-4911. 2. B. Goblirsch, R. C. Kurker, B. R. Streit, C. M. Wilmot, J. L. DuBois, 2011. J Mol Biol, 408(3), 379-398. 3. S. Kanitz, Y. Franco, V. Patrone, M. Caltabellotta, E. Raffo, C. Riggi, D. Timitilli, G. Ravera, 1996. Environ Health Persp, 104(5), 516-520. .

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Current Synchrotron-based Research in Portugal 2014 Structural Biology

The crystal structure of tungstate binding protein TupA from Desulfovibrio alaskensis G20 Raquel S. C. Cordeiroa, Ana R. Otrelo-Cardosoa, Rashmi R. Naira, Teresa Santos-Silvaa & Maria G. Rivas a,b a

REQUIMTE/CQFB, Departamento de Química, Faculdade de Ciências e Tecnologia, Universidade Nova de Lisboa, Portugal. b Departamento de Física, Facultad de Bioquímica y Ciencias Biológicas, Universidad Nacional del Litoral, Argentina. Email of corresponding author: rs.cordeiro@campus.fct.unl.pt

Keywords: TupA, Tungsten, Molybdenum, Crystallization, X-ray diffraction Molybdenum (Mo) and tungsten (W) are chemically analogous elements that are found in the environment.1 In biological systems, Mo and W are incorporated into the active site of enzymes through binding to a pyranopterin moiety.2 The Mo and W enzymes have an important physiological role, including the catalysis of some main reactions in the metabolism of carbon, nitrogen and sulfur.3 These elements are incorporated into the cell through specific transport systems, one of them known as TupABC. This is composed of a protein located in periplasm (TupA), a membrane protein (TupB) and a protein confined in the cytoplasm (TupC) responsible for the hydrolysis of ATP, thus generating the energy needed to transport the oxyanions to the cell.4 TupA from Desulfovibrio alaskensis G20 has been successfully crystallized using PEG3350 as precipitating agent and a complete dataset was collected at ID 23-1 beamline of the ESRF (European Synchrotron Radiation Facility). The crystals diffracted up to 1.4 Å resolution and belong to the P21 space group with the cell constants a=52.25 Å, b=42.50 Å, c=54.71Å and β=95.43˚. In order to understand the binding mode and the metal specificity of the protein, site directed mutagenesis has been carried out and three different mutants have been designed, substituting arginine 118 to a lysine (R118K), a glutamine (R118Q) and a glutamate (R118E). Native polyacrylamide gel electrophoresis was performed for a qualitative analysis of the effect of the mutations for tungstate and molybdate binding. Crystals of the mutated forms of TupA will also be prepared in the presence and absence of ligands. The structural and biochemical characterization of the proteins involved in ion transport will contribute to the understanding of the specificity of the metal uptake process and its effect in the cell. References 1. J.P. Smart, M.J. Cliff, D.J. Kelly. 2009. Mol Microbiol, 74, 742-757. 2. G. Schwarz, P. Hagndoorn, K. Fischer. 2007, Molecular Microbiology of Heavy Metals, 6, 423-451. 3. E. Aguilar-Barajas, C. Díaz-Pérez, M. Ramírez-Díaz, H. Riveros-Rosas, C. Cervantes. 2011. Biometals, 24, 687-77. 4. S. Mota, O. Valette, P. González, D. Brondino, J. Moura, I. Moura, A. Dolla, M. Rivas. 2011, J Bacteriol, 193, 2917-2923.

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Current Synchrotron-based Research in Portugal 2014 Structural Biology

Characterisation and crystallisation of Notch ligands and generation of bio-therapeutic agents Margarida Silvaa, P.M. Matiasa,b, T.M. Bandeirasa,b & A. Barbasa a

b

iBET - Instituto de Biologia Experimental e Tecnol贸gica, Oeiras, Portugal; ITQB - Instituto de Tecnologia Qu铆mica e Biol贸gica, Universidade Nova de Lisboa, Oeiras, Portugal Email of corresponding author: margaridasilva@ibet.pt

Keywords: Notch signalling, Breast cancer, Phage display, Thermofluor, Protein crystallization. Cell-to-cell communication is required for many biological processes and one of the most common systems utilized by a wide range of eukaryotes is the Notch-signalling pathway [1]. High level expression of Notch receptors and ligands, and their increased activation in several breast cancers and early precursors, place Notch signaling as a key player in breast cancer pathogenesis [2]. Studies have shown that the Notch ligands expression is undetectable in normal breast tissues, but moderate to high expression has been detected in breast cancer [3], placing Notch signaling inhibition as a novel therapeutic target. The structural models of Jag1 ligand and Notch1 show intrinsically disordered proteins, clearly suggesting Notch signaling proteins to have similar secondary structure disorder features. This explains why no 3D structure of the full-length extra-cellular domains of Jagged1 (Jag1), Jagged2 (Jag2) and Delta-like-1 (Dll1) Notch 1 ligands has been determined so far. Our aim is to generate different ligand protein constructs and develop specific functionblocking antibodies, using phage display technology. Moreover, we propose to crystallize the Notch1 ligands (Jag1, Jag2 and Dll1) alone and in complex with the phage display selected antibodies, and biophysically characterize the antibody-ligand complex interactions. Proteins constructs were purified from refolded inclusion bodies or mammalian cell culture supernatants, and purity was confirmed to be higher than 95% by SDS-PAGE [4]. Protein thermal stability was assessed by thermal-shift assays, and nano-scale crystallization trials were set up in 96-well plates using several commercial available screens. Structural models are very useful to allow improvement of the understanding of biological pathways. Therefore, crystallization of the Notch 1 ligands and the study of their interaction with specific antibodies will allow determining the molecular basis of antagonistic activity in the Notch 1 signaling pathway. References 1. R. Kopan. 2012. Cold Spring Harbor Perspectives in Biology, 4, a011213. 2. Z .Wang, Y. Li, FH. Sarkar. 2010. Curr Protein Pept Sci, 11(6), 398-408. 3. S. Mittal, D. Subramanyam, D. Dey, R.V. Kumar, A. Rangarajan. 2009. Mol Cancer, 8, 128. 4. M.M. Silva, P. M. Matias, et al. 2013. Eur Biophys J Biophy, 42, S189-S189.

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Current Synchrotron-based Research in Portugal 2014 Structural Biology

Implementation of the Microarray Technology for studying molecular interactions of the bacterial Cellulosome V.G. Correiaa, D. Ribeiroa, M. J. Romãoa, A.L. Carvalhoa, B.A. Pinheiroa & A.S. Palmaa. a

REQUIMTE/CQFB, Departamento de Química, FCT, UNL, 2829-516 Caparica, Portugal Email of corresponding author: vivianacorreia@campus.fct.unl.pt

Keywords: Microarrays, cellulosome, cohesins, dockerins, CBMs The bacterial cellulosome, an extracellular multi-enzyme complex of Carbohydrate Active enZYmes (CAZYmes) and structural proteins, plays a pivotal role in the degradation of plant cell wall recalcitrant polysaccharides by these microorganisms. The cellulossome assembly relies on the interaction between non-catalytic proteins – dockerins and cohesins. Dockerins are found in cellulosomal enzymes and each recognizes one of the various cohesin domains located in a macromolecular structural scaffoldin. Enzymes of this extensive consortia are often appended to other non-catalytic domains – CBMs (carbohydrate binding modules), which participate in pivotal protein:carbohydrate interactions, like the anchoring of the entire complex to crystalline cellulose or the targeting of specific substrates by the catalytic module [1, 2]. Sequencing of bacterial genomes revealed the presence of unknown cellulosomes in several cellulolytic microorganisms and as a result, a large number of novel dockerin- and CBM-bearing proteins. For studying these novel proteins and their key interactions, we reasoned that the high-throughput screening feature of the microarray technology could be combined with X-ray crystallography to derive structural information at the molecular and atomic levels. The use of protein and carbohydrate microarrays enables the screening of a diverse and wide range of protein:protein and protein:carbohydrate interactions on the same chip using only minute amounts of sample [3-6]. Currently, we are establishing both the protein and carbohydrate microarray methodology in our laboratory in combination with highthroughput features of novel cloning and protein purification strategies. The work was supported by the Portuguese Science and Technology Foundation (FCT-MEC) through grants PEst-C/EQB/LA0006/2011 and PEst-C/EQB/LA0006/2013 (to Associate Lab REQUIMTE) and through the projects grants EXPL/BBB-BEP/0506/2012, PTDC/QUIQUI/112537/2009 and RECI/BBB-BEP/0124/2012. References 1. B.A. Pinheiro, J.L.A. Brás, S. Najmudin, A.L. Carvalho, L.M.A. Ferreira, J.A.M. Prates, C.M. Fontes. 2012. Biocatal biotransfor, 30(3), 309-315. 2. C.M. Fontes, H.J. Gilbert. 2010. Annu Rev Biochem, 79, 655-681. 3. A.S. Palma, T. Feizi, R.A. Childs, W. Chai, Y. Liu. 2014. Curr Opin Chem Biol, 18C, 87-94. 4. Y. Liu, A.S. Palma, T. Feizi. 2009. Biol Chem, 390(7), 647-656. 5. D.A. Hall, J. Ptacek, M. Snyder. 2007. Mech Ageing Dev, 128(1), 161-167. 6. T. Feizi, W. Chai. 2004. Nat Rev Mol Cell Biol, 5(7), 582-588.

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Current Synchrotron-based Research in Portugal 2014 Structural Biology

The Crystal Structure of the ER Lectin Malectin with its carbohydrate ligands Benedita Pinheiroa, Maria J. Romãoa, Ana L. Carvalhoa, Angelina S. Palmaa a

REQUIMTE/CQFB, Departamento de Química, Faculdade de Ciências e Tecnologia, Email of corresponding author: b.pinheiro@fct.unl.pt

Keywords: N-Glycosylation, endoplasmic reticulum, lectin A new membrane-anchored lectin (glycan-binding) protein of the ER, named malectin, has been identified [1]. Malectin is highly conserved in animals and plays a role in the Nglycosylation pathway and quality control of nascent glycoproteins [2, 3]. However, its mechanism of action in the ER is not entirely known. Structure determination of malectin by NMR showed that the luminal part is a glycan-binding domain that recognizes glucose oligomers and other related glucose di- and oligosaccharides as ligands [1]. Glycan microarray analysis revealed a unique selective binding to a Glc2-high mannose N-glycan oligosaccharide, of the type that occurs in the ER of eukaryotic cells as intermediate in the early steps of the N-glycosylation pathway of nascent proteins [1, 4]. We have been able to crystallize and solve the crystal structure of this newly identified lectin, complexed with its glucose disaccharide ligands maltose (Glcα1,4Glc), nigerose (Glcα1,3Glc) and with the pentasaccharide Glc2Man3 (Glcα1,3Glcα1,3Manα1,2Manα1,2Man), which represents the D1 arm of the di-glucosylated high-mannose N-glycan. These studies elucidate at molecular level the ligand for malectin, the Glcα1,3Glcα1,3Man on N-glycan D1 arm, and reveal the participation of the Man1 residue through a newly identified water-mediated hydrogen bonding network. The work was supported by the Portuguese Science and Technology Foundation (FCT-MEC) through SFRH/BPD/68563/2010 (to the first author), PEst-C/EQB/LA0006/2013 (to Associate Lab REQUIMTE) References 1. T. Schallus, C. Jaeckh, K. Féeher, A.S. Palma, Y. Liu, J.C. Simpson, M. Mackeen, G. Stier, T.J. Gibson, T. Feizi, T. Pieler, C. Muhle-Goll. 2008. Mol Biol Cell, 19, 3404-3414. 2. C. Galli, R. Bernasconi, T. Soldà, V. Calanca, M. Molinari. 2011. PLoS One, 26, e16304. 3. Y. Chen, D. Hu, R. Yabe, H. Tateno, S.Y. Qin, N. Matsumoto, J. Hirabayashi, K. Yamamoto. 2011. Mol Biol Cell, 22(19), 3559-3570. 4. A.S. Palma, Y. Liu, C. Muhle-Goll, T.D. Butters, Y. Zhang, R. Childs, W. Chai, T. Feizi. 2010. Methods Enzymol, 478, 265-86.

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Current Synchrotron-based Research in Portugal 2014 Structural Biology

Studies on the stability and oligomerization states of the helicases RuvBL1 and RuvBL2 Sara T.N. Silvaa, Tiago M. Bandeirasb & Pedro Matiasa,b a

Instituto de Tecnologia Química e Biológica, Industry and Medicine Applied Crystallography Laboratory b Instituto de Biologia Experimental e Tecnológica, Proteomics Laboratory Email of corresponding author: saras@itqb.unl.pt

Keywords: ATPases, helicases, RuvBL1, RuvBL2, Pontin, Reptin. RuvBL1 and RuvBL2 are highly conserved ATPases known to have a key role in many cellular pathways, and cause cancer when deregulated. They use chemical energy obtained through ATP hydrolysis to exert helicase activity. To date, only the 3D structures of the fulllength RuvBL1 hexamer [1] and the RuvBL1∆DII/RuvBL2∆DII dodecameric complex [2] have been determined. The main objectives of this work are the attainment of diffractingquality crystals of full-length RuvBL2, gain insight into the chemo-mechanical coupling between ATP hydrolysis and DNA unwinding, and to study stability and oligomerization dependence on tag presence and position. During the process of RuvBL2 purification we have used Thermal Shift Assays (TSA) as an essential technique to find the conditions that improve stability of the tested proteins, which led to the addition of ADP as a stabilising cofactor. Once the optimal conditions were determined, we could assess the influence of tags on protein stability, and found that positioning the tags on the N-terminus destabilizes RuvBL2, when compared to positioning on the C-terminus. Removal of the tag provides additional stability. Size-Exclusion Chromatography (SEC) coupled to Multi-Angle Laser Light Scattering of RuvBL2-ADP has shown that this protein is 100% hexameric at various concentrations, even after dialysis into an ATP-containing buffer. Whether there is nucleotide exchange after the hexamer is formed is still unclear, since the 3.4 Angstrom preliminary structure obtained so far shows no nucleotide molecule in the binding pocket. In contrast to RuvBL2, RuvBL1 can be isolated as a monomer at low concentrations, and as a hexamer at more than 6 mg/mL, which allows for the study of the oligomerization process. SEC shows that in the presence of AMP/PNP, a non-hydrolysable analogue of ATP, RuvBL1 is monomeric even at high concentrations, suggesting that ATP hydrolysis is a crucial step in the oligomerization process. After ssDNA is added to the RuvBL1-AMP/PNP complex, SEC shows the formation of a small proportion of protein-DNA complex, suggesting that this binding may not be dependent on ATP hydrolysis. Crystallisation of RuvBL1-DNAAMP/PNP complex is also in course. References 1. P.M. Matias, S. Gorynia, P. Donner and M.A. Carrondo, 2006. J. Biol. Chem., 281, 38918-38929. 2. S. Gorynia, T. Bandeiras, F. Pinho, C. McVey, C. Vonrhein, A. Round, D. Svergun, P. Donner, P. Matias, M. Carrondo, 2011. J. Struct. Biol. 176, 3, 279-291.

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Current Synchrotron-based Research in Portugal 2014 Structural Biology

Preparation and X-ray crystallographic analysis of molybdenum-substituted rubredoxin crystals Francisco Leisicoa, Biplab K. Maitiaa, José J. G. Mouraa, Maria J. Romãoa & Teresa Santos-Silvaa a

REQUIMTE-CQFB, Departamento de Química, Faculdade de Ciências e Tecnologia, Universidade Nova de Lisboa, 2829-516 Caparica, Portugal Email of corresponding author: franciscoleisico@fct.unl.pt

Keywords: Molybdenum, X-ray crystallography, Rubredoxin, Metal coordination sites, Molybdoenzymes Molybdoenzymes catalyse a variety of oxidation/reduction reactions promoting a diversity of biological functions at the metabolism of carbon, sulphur and nitrogen [1]. Specific characteristics of active site coordination geometries determine function and specificity of these proteins, which makes X-ray crystallography an interesting approach for their study. The rich chemistry of multisulphur transition metal systems admits chemical versatility and molybdoenzymes exploits this flexibility in their biological functions [2]. The investigation of molybdenum (V) coordination chemistry using proper systems featuring metal interactions is relevant to the enzyme catalytic functionality. Sulphur-rich metal coordination sites are characteristics in iron-sulphur centers and rubredoxins are the simplest iron-sulphur protein, containing a single iron atom tetrahedrally coordinated by the sulphur atoms of four cysteine residues [3]. The iron atom in rubredoxin can be substituted by others transition metals as previously described in the literature [4]. Molybdenum-substituted rubredoxin may represent a useful model system to study molybdenum chemistry in protein active sites. A substituted-molybdenum form of rubredoxin was crystallized after testing different crystallization conditions. Crystals of 0.1x0.01x0.01 mm3 size were obtained and diffracted beyond 1.3 Å resolution. Crystallization conditions continue under optimization in order to achieve crystals of the reduced form of the protein and also in the presence of different sulphur based ligands. The data collected will be used to molecular structure determination and consequently, postulate features of molybdoenzyme active sites. References 1. R. R. Mendel, T. Kruse. 2012. Biochim Biophys Acta 1823, 1568–1579. 2. C. Iobbi-Nivol, S. Leimkühler. 2013. Biochim Biophys Acta, 1827, 1086–1101. 3. C.J. Chen, M.Y. Liu, Y.T. Chen, J. LeGall. 2003. Biochem. Biophys. Res. Commun, 308, 684–688. 4. A. Thapper et al. 2013. J Inorg Biochem, 127, 232-237.

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Current Synchrotron-based Research in Portugal 2014 Structural Biology

X-ray crystal structure determination of Escherichia coli nitric oxide reductase flavorubredoxin P. Borgesa, R. G. Davida, J.B. Vicentea, T.M. Bandeirasa, , M. Teixeiraa , C. V. Romãoa & C. Frazãoa a

Instituto de Tecnologia Química e Biológica (ITQB-UNL) Oeiras, Portugal Email of corresponding author: pborges@itqb.unl.pt

Keywords: Flavorubredoxin, oxidoreductase, E.coli, nitric oxide detoxification, electron transport. Flavodiiron proteins (FDPs) are widespread in Archaea and Bacteria [1], in which mostly are anaerobic organisms. Furthermore, the presence of these proteins have been reported also on Eukarya organisms, such as the pathogenic protozoa Trichomonas vaginalis, Entamoeba histolytica and Giardia intestinalis [2]. FDPs are part of the microbial response mechanisms against oxygen and/or nitric oxide. They were classified into four classes according to their structural domains composition, but for the moment only Class A members were structurally characterized. E.coli flavorubredoxin from Class B is so far the most extensively characterized FDP, in spectroscopic and kinetics terms. In vivo and in vitro studies on E.coli FDP revealed a nitric oxide reductase (NOR) function. It was then proposed to be part of the E.coli protecting mechanism against nitrosative stress under anaerobic growth conditions, i.e, it effectively removes NO by its reduction to nontoxic N2O [3-5]. Hereby we report the crystallographic X-ray structure of E.coli FDP. Crystallization trials led to good diffracting crystals and a 1.76 Å resolution diffraction data set was obtained at ESRF. The crystal belonged to hexagonal space group P622, unit-cell parameters a = 150.27, b = 150.27 and c = 95.27 Å. The structure was solved by molecular replacement and is currently under refinement in order to provide insights into the molecular function of this flavorubredoxin References 1. A. Wasserfallen, S. Ragettli, Y. Jouanneau, T. Leisinger, 1998. Eur.J.Biochem., 254, 325-332. 2. A. Di Matteo, F.M Scandurra, F. Testa, E. Forte, P. Sarti, M. Brunori, A. Giuffre. 2008. J. Biol. Chem., 283, 4061-4068. 3. A.M. Gardner, R.A. Helmick, P.R. Gardner. 2002. J. Biol. Chem., 277, 8172-8177. 4. C.M. Gomes, A. Giuffre, E. Forte, J.B. Vicente, L.M. Saraiva, M. Brunori, M. Teixeira. 2002. J. Biol. Chem., 277, 25273-25276. 5. J.B. Vicente, F.M. Scandurra, J.V. Rodrigues, M. Brunori, P. Sarti, M. Teixeira, A. Giuffre. 2007. FEBS Journal, 274, 677–686.

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Current Synchrotron-based Research in Portugal 2014 Structural Biology

Structural and Functional Insights into centriole biogenesis P. E Santoa & T. M. Bandeirasa, b a

b

iBET - Instituto de Biologia Experimental e Tecnológica, Oeiras, Portugal ITQB - Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, Oeiras, Portugal Email of corresponding author: psanto@itqb.unl.pt

Keywords: Cell Cycle, Centrioles, BLD10, Protein Crystallization The centrosome is the principal microtubule-organizing centre of most animal cells, regulating cell shape and polarity in interphase, and spindle pole organization during mitosis [1]. Structurally, the centrosome comprises a pair of centrioles and their pericentriolar material (PCM). Centrioles are small cylindrical structures, made of nine microtubule (MT) triplets organized in a very well conserved, radially-symmetric manner. Centrioles are also essential to nucleate the formation of cilia and flagella, structures involved in cell motility and sensory functions [1] that are involved in human diseases, including ciliopathies and sterility. Their motility is often controlled by a central MT pair localized within the ciliary MT-based skeleton, the axoneme [2]. BLD10/CEP135, a conserved player in centriole and flagella biogenesis, can bind and stabilize MT’s and is required for the early steps of central MT formation [3]. In this work, we divided Drosophila melanogaster BLD10 in three constructs. The Nterminal which we purified to homogeneity and was shown to bind MT’s [4]. The middle region of BLD10, which proved to be not amenable for purification, was submitted to a limited digestion proteolysis approach, in order to generate a soluble smaller fragment which may crystallize. We were able to generate a highly stable domain, as seen by Circular Dichroism (CD), and efforts to crystallize and determine its structure are ongoing. The Cterminal region is purified as 3 different oligomers after final size exclusion, which reequilibrate in solution when separated from each other. We managed to bypass this reequilibration and found two promising crystallization conditions. The first produced well defined rectangular shaped crystals which diffracted at up to 6 Å, while the second condition is still under optimization. Future work will focus on crystallization of all BLD10 constructs, as well as, biophysical studies, including stability assessment by CD or Thermofluor, SAXS studies to determine the different oligomers structure and MT binding assays. References 1. M. Bettencourt-Dias, D.M. Glover. 2007. Nat Rev Mol Cell Biol, 8, 451-463. 2. M. Filegauf, J. Wahrman. 2007. Nat. Rev, 8, 880-893. 3. Carvalho-Santos, et al. 2010. J. Cell. Sci, 123, 1414-1426. 4. Carvalho-Santos, et al. 2012. Dev. Cell, 23, 412-424.

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Current Synchrotron-based Research in Portugal 2014 Structural Biology

IscR is a unique [Fe-S] cluster-containing transcription factor Joana A. Santosa, Noelia Alonso-Garcíaa, Sandra Macedo-Ribeiroa & P. J. B. Pereiraa a

IBMC – Instituto de Biologia Molecular e Celular, Universidade do Porto, Porto, Portugal Email of corresponding author: ppereira@ibmc.up.pt

Keywords: Rrf2-like regulator, transcription regulation, helix-turn-helix motif, DNA recognition The ubiquitous iron-sulfur (Fe-S) cluster-containing proteins are involved in countless biological routes and play crucial roles for the functioning of both prokaryotic and eukaryotic cells [1]. The transcription factor IscR, of the helix–turn–helix family, was first implicated in repressing expression of the ISC (Iron Sulfur Cluster biogenesis) pathway and was shown to contain a [2Fe–2S] cluster [2]. Promoters controlled by IscR belong to two distinct sequence groups and the [2Fe-2S] cluster of IscR was shown to be essential for regulation of type-1 promoters (isc, yadR, yhgI), while apo-IscR is responsible for the regulation of type-2 promoters. The proposed model for IscR action consists on a mechanism for Fe-S biogenesis fine-tuned by the cellular Fe-S cluster status. Despite recent advances in understanding the regulation of isc and suf operons by environmental signals, the features of IscR target-site recognition and the structural changes that alter the DNA binding specificity of IscR upon ligation of the [2Fe-2S] cluster are unknown. To understand how IscR recognizes two different DNA motifs, we solved the three-dimensional structures of free apo-IscR and of its complex with a type-2 target sequence, the hya promoter from the hydrogenase-1 operon. These experimental models revealed some of the molecular details of the unusual environmentally modulated recognition of two distinct promoter consensus sequences by IscR, using a single predicted helix–turn–helix DNA binding motif. To our knowledge, IscR is the only transcription factor that is active in both the apo and holo-forms and which displays cofactor-mediated modulation of its DNA binding specificity. This work was funded by Fundação para a Ciência e a Tecnologia (Portugal) through grant PTDC/BBB-BEP/2127/2012 (EU-FEDER funding through COMPETE FCOMP-01-0124FEDER-028116) and PhD fellowship SFRH/BD/66461/2009 to JAS. References 1. X. M. Xu, S. G. Møller, 2011. Antioxid Redox Signal, 15, 271-307. 2. C. J. Schwartz et al., 2001. Proc Natl Acad Sci USA, 98, 14895-14900.

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Current Synchrotron-based Research in Portugal 2014 Structural Biology

Crystal structure of a widespread unusual acidophilic c-type cytochrome José A. Britoa, Kevin Denkmannb, Inês A. C. Pereiraa, Christiane Dahlb & Margarida Archera a

Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, Avenida da República, 2780-157 Oeiras, Portugal b Institut für Mikrobiologie & Biotechnologie, Rheinische Friedrich-Wilhelms-Universität Bonn, Meckenheimer Allee 168, D-53115 Bonn, Germany Email of corresponding author: archer@itqb.unl.pt

Keywords: X-ray crystallography, thiosulphate dehydrogenase, c-type cytochrome, sulphur metabolism, haem coordination, ligand switching The ability to perform the very simple oxidation of two molecules of thiosulphate to tetrathionate is wide spread among prokaryotes. Despite the widespread occurrence of tetrathionate formation, and its well-documented significance within the sulphur cycle, little is known about the enzymes catalysing the oxidative condensation of two thiosulphate anions. To fill this gap, the thiosulphate dehydrogenase (TsdA), enzyme from Allochromatium vinosum, was recombinantly expressed, purified and characterized. The protein is a periplasmic, monomeric, 243 amino acids c-type cytochrome with an enzyme activity optimum at pH 4.0. Two typical haem c binding sites of the characteristic sequence CXXCH are present in the sequence predicting two covalently bound haems in the protein. EPR spectra suggested quite a lot of structural flexibility in TsdA that affected both haems. We crystallized the protein and solved its crystal structure by Single Anomalous Dispersion (SAD) method using the Fe-haem anomalous signal. The protein crystallized in space group C2 with PEG 3350 as precipitant and one molecule in the asymmetric unit. Initial crystallization trials rendered multiple, urchin-like crystals with no diffraction ability. Using iodide as an additive worked as a “silver bullet” allowing to obtain single crystals that diffract to 1.4 Å resolution. The X-ray structure showed an all-alpha structure with structural similarities to the Rhodovulum sulfidophilum’s SoxAX (PDB code 2OZ1), and the low-redox-potential cytochrom c6 from Hizikia fusiformis (PDB code 2ZBO). Moreover, we could observe a ligand switching mechanism that allows a His/Lys and His/Met coordination in haem 2 corroborating the EPR data obtained earlier. References K. Denkmann et al., 2012. Environ Microbiol, 14(10), 2673-2688. J. A. Brito et al., 2014, in preparation

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Current Synchrotron-based Research in Portugal 2014 X-ray imaging

Synchrotron radiation topography study of temperatureinduced phase transformation in ferroelectric crystals Jingzhong XIAO∗a, Peiping Zhub, Wanxia Huangb, Qingxi Yuanb a

b

Department of Physics, University of Coimbra, 3004-516 Coimbra, Portugal Beijing Synchrotron Radiation Laboratory, Institute of High Energy, Beijing 100039, China Email of corresponding author: xiao@pollux.fis.uc.pt

Keywords: ferroelectrics; phase transition; synchrotron radiation. By real time synchrotron radiation topography method, dynamical behavior of ferroelectric domains as a function of temperature in 0.92Pb(Zn1/3Nb2/3)O3-0.08PbTiO3 crystals is investigated. A sequence of temperature-induced monoclinic phase transformation is observed, and verified by combining this method with temperature-dependence Raman scattering, capacitance measurement, and X-ray diffraction. This phase evolution picture seems remarkably unique as the monoclinic phase is able to coexist with cubic phase well above TC. References S.–E.. Park, T. R. Shrout. 1997. J. Appl. Phys. 82, 1804. J. Kuwata, K. Uchino, S. Nomura. 1982. J. Appl. Phys. 21, 1298.

Corresponding author. E-mail address: xiaojz66@hotmail.com, jzxiao@fudan.edu.cn (Tel: 00351-234-370217; Fax: 00351-234-425300)

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Current Synchrotron-based Research in Portugal 2014 X-ray imaging

New technologies, old fossils: evolution and preservation of Permian dicynodonts from Mozambique Rui M.S. Martinsa,b, Rui Castanhinhab,c, Ricardo Araújoa,b,d,e, Luís C. Júniorf, Gabriel Martinsc, Felix Beckmanng, Fabian Wildeg , Norbert Schellg & Jörg Fröbischd a

Instituto Superior Técnico/Campus Tecnológico e Nuclear, Bobadela, Portugal b GEAL - Museu da Lourinhã, Lourinhã, Portugal c Instituto Gulbenkian de Ciência, Oeiras, Portugal d Museum für Naturkunde - Leibniz Institute for Evolution and Biodiversity Science, Berlin, Germany e Southern Methodist University, Dallas, Texas, United States of America f Museu Nacional de Geologia, Maputo, Moçambique g Helmholtz-Zentrum Geesthacht, Geesthacht, Germany Email of corresponding author: rmsm@fct.unl.pt Keywords: Permian dicynodonts, Tomographic and geochemical analytical techniques, Three-dimensional visualization and segmentation, Anatomy and evolution Dicynodontia represents the most diverse tetrapod group during the Late Permian. They survived the Permo-Triassic mass extinction and are as basal therapsids important for the understanding of the early evolutionary history of the mammalian lineage. Although extensively studied, several aspects of dicynodont paleobiology such as: neuroanatomy, inner ear morphology and internal cranial anatomy remain obscure. PALEOTECH project proposes two central research components pertaining to the Permian dicynodonts from Mozambique: 1) Evolution, which requires anatomical description through micro-computed tomography techniques allowing the digital visualization of the morphological traits encumbered by rock matrix (see Fig. 1). 2) Preservation, which through a wide array of techniques, including synchrotron radiation-based X-ray diffraction, allow us to understand the geochemical basis underpinning preservation of fossils with roughly 257 Ma, and assess the extent of geochemical postmortem phenomena on morphology.

Figure 1. Niassodon mfumukasi holotype [1]: photo (left) and tomographic data (right).

References 1. R. Castanhinha, R. Araújo, L.C. Júnior, K.D. Angielczyk et al. 2013. PLoS ONE, 8, e80974.

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Current Synchrotron-based Research in Portugal 2014

Unveiling the crystal structure of a dapsone bio-inspired coordination network from ESRF data V. Andréa,b, S. Quaresmaa, M. Martinsa & M. T. Duartea a

Centro de Química Estrutural, Instituto Superior Técnico, Universidade de Lisboa, Av Rovisco Pais, 1049-001 Lisboa, Portugal b CICECO, Universidade de Aveiro,3010-193 Aveiro, Portugal Email of corresponding author: vaniandre@tecnico.ulisboa.pt

Keywords: BioMOFs, metallopharmaceuticals, dapsone, synchrotron radiation We are engaged in a project that proposes to synthesize "bio-inspired" metal organic frameworks, BioMOFs [1-4] using non-toxic metals, by different approaches: (a) having active pharmaceutical ingredients as linkers, what would be attained by the direct coupling of the bioactive molecule to the metal; (b) having the drug carried within the pores as guest with suitable pore sizes; (c) enclosing in the same BioMOF drugs acting both as linkers and guests. This first approach avoids the need of large pore sizes, since the release of the API is achieved by material degradation, without side effects due to the release of the linker. Up to now only one of these materials has been synthesized and characterized, BioMIL-1. These frameworks will be very useful in the transport and release of APIs which is quite often a problem in drug development [5]. Dapsone was one of the first compounds we have used in approach (a). It is a medication used in multidrug therapy for the treatment of leprosy and malaria; it is moreover used in prophylaxis against Pneumocystis pneumonia in HIV patients; also applied in the treatment of mild to moderate acne and other rashes like dermatitis herpetiformis. Mechanochemistry [6], a green synthetic technique, has been successfully applied to coordinate dapsone with several metals, such as Bi, Zn, Fe and Ni. These forms were identified by X-ray powder diffraction and despite the many efforts made to crystallize them no single crystals suitable for single crystal X-ray diffraction analysis were obtained. Data collected at ESRF in September 2013 was very important for this project and particularly for the studies with dapsone and the structural characterization of the dapsone:Ni complex is being carried out. SS-NMR spectroscopy may also be helpful in this characterization. References 1. S. Keskin, S. Kizilel. 2011. Ind Eng Chem Res, 50, 1799-1812. 2. C. Janiak, J. K. Vieth. 2010. New J Chem, 34, 2366-2388. 3. J. Y. An, S. J. Geib, N. L. Rosi. 2009. J Am Chem Soc, 131, 8376. 4. R. Babarao, J. W. Jiang. 2009. J Phys Chem C, 113, 18287-18291. 5. McKinlay, A.C. et al. 2010. Angew. Chem. Int. Ed., 49, 6260 – 6266. 6. Delori, A. et al. 2012. Cryst Eng Comm, 14, 2350.

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Current Synchrotron-based Research in Portugal 2014  
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