Newsletter No. 2 July 2013 Greetings from the Coordinator

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Jul 2, 2013 ... structural biology approaches and to select the facilities of your choice, also allowing more transparency and ultimately facilitating procedures ...
Newsletter No. 2 July 2013

For Life Science in Europe

Greetings from the Coordinator Matthias Wilmanns takes stock at half time Credit DESY

BioStruct‐X is an EU funded project for transnational access to synchrotron and emerging Free Electron Laser facilities for applications in structural biology. Now at half time we are starting to see the first fruits of our labours! For example you can read about some of the first scientific highlights on page 3. The field of X‐ray imaging is growing fast and the recent workshops organised in the field (page 6) show just how crucial these courses are for the new users. Work on multi circle goniometry has also shown how patchy the knowledge and expertise is here (page 6). At the time of writing we are approaching the next deadline for Transnational Access proposals, and we are very pleased to be able to announce that access to WP5 platforms is available in this round of applications. Read more about these opportunities on page 2.

What are the key tasks for the remaining second phase of the BioStruct‐X project? Upon request by the European Commission we have established a web-based portal for project submission and a central Project Evaluation Committee. The motivation for its implementation has been to create an incentive for combined use of different structural biology approaches and to select the facilities of your choice, also allowing more transparency and ultimately facilitating procedures, both from the facilities and from the research client’s point of view. We do admit that the latter has become more complex than originally foreseen, and therefore we believe that the key task for the remaining project will be to review, and where needed simplify presently established procedures. This effort should allow us to approach the overall goal of the project to facilitate transnational access to the participating facilities for a large scientific research community across Europe reducing bureaucracy and allowing them to focus on what they are supposed to work: succeeding with the best possible research data, inspired by the technical opportunities provided by the BioStruct-X partner facilities. Contents: Greetings from the Coordinator Work Package 5 gets going Scientific Highlight – Work Package 7 Centre Feature – ALBA Centre Feature – ITQB Centre Feature – EMBL Hamburg Networking News Upcoming events Upcoming BioStruct-X Meetings

BioStruct-X in numbers: 1 2 3 4 5 6 7 7 8

Start: September 2011 Duration: 48 months Total Budget: 9 Million Euro Number of partners: 19 Number of installations: 44 Number of projects submitted in year 1: ca. 500 Number of projects supported after call 1: 334

Work Package 5 gets going Work package 5 (WP5) is up and running and ready for applications….. Within the BioStruct-X WP 5 different, highly complementary automated platforms located at five partner facilities (EMBL Hamburg, EMBL Grenoble, DIAMOND, University of Oxford and PSI) are providing access for i) protein expression of challenging proteins, including multi-subunit complexes, novel/low homology targets, and membrane proteins; ii) high throughput (HTP) crystallisation in micro and nanodrop formats and in lipidic cubic phase (LCP) for membrane proteins. EMBL Hamburg The sample preparation and characterization (SPC) and the High-Throughput Crystallization (HT-X) facilities are part of the integrated facility for structural biology at EMBL@PETRA3. Users will be able to process samples for purification and optimization, crystallization and structure determination to leave with a comprehensive structural understanding of their system. EMBL Grenoble At EMBL Grenoble two protein production platforms provide two complementary services, ESPRIT and MultiBac. ESPRIT uses advanced robotics to find soluble constructs in random libraries of typically 28,000 fragmented constructs of a target gene in E. coli. MultiBac permits production of large eukaryotic multisubunit protein complexes by using recombinant multi-gene baculoviruses to infect insect cell cultures. In addition, High-Throughput Crystallization platform offers completely automated nanovolume crystallization screening. PSI The PSI Protein Production Facility offers membrane protein expression and purification in mammalian cells for structural biology. The facility offers access to sophisticated equipment and technologies and provides advice and technical assistance in sample preparation. The facility provides small-scale expression and analysis of constructs, upscaled mammalian cell expression, purification and analysis in various detergents. University of Oxford Strubi facility at University of Oxford is dedicated to protein expression in mammalian cells for structural biology. It offers streamlined methodologies for transient expression in 293T cells, first for rapid small-scale screening of multiple constructs, and then for scale-up of selected constructs as well as technologies for controlling glycosylation in secreted proteins and co-expression to produce oligomeric protein complexes for structure determination. DIAMOND The membrane protein lab (MPL) at Diamond Light Source is a research and training state-of-the art facility for scientists interested in the solving the 3-dimensional structures of membrane proteins by X-ray crystallography. The Fluidigm Topaz crystallization system is available for free interface diffusion (FID) crystallization of very small quantities of protein in microfluidic chips as well as the crystallization of membrane proteins using the MemGold screen.

Mode of Access Due to the limited amount of access available for certain platforms within this Work Package (WP), BioStructX introduces a modified mode of access for WP5 applications. Scientist interested in applying for access to WP 5 platforms should contact the platform leader for technical feasibility evaluation prior to submitting an application to BioStruct-X. In addition, a rapid mode of access allowing evaluation in between the application deadlines has been established for applications to High-Throughput Crystallization platforms. For more detailed description of the platforms, technical specifications and contact details of platform leaders, please visit BioStruct-X website.

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Scientific Highlight – WP7 Automated multipronged sample characterization at biological SAXS beamlines To obtain data with improved quality as well as to benefit from an on-line data validation tool, a multi-detector size exclusion chromatography (SEC) system was implemented at the PETRA-III BioSAXS beamline in Hamburg in collaboration between the EMBL-Hamburg and Malvern partners. The integrated purification system allows immediate data acquisition after the individual components elute from the column such that one may purify the samples on the fly, separate different fractions but also observe transient complexes. Yet more importantly, the analysis of the SAXS data is greatly facilitated as the sample is characterized biophysically by complementary techniques. In particular, determination of the concentration as well as the molecular weight are provided by the Malvern Viscotek TDA (Triple Detector Array) which incorporates a refractometer (RI), static light scatterer (SLS) and ultraviolet absorption spectrometer (UV) pipeline (Figure 1). Figure 1. The FPLC setup at the P12 beamline of the EMBL in Hamburg (left), the elution profile from the Malvern TDA (top right) and the overall parameters (forward intensity (I0) and the radius of gyration Rg) extracted from the individual SAXS frames (bottom right). The analysis system automatically determines the pure buffer region (indicated in grey) and subtracts it from the scattering data of the peak fractions. Shown is the test run from a commercial bovine serum albumin (BSA) sample, where the fractions of dimeric and monomeric BSA are separated and their shapes are determined by the automated pipeline (shown at the bottom row as transparent envelopes superimposed with the known crystal structures).

With the latter the confidence in the model derived from the SAXS data is immensely increased. After thorough testing during the second half of 2012, this method is routinely offered to the SAXS User community as of February, 2013. The high demand for such a setup is mirrored in the large number of Users who have opted for this additional feature in the latest proposal round at the EMBL. It is expected that many projects will profit from this multipronged characterization and much effort was therefore put in its integration into the existing automated pipeline. Not only does the 'Malvern box' generate a signal that triggers data collection at the appropriate point of time, but the collected biophysical data is correlated with the X-ray scattering frames at the end of each run. Thus, within less than 30 min after data collection the User receives the overall parameters and even the tentative ab inito low resolution models from the peak fractions (Figure 1). The first test users were very thrilled about this feature, as they could, therefore, make the best use of their allocated beam time. Future developments to the system include expansion of the temperature range, automatic sample injection and inclusion of the biophysical parameters in the data analysis. 3

Centre Feature – ALBA Synchrotron Light Source, Spain Located 20 km from Barcelona, the ALBA Synchrotron is the most important scientific infrastructure ever built in Spain. ALBA is the last synchrotron light source that started operations, with seven different beamlines completely operative in 2013. It is expected to operate 4.600 hours in 2013, planning to reach 6.000 hours in 2015. ALBA is managed by a Consortium, CELLS, which is co-financed by the Spanish and the Catalan Governments. Aerial view of ALBA Synchrotron Light Source

The ALBA Synchrotron Light Source is participating in BioStruct-X project with two beamlines for biological applications: BL09-MISTRAL and BL13-XALOC. Both beamlines have already received funded experiments from BioStruct-X project, and are prepared to fund and host more.

EndStation of Beamline 09-MISTRAL

EndStation of Beamline 13-XALOC

BL09-MISTRAL is a soft X-ray full field transmission microscopy beamline devoted to cryo nano-tomography of cells in the water window energy range and spectroscopic imaging from 270 eV to 851.5 eV. The microscope is equipped with two objective lenses, a 40 nm and a 25 nm Fresnel zone plates, and a cryo-fluorescence microscope off-line for correlative microscopy. It opened for users operation in February 2013. This beamline was also in charge of the organization of the first BioStruct-X workshop on “Cryo X-Ray Imaging for Biological Applications”, held on March 22nd at ALBA. Nearly 50 participants from all over the world took part in this workshop. Addressed to structural biology researchers, BL13-XALOC is an automated MX beamline that started user operation in 2012 with state-of-the-art equipment. The photon source is an invacuum undulator with full tunability in the 5-23 keV range. The end-station includes a high accuracy diffractometer, a removable mini-kappa mount, a CATS automated sample mounting robot and a Pilatus 6M pixel detector. Resulting beam at sample position is very stable, and has a dimension of 52×5.7 µm2 FWHM (h×v). With these features, the beamline copes with a broad variety of crystal sizes and unit cell parameters and allows both wavelength dependent and independent experiments.

Since the aperture to users, this beamline has allowed solving a large variety of structures that include membrane proteins and large unit cell crystals. The anomalous diffraction from many absorption edges (S-K, Cd-K, I-K, Fe-K, Pt-K, Ni-K, Pt-L3, Ir-L3, and Se-K, among others) has also been used extensively.

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Centre Feature – EMBL Hamburg, Germany EMBL Hamburg: an integrated pipeline from bench to beamline EMBL Hamburg operates an integrated infrastructure for life science applications using synchrotron radiation at the PETRA III storage ring, on the German Electron Synchrotron Site (DESY) in the west of Hamburg, Germany. Alongside three beamlines for Macromolecular Crystallography and Small Angle X-ray Scattering, users can also use facilities for sample preparation and characterisation and computational facilities for X-ray data processing and evaluation. An integrated pipeline from bench to beamline all under one roof.

PETRA III hall

Working at the SPC facility

BioSAXS sample changer

Aerial view of the DESY site with the PETRA III hall to the lower left

EMBL Hamburg operates an Integrated structural biology service facility situated at the PETRA III storage ring on the DESY campus in Hamburg, Germany. Users can apply for access to a package of structural biology services and expertise all under one roof. The sample preparation and characterisation (SPC) facility is equipped to perform quality assessment and optimization on macromolecular samples and provides users with tools to characterise their samples, carry out quality control measurements and sample optimisation protocols before moving next door to the EMBL Hamburg beamlines.

Entrance to the EMBL beamline area

EMBL Hamburg operates three beamlines at the PETRA III synchrotron ring on the DESY Campus, designed to accommodate a range of scientific experimental demands. The BioSAXS beamline P12 is optimized for low background data collection from macromolecular solutions and kinetic experiments with high temporal resolution. The endstation is equipped with photon counting Pilatus detectors and with a robotic sample changer. P12 allows for fully automated and remote data acquisition and processing using the Beamline Meta Server software.

The two Macromolecular Crystallography beamlines P13 and P14 complement each other in the experiments they are optimised for. P13 is particularly suited for phasing experiments on crystals with large unit cells, while P14 has a small beam size in combination with the high-precision vertical rotation axis and provides excellent conditions for diffraction experiments with small crystals. P14’s experimental endstation is equipped with a sample mounting system known as MARVIN. For more information about BioStruct-X facilities at EMBL Hamburg see http://www.biostruct-x.eu/content/embl-hamburg-hamburg-germany

P13 MX experimental station with PILATUS detector

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Centre Feature – ITQB, Portugal The Instituto de Tecnologia Química e Biológica (ITQB) in Oeiras, Portugal is one of the four BioStruct-X Training, Information and Dissemination (TID) centres providing decentralised activities. BioCrys was the first BioStruct-X supported training event at the ITQB and focused on basic Macromolecular Crystallography and was especially aimed researchers in the early stage of their crystallographic career. The course was organised by Maria Arménia Carrondo and Thomas Schneider. The BioCrys Course on 'Fundamentals of modern methods in biocrystallography, took place at the ITQB in Oeiras from 20-27 October 2012. The course was supported by FEBS, IUCr, Biostruct-X and IUBMB. It brought together 38 students from 19 different countries and 20 tutors from 9 different countries. The aim of the course was to introduce the participants to concepts of Macromolecular Crystallography, and illustrate these with tutorial and practical sessions. This is particularly important as the field is developing very rapidly and many young researchers do not receive a formal education in Crystallography. While learning the efficient use of automated tools in crystallography, it is also critical to understand the underlying concepts for tackling difficult problems of structural biology, such as dealing with membrane proteins that are not amenable to the current generation. Additionally and with the advent of free electron lasers, new experimental schemes are being developed and it will be mandatory to have a good understanding of the basics of crystallography to effectively exploit the up-coming laser sources in structural biology. Main topics covered by the course included; Basics of crystallography in real and reciprocal space, X-ray sources and X-ray detectors, Crystal cooling and data collection, Recent software and methodological advancements in data processing and integration, MAD and SADphasing, molecular replacement, phase improvement and the use of maximum likelihood, model building, refinement and validation. Other topics also covered were sample preparation and characterization and two special lectures on crystallographic studies of membrane proteins, challenges and advantages and on the future use of X-ray free electron lasers in Structural Biology.

Participants attending a lecture

Students in a tutorial computing session

Course participants and tutors

During the entire course, a box was available to which specific 'burning questions' about crystallography could be submitted anonymously. These questions were later answered by the tutors in a dedicated session. This system proved to be very popular in this and in the previous editions of these courses and gave valuable feedback to the speakers and tutors. The participants were requested to fill one questionnaire with two questions for each lecture or tutorial where its ‘Usefulness’ and ‘Understandability’ were evaluated on a scale from 1 to 5. Additionally, and although the program was extensive, there was plenty of opportunity for all participants to interact outside the formal program of the course. In particular, the fact that all participants were staying and had dinner in the same hotel close to ITQB in Oeiras, Hotel Praia-Mar, provided a good environment to mix and to talk to each other.

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Networking News The aim of the networking work package (WP10) is to foster a culture of cooperation between the BioStructX participants and the scientific communities that benefit from the research infrastructures and, in so doing, help to develop an efficient and attractive European Research Area. Key tasks are to promote the dissemination of good practices, promote the clustering of and coordinated actions between related projects, coordinate with international related initiatives, support the deployment of global and sustainable approaches in the field, and to support training for new users. The last six months has seen a number of training events organised within WP10 – including two events under 10.7 (Training and networking in the field of multi-circle goniometry). The first event in November 2012 was a Workshop on Advanced Data Collection with Multi-Axis Goniometer and Single-Photon Counting Detector. In a previous meeting under task 10.7 it had become apparent that the uptake, by the user community, of possibilities of multi-circle goniometry was patchy and dependent on the commitment of a particular site or local contact to suggest how best to exploit the goniometers for specific problems. For this reason the organisers set up the workshop in November at the PSI in Switzerland to present the possibilities opened up by multi-circle goniometers at different sites, and to discuss and consolidate experience using multi-circle goniometry for different experimental problems. As well as proving to be an excellent introduction to the evolving needs of the user community, several issues were also identified for the next networking event, a technical discussion which took place in April 2013 in Lund, Sweden between sites committed to a better exploitation of multi-circle goniometry. A number of events have also taken place under the task 10.4.2 (User training for crucial steps in the 3D tomographic X-ray Imaging pipeline). In December 2012 a one day workshop took place at the Oxford Particle Imaging Centre of the Wellcome Trust Centre for Human Genetics, focussing on aspects of Soft X-ray microscopy sample preparation and pre-characterization. The aim of the workshop at Alba in March 2013, was to bring together experts and users of all levels to exchange expertise and knowledge on aspects of Cryo X-ray Imaging for Biological Applications, in particular to strengthen the technique potentials and help disseminate its possibilities. In the relatively new, but growing field of X-ray Imaging the need seems apparent for user orientated workshops and dissemination activities. Future activities are planned within this task, and BioStruct-X is also pleased to announce the HERCULES Advanced Course on Synchrotron X-ray Imaging for Biology scheduled to take place at the ESRF in September. News and reports of all networking events can be found on the BioStruct-X webpages under www.biostruct-x.eu/node/102

Upcoming Events 16th - 20th September 2013 HERCULES Advanced Course HSC15: Synchrotron X-ray Imaging for Biology, ESRF, France 19th - 20th September 2013 ESUO Annual Meeting 2013, PSI, Switzerland

HERCULES, March 2013

ITQB, Portugal, October 2012

PSI, Switzerland, November 2012

Oxford, December 2012

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Join us for the 2nd BioStruct-X Annual Meeting in Hamburg! The second BioStruct-X Annual meeting will take place in Hamburg from the 5th – 6th September 2013. The programme will include updates from the work packages and opportunities for networking with colleagues from across Europe. For more information and to register please go to the BioStruct-X webpages. The dates have been chosen to run just before the 11th International Conference on Biology and Synchrotron Radiation (BSR) which offers a diverse and exciting programme of topics and speakers and will take place in the centre of the city of Hamburg from the 8th – 11th September. Participants of the BSR will also be able to enjoy a tour of the state-ofthe-art facilities on the DESY campus, and an extensive social programme. So why not make the most of your visit and take the opportunity to enjoy Hamburg’s sights alongside a vibrant and varied scientific programme?! Check out the BSR webpages (www.bsr2013.org) for more information and to register.

Upcoming BioStruct-X meetings •2nd Annual Meeting, 5th and 6th Sept. 2013, Hamburg, DE •Executive Committee Meeting, 5th Sept 2013, Hamburg •General Assembly, 6th Sept. 2013, Hamburg, DE •PEC Meeting, 7th Sept. 2013, Hamburg, DE

Next application deadline: August 15th2013 How to apply for Access Interested researchers are invited to visit the BioStruct-X website (www.biostruct-x.eu) where they will find all relevant information about the participating facilities and platforms as well as information about the application process. Proposal submission is done entirely online. Submitted proposals will be evaluated by the Project Evaluation Committee (PEC).

Contact BioStruct-X Coordinator Matthias Wilmanns BioStruct-X Project Manager Ivana Custic Tel: +49 (0)40 89902 124 Email: [email protected] EMBL Hamburg, c/o DESY Building 25a Notkestrasse 85 22603 Hamburg Germany Newsletter editors: Ivana Custic, Rosemary Wilson, (EMBL Hamburg, Germany)

Newsletter contributors: Maria Armenia Carrondo (ITQB), Dmitri Svergun (EMBL Hamburg)

www.biostruct-x.eu

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