Community Research and Development Information Service - CORDIS

FP7

BIOSTRUCT-X Report Summary

Project ID: 283570
Funded under: FP7-INFRASTRUCTURES
Country: Germany

Final Report Summary - BIOSTRUCT-X (Transnational access and enhancement of integrated Biological Structure determination at synchrotron X-ray radiation facilities)

Executive Summary:
The overall aim of BioStruct-X was to provide a consolidated platform that brings together the X-ray based methods in structural biology. In doing so, BioStruct-X created a single gateway to Europe’s leading synchrotron facilities and associated infrastructures, ultimately to the benefit of the overall user community. By organising the leading European Synchrothron facilities in a pan-European Consortium composed of 19 partners, BioStruct-X pioneered integrated European infrastructure provision allowing diverse structural biology synchrotron activities to be organised by structural biologists for the scientific community.

Through its website, BioStruct-X developed and deployed project tools that enabled the offer of a unified portal for simultaneous multi-site user project applications and their evaluation by a centralized project review committee – without precedent in the history of European transnational access provision initiatives. This was the first step towards the provision of a broader integrated European infrastructure access in structural biology thereby facilitating the studies of macromolecular structure, dynamics and function. Through BioStruct-X transnational access activities 1156 scientific projects and 1991 users (of those, 39% female scientists and 48% users younger than 35 years of age) received support that already resulted in over 340 publications in peer-reviewed journals (http://www.biostructx.eu/content/publications).

Transnational access activities in BioStruct-X were directly enhanced and tightly supported by four complementary joint research activities, focusing on developing methods and instruments important for the field. Several software tools, prototypes and kits have been designed, developed and tested and are now available for the scientific community.

The highly interactive and collaborative nature of the BioStruct-X project is demonstrated by its portfolio of networking activities that took place across Europe. Through a variety of user-oriented and integrated networking activities the BioStruct-X project fostered a culture of cooperation between the BioStruct-X participants and the scientific communities that benefit from the research infrastructures. A total of 36 networking events focused on training of the user communities, awareness raising and information and dissemination of project results were supported in BioStruct-X thereby directly helping to develop an efficient and attractive European Research Area (ERA). To further strengthen the ERA in the field of structural biology, BioStruct-X integrated its activities with the ESFRI project INSTRUCT and actively reinforced links with other related I3 initiatives in Europe.

In summary, BioStruct-X has brought together leading European research organisations in a successful effort to build a broad platform of infrastructures addressing all stages of biological structure determination, from protein production of sufficient quantity and quality for structure analysis, to sample production and data collection by a variety of X-ray methods (macromolecular crystallography, small-angle X-ray scattering, X-ray imaging). In doing so, BioStruct-X became a role model for related projects within the ESFRI roadmap and future I3-related projects in HORIZON 2020.

Project Context and Objectives:
BioStruct-X is a pan-European initiative that groups 19 research organisations from 11 EU member and associated states aiming to provide integrated support for X-ray based structural biology applications. The ultimate goal of the BioStruct-X project is to establish a centralised Europe-wide infrastructure platform aiming to provide the scientific community with an integrated and centrally coordinated offer of facilities suitable for structural studies on the most challenging biological projects. In the context of contributing to the structuring and the development of the European Research Area, BioStruct-X through its networking and joint research activities actively promotes coordinated actions with industry, related I3 and ESFRI projects by strengthening the links between the relevant stakeholders and developing a sustainable vision of future integration on a pan-European scale.
More specifically, the overall objectives of the BioStruct-X workplan are:
- To provide supported transnational access (TNA) to researchers across Europe, based upon scientific excellence of the projects proposed, to leading research services in X-ray based structural biology, including protein production, HTP crystallisation and structural biology-oriented synchrotron radiation beamlines, with applications in macromolecular X-ray crystallography (MX), small angle X-ray scattering (SAXS) and X-ray imaging (XI). The selected facilities offer options for automatic on-line and post-experiment data analysis and for remote experiment control.
- To encourage combined and hybrid approaches in structural biology, using facilities from multiple sites, thus enforcing a future concept of integrated structural cell biology.
- To reinforce TNA to these facilities and to promote the future availability of emerging facilities (XFEL) by an ambitious focused R&D programme, via four joint research activities (JRAs).
- To provide broad user training and dissemination of new scientific advances via a programme involving both specific TNA facility partners dedicated to these activities and decentralised so called TID centres at non-facility sites.
- To liaise with relevant communities, specifically INSTRUCT, other relevant I3 projects, ESUO, and industrial communities.
- To enforce the goals by the establishment of an efficient and transparent management structure.

Project Results:
In the following section we describe the main project achievements grouped according to the project’s Workpackages (WPs) and including a short description of the main activities and results that took place during the fifty four months of BioStruct-X project.

WP1 – Management

The BioStruct-X Consortium is composed of 19 partners from 11 EU member and associated states, who lead in facility provision and research in structural biology using high brilliance X-rays, provided by state-of-the art synchrotrons and a free electron laser. The Consortium coordination and project management activities were carried out by EMBL Hamburg with the support of the project’s Executive Committee, the Scientific Advisory Board, SAB - composed of internationally renowned experts in the structural biology field and the General Assembly, that includes all BioStruct-X partners and three elected User representatives, as the main decision making body.
The objectives of the Management WP were to provide maximum support at the level of the consortium for all project objectives, to monitor the progress of each work package, coordinate project activities, implement quality control by defining appropriate project standards and manage the targeted dissemination of knowledge arising from the project. Furthermore, the management team was responsible for the design and the development of the project’s website, setting up the infrastructure for centralized TNA proposal submission, evaluation and management and additionally provided operational support and assistance to the centralized BioStruct-X Project Evaluation Committee. The project website was maintained by the management team and served as a dissemination platform for the project's target groups and a communication platform for the partners in the Consortium.

The corporate identity “tool-kit“ including project dissemination materials for scientific purposes and for promotion of TNA have been prepared, regularly updated and distributed among partners and within the scientific community. During the course of the project, consortium-wide communication was assured by regular updates and organization of dedicated bilateral and/or project activity-specific meetings as well as annual meetings where the progress of all project activities was discussed in detail and any corrective actions were established. The timely preparation and submission of project deliverables, periodic project reports and the management and distribution of the EC financial contribution was successfully coordinated by the management team.
Moreover, the establishment of tight communications with related I3 projects (such as Calipso and BioNMR), with the Umbrella initiative and with the ESFRI project INSTRUCT was of great importance for the success of BioStruct-X. The organization of joint networking events directed primarily to the exchange of expertise and experiences ultimately resulted in a pilot design of the standardized proposal format, the development of single sign on authentication system and the infrastructure for bilateral data flow that was successfully put in place by the end of the project.

Achieved deliverables:
D1.1 Reports on Kick-off Meeting and project meetings - Report (R)
D1.2 Project Website - Other (O)
D1.3 Corporate identity "tool-kit" - Other (O)
D1.4 SWOT analysis - Report (R)

Transnational Access Activities (TNA)

BioStruct-X provided integrated transnational access via 44 installations in four key areas of structural biology: macromolecular X-ray crystallography (MX), small angle X-ray scattering (SAXS), X-ray imaging (XI), protein production and high-throughput crystallisation (PP&HTX). Applications for transnational access support by BioStruct-X were carried out either via submission of Block Application Group (BAG) proposals submitted for multiple projects by local regional or national research consortia or via Single Project (SP) proposals. The transnational access provision started in March 2012. During the course of the project, 2867 project proposals were submitted and evaluated by the centralised BioStruct-X Project Evaluation Committee (PEC). A total of 2774 projects were approved for funding and 1156 projects were carried out using BioStruct-X support.

WP2 –TNA to Biological Small Angle X-ray Scattering (SAXS) Beamlines

In BioStruct-X, four synchrotron facilities from Germany, France, UK and Sweden have provided transnational access to European users performing Small Angle X-ray Scattering (BioSAXS) experiments in workpackage 2. In total 5 beamlines have been offered for use during the course of BioStructX (X33, P12, SWING, B21 and I911-4), plus a single project ran on I22 at Diamond (UK) during PR2. During PR1, the beamline EMBL beamline X33, from the old DORIS III ring at DESY in Hamburg, was closed, and almost immediately taken over by the high brilliance P12 on the upgraded storage ring PETRA III. The beamline B21 at Diamond (UK) was commissioned during PR2 and started to host users only within PR3, with a spectacular amount of delivered beamtime. The MAXlab facility (Lund, Sweden) closed down during PR3 discontinuing access to the I911-4 beamline accordingly.
The project began with the beamlines already having dedicated environments, e.g. high-throughput automated collection and analysis at PETRA III, Size Exclusion Chromatography on-line with SAXS at Soleil. Since then, the user demand and the scientific expectations from the Bio-SAXS technique have been such, that the three beamlines now remaining in operation (P12, SWING, B21) were pushed to further enhance their set-ups and now all of them offer these technologies, in addition to other more specific environments (High-Pressure, Stopped-Flow, microfluidics). The new coSAXS beamline presently being built at MAX-IV, a successor of MAXlab in Lund, will further profit from this experience and developments (coSAXS is to be commissioned by autumn, 2017).

Clearly the input from the BioStruct-X users, thanks to the experience acquired from several beamlines all along the project, has strongly contributed to enhance the quality of the experiments at all partner sites. The periodic BioStruct-X report meetings were also a unique opportunity to share ideas on future developments.
The success of the BioStruct-X is directly reflected in the tremendous rise of the delivered beamtime all along its duration. Of remarkable note is the high number of projects from Eastern European countries that could be accepted, which was a crucial support to develop BioSAXS expertise in those countries.

Scientific highlight
Euripedes de Almeida Ribeiro Jr., Nikos Pinotsis, Andrea Ghisleni, Anita Salmazo, Petr V. Konarev, Julius Kostan, Björn Sjöblom, Claudia Schreiner, Anton A. Polyansky, Eirini A. Gkougkoulia, Mark R. Holt, Finn L. Aachmann, Bojan Žagrović, Enrica Bordignon, Katharina F. Pirker, Dmitri I. Svergun, Mathias Gautel, Kristina Djinović-Carugo. The Structure and Regulation of Human Muscle α-Actinin. (2014), Cell, 159, 1447–1460
Project title: Structural Biology of Muscle Z-disk
Discipline: Molecular and cellular biology
Project summary:
The spectrin superfamily of proteins plays key roles in assembling the actin cytoskeleton in various cell types, crosslinks actin filaments, and acts as scaf-folds for the assembly of large protein complexes involved in structural integrity and mechanosensation, as well as cell signaling. α-actinins in particular are the major actin crosslinkers in muscle Z-disks, focal adhesions, and actin stress fibers. α-actinin particular isoform 2 is an antiparallel homodimer of more than 200 kDa, comprising an N-terminal actin-binding domain (ABD), a central domain of four spectrin-like repeats (SRs), and a C-terminal calmodulin-like domain (CAMD) with two pairs of EF hand motifs (EFs) A number of structural and biophysical techniques was synergistically employed to characterize the α-actinin-2 dimer from striated muscle. X-ray crystallography revealed a high resolution model with a modular architecture of α-actinin-2, yet the structure is more than just ‘‘the sum of its parts’’; important intra- and inter-molecular contacts lock the molecule in a closed conformation that is crucial for dynamic regulation. SAXS was used to elucidate the solution structures of the wild type protein and an open state WEEK mutant for a better understanding of the structural differences between closed and open conformations.
Innovation value:

The structures depicted in Figure 1 provide insight into the phosphoinositide-based mechanism controlling its interaction with sarcomeric proteins such as titin, lay a foundation for studying the impact of patho-genic mutations at molecular resolution, and are likely to be broadly relevant for the regulation of spectrin-like proteins.

Figure 1. Solution Structure of α-Actinin-2 and the NEECK Mutant Derived from SAXS. (A) Experimental SAXS data of WT (black) and the NEECK mutant (green) of α-actinin-2. SAXS curves are computed from a rigid-body (RB) model for WT (gray) and NEECK (black). The logarithm of scattering intensity (I) is plotted as a function of the momentum transfers (s, in reciprocal Angstroms). Successive curves are displaced by one better visualization. Distance distribution functions (inset) P(r) for WT and NEECK assume slightly different shapes. RB modeling fits the experimental WT data with discrepancy 1.25 (gray line) and experimental NEECK data with dic 1.14 (dashed black line). The fit discrepancy for NEECK increased to 1.32, assuming a helical neck (solid black line). (B) Characterization of hydrodynamic properties of α-actinin WT and the NEECK mutant by SEC-MALLS. The lines across the protein elution volume show the molecular masses (MWs) of proteins. SEC-MALLS shows that NEECK has the same molecular weight as WT α-actinin-2 but a higher Stokes radius Rs (inset; data are represented as mean ± SD of three experiments), corroborating the open conformation for NEECK suggested by SAXS (C). AU, arbitrary units. (C) RB model of NEECK in solvent-accessible surface representation. The neck region was modeled as a flexible linker between the rigid bodies of ABD and rod, with no contact restraint.

WP3 – TNA to Macromolecular Crystallography (MX) Beamlines

In BioStruct-X workpackage 3, a total of 24 beamlines from 11 synchrotron facilities offered transnational access to users performing macromolecular crystallography (MX) research. These 11 facilities are geographically widely spread across Europe from the south (Elettra, Alba) to the north (MAXlab). The wide range of different specialities of offered beamlines, from high throughput beamlines for projects that require data collection on many samples or screening tools to highly advanced microfocus beamlines to measure on micron sized crystals, as well as beamlines with relatively long wavelengths, versatile goniometry and more, allowed the most demanding scientific projects to be carried out successfully.
During the project there have been several changes to the portfolio of offered installations. Early in the project, the old DORIS III ring at DESY, Hamburg Germany closed. This meant that 3 beamlines that were initially offered during PR1 of the project, 2 of which were MX beamlines, closed and no longer provided access. Another beamline at MAXlab (I911-5) also closed during PR1. However, during PR2 the upgraded storage ring PETRA III at the DESY site opened and new beamlines became accessible to users for transnational access. These beamlines offered new possibilities for microfocus and serial crystallography as well as phasing. The MX beamline at Alba (Barcelona, Spain) also became fully operational during PR2 and has become a valuable resource for the MX community in southern Europe. Finally, during PR3 the MAXlab facilty (Lund, Sweden) closed down.
While the project had a slow start, it started to gain momentum at the end of PR1 and during PR2 and PR3 delivered as set out in the project proposal. An amendment that reflected the problems encountered was made after project’s mid-term review and the project has run smoothly within the terms of the new amendment.

The success of BioStruct-X is directly reflected in the overall access units that were provided to the users – over 34 000 beamtime hours in total.
Furthermore, another big success of the project is the fact that BioStruct-X has been able to attract new communities to the MX beamlines. Prior to BioStruct-X, structural biology in the Baltic States (Estonia, Lithuania and Latvia) was at a very low level of activity. In BioStruct-X, access was provided to users from Lithuania and Latvia during PR3 and this has been the first time that these users visited the infrastructures. The fact that these experiments carried out using BioStruct-X support already led to publications (e.g. Kalnins et al 2015 and Leitans et al 2015) only emphasises further the overall importance and success of the project.

Another success from WP3 activities in BioStruct-X is the introduction of remote access experiments, in particular at the Diamond synchrotron. At the start of BioStruct-X, remote access was not at all introduced in Europe in strong contrast with equivalent facilities in the US where remote access is a major mode of access. Only at the Diamond synchrotron some remote access was employed for about 3% of the access time while the majority of the visits was still onsite in 2011. This has dramatically changed later on at Diamond and during 2015 remote access has been the mode of operation for 44.7% of the time, a mixed mode operation where remote access is shared with some people on site is used for 21.3%, while full on-site operations stands for 34% of the time. This means that operations where remote access is involved has become the main mode of operations during the BioStruct-X project at Diamond. For users this is beneficial as it reduces stress and costs related to travelling and for the facilities it means that beamtime can be scheduled more efficiently with less dead-time. Other facilities, such as Soleil and MAXlab started testing remote access protocols and it can be foreseen that the importance of remote access will increase within the coming years.

Finally, the access to highly sophisticated crystallography beamlines such as offered in WP3 has led to many scientific results and the full impact of BioStruct-X will only become apparent over the coming years. The number of publications resulting from data collected at the several beamlines in WP3 has been steadily rising and it is foreseen that papers will continue to be published after the finish of the project.

Scientific highlight
Bokhove M, Nishimura K, Brunati M, Han L, De Sanctis D, Rampoldi L, Jovine L. A structured interdomain linker directs self-polymerization of human uromodulin. Proceedings Of The National Academy Of Sciences. 2016;113 (6).
Project title: A structured interdomain linker directs self-polymerization of human uromodulin
Discipline: Molecular and cellular biology
Project summary:
Significance: Urinary tract infection is the most common nonepidemic bacterial infection in humans, with 150 million cases per year and a global health care cost above $6 billion. Because the urinary tract is not protected by mucus, mammals produce a molecular net that captures pathogenic bacteria in the urine and clears them from the body.
Bokhove et al used beamlines at Diamond light Source (I02) and ESRF (ID29) to solve the structure of its building block, glycoprotein uromodulin, which provides insights into how the net is built, and how it is compromised by mutations in patients with kidney diseases. Their work also explains nonsyndromic deafness due to mutations affecting the tectorial membrane, a similar filamentous structure in the human inner ear.

Figure 2. Structure of the protease-resistant core of human UMOD. (A) Overall UMODpXR architecture, with molecule A colored as in Fig. 3.9 and molecule B in green. N-glycans and Cys are depicted in a ball-and-stick representation. (Right) Possible orientation relative to the plasma membrane due to GPI anchoring is depicted. (B) Close-up view of EGF IV and its connection to ZP-N. An anomalous difference map calculated with Bijvoet differences collected at λ = 1.8 Å and contoured at 3.5 σ is shown as a yellow mesh.

WP4 – TNA for X-ray Imaging (XI) Beamlines

The provision of TNA access in WP4 started in March 2012. At that time, only one beamline (HZB) out of four involved in this workpackage was operational for users. The other beamlines were in commissioning (ALBA), design phase (SOLEIL & DESY) or even in the planning phase (DIAMOND). At the end of the project, only one beamline is operational for users (ALBA) as HZB suffered a relocation, and SOLEIL and DESY had several delays in the commissioning. The instrumentational timelines as well as the timeline of the project and the novelty of the X-ray imaging techniques in structural biology did not facilitate an easy and smooth start of the TNA. In addition, the users’ community had to be built up requiring time and effort in the dissemination of the X-ray imaging capabilities to the biologists’ community by the scientists involved in this TNA. Despite the above listed difficulties, the overall access provision as well the interest of the scientific community for X-ray imaging methods increased gradually along the project so that at the end of the project the new ALBA beamline MISTRAL has over-delivered.
The HZB beamline has been the first beamline in Europe of its kind allowing for cryo soft X-ray nanotomography and has been operational for users since the beginning of the BioStruct-X project. During the project and in the framework of JRA WP8, a high-quality in-line light microscope with fluorescence capabilities has been successfully implemented. This allowed the users to perform correlative microscopy under cryogenic conditions. Also in the frame of JRA WP8, first phase contrast imaging in the water window energy range has been performed. The Nanoscopium beamline at SOLEIL was under construction at the onset of the project in September 2011. Unfortunately, due to the unforeseen technical difficulties of the modifications of the undulator source, which is related to the construction of the adjacent canted tomography beamline, the first user experiments could be scheduled only in February 2016. The cryo soft X-ray tomography beamline at Diamond was constructed and commissioned during the BioStruct-X project and the first users collected data in December 2015. Given this timeline Diamond was not included in the TNA scheme of BioStruct-X. However, the project benefited enormously from the cryo soft X-ray tomography community interactions that the project enabled.
In summary, it is worth noticing that the BioStruct-X project has directly enabled the soft X-ray tomography community to grow and strengthen, as the technique was scarcely used by the biologist community some years ago (only 2 beamlines in Europe are as of today dedicated to cryo SXT for biological applications and operational for users and one more beamline coming soon online at Diamond Light Source).

Scientific highlight
Sanja Sviben, Assaf Gal, Matthew A. Hood, Luca Bertinetti, Yael Politi, Mathieu Bennet, Praveen Krishnamoorthy, Andreas Schertel, Richard Wirth, Andrea Sorrentino, Eva Pereiro, Damien Faivre & André Scheffel. A vacuole-like compartment concentrates a disordered calcium phase in a key coccolithophorid alga. Nat. Comm. 7, Article number:11228, doi:10.1038/ncomms11228. April 2016.
Project Title: Imaging intracellular calcium accumulation in calcifying marine algae using cryo-X-ray tomography
Discipline: Molecular and cellular biology

Project Summary:
Coccolithophores are widespread marine algae that produce biogenic calcite in the form of minute calcitic scales, known as coccoliths. The coccolith is formed intracellularly, inside a membrane-bound compartment, and upon maturation it is extruded to the cell exterior where many coccoliths are covering the cell surface. This biomineralization process draws considerable attention since coccolithophores may play an important role in the response of the oceanic ecosystem to predicted global climate change, and since similar changes in the past were recorded in the chemical composition of coccoliths that accumulated in ocean-floor sediments. Following dynamic cellular processes in vivo, such as coccolith synthesis, is extremely challenging. This is because of the experimental difficulties involved in following inorganic small molecules at the spatial and temporal resolution needed to trace intermediate and short-lived phases in the crystallization pathway. Therefore, the intracellular pathways responsible for the transport of the constituent ions from seawater to the growing coccolith are mostly unknown.

We used synchrotron soft-X-ray tomography at cryogenic conditions (Mistral beamline at ALBA) in order to map the intracellular calcium in coccolith producing cells of the model coccolithophore species Emiliania huxleyi. The cells were rapidly frozen and maintained at cryogenic conditions to preserve their intracellular organization. Single cells were imaged with the X-ray microscope at a resolution of 50 nm. Two types of data sets were acquired. The first is a tilt-series at the ‘water window’ energy range. At this X-ray energy the best contrast between carbon-rich intracellular membranes and the water-rich cytoplasm is achieved so the data can be used for 3D reconstruction of cells. The second data set was an energy scan around the Ca L-edge. From these data a complete X-ray Absorption Near-Edge Spectroscopy (XANES) spectrum can be extracted for each pixel in the image, providing information on the concentration of calcium inside intracellular organelles and spectroscopic information on the crystallinity of this Ca-rich phase. In the tomograms of cells all major organelles were visible, as well as intracellular membrane-bound coccoliths in status nascendi. To our surprise, the cells contained distinct intracellular compartments packed with highly absorbing material, which the spectroscopic data showed to be rich in calcium. The XANES spectra collected from multiple Ca-rich compartments were clearly different from the spectra of coccolith calcite and exhibited characteristics of disordered local environment around the calcium atoms.

The here presented data provides the first insights on the spatial distribution of calcium in coccolithophorid cells. We discovered high amounts of calcium to be concentrated in membrane-bound compartments that are separate from the coccolith producing compartment and we propose that this calcium pool is used for coccolith calcite formation. Inside the compartment calcium is stored as a disordered phase. This finding makes it tempting to speculate that a major fraction of the coccolith calcium is transported as intermediate calcium phase to the site of calcite formation. What is the exact chemical composition of these intermediate phases, and how material is allocated in and out of the compartments is still elusive.
Figure 3. A) Virtual slice of the reconstructed tomogram showing 3 E. huxleyi cells. Immature coccoliths and calcium-rich bodies are marked by arrowheads and arrows, respectively. B) Segmented volume of a tomogram with the nucleus in red, the chloroplast in yellow, the intracellular coccolith in violet and the calcium-rich bodies in green. C) Soft X-ray projections recorded below the Ca L3 edge (top), at the edge (middle) and the absorption difference (bottom). D & E) XANES spectra at the Ca L3,2 edge of the different features shown in D. Scale bars: 1 µm

WP5 – TNA for Protein Production & HTP Crystallisation

In BioStruct-X WP5, user access was provided to 9 complementary, highly automated platforms for protein production and crystallization located at 4 synchrotron sites in Europe (France, Germany, United Kingdom and Switzerland). The activities within WP5 started on April 1st 2013 following the end of the I3 project P-CUBE. Throughout the project duration, the WP5 participating partners delivered 2624 HTX units, 111.8 SPC units, 10 ESPRIT, 12 Multibac units and 50 protein production units.

The WP5 activities have made a major contribution to the success of the BioStruct-X consortium. The total amount of access delivered through the activities of this workpackage is impressive taking into account its limited duration within the BioStruct-X project. This effort has been dedicated to support a total of 227 users originating from 19 countries including Austria, Belgium, Czech Republic, Denmark, Finland, France, Germany, Greece, Ireland, Israel, Italy, Norway, Poland, Portugal, Spain, Sweden, Switzerland, Turkey and UK. The projects supported span a wide variety of research areas including life sciences and biotechnology, medicine, molecular and cellular biology and plant biology among others.
User demand has been very high at most of the platforms within WP5. Three of the platforms have experienced a particularly high level of demand. This is the case for the integrated crystallization services provided by the HTX lab (EMBL-GR), the sample characterization services provided by the SPC facility (EMBL-HH) and the Oxford Mammalian expression platform (UOXF). The scientific staff at these facilities has made a major effort to extend the number of TNA units available in order to accommodate the strong demand. Thereby the HTX facility (EMBL-GR) has provided 1652 TNA units instead of the 500 stipulated. The SPC facility (EMBL-HH) has provided 111.8 instead of 100 and the Oxford Mammalian expression facility has supported 35 projects instead of 20 initially planned. This has been possible by one or several of the following mechanisms: i) Developments at the facility leading to increased efficiency and lower per unit costs ii) reallocation of TNA funds from other facilities with lower costs than planned. Finally, a small number of facilities have experienced lower demand than anticipated. These include the Oxford Crystallization facility (UOXF) where following the demand, efforts have been shifted to the Mammalian expression facility (UOXF) and the Membrane protein expression and membrane protein crystallization facilities at Diamond. The latter two facilities are exclusively dedicated to supporting projects targeting the study of membrane proteins and the low demand is likely due to the fact that membrane proteins are very difficult to work with and produce insufficient amounts for crystallization experiments. Hence despite the strong interest on this type of platform for the user community the access barrier is very high, resulting in a lower number of projects than anticipated reaching this state. Nevertheless these two facilities combined have provided a total of 35 Units of access. Due to administrative problems which make it unlikely to recover the TNA access costs Diamond has provided these units on a good-will basis.

In summary, access to protein production and high throughput crystallization facilities supported through WP5 activities has facilitated a rapid progression of many projects through the early phases to data collection stage, which in a majority of the cases was supported by other BioStruct-X work packages (like WP2 and WP3). WP5 has contributed a sizable part of the overall TNA units provided by the BioStruct-X consortium and it has had a major impact in the European research landscape by contributing to advance the research programs of a large number of scientists some of which were new users of synchrotron facilities.

Scientific highlight
Krossa S, Faust A, Ober D, Scheidig AJ. Comprehensive Structural Characterization of the Bacterial Homospermidine Synthase-an Essential Enzyme of the Polyamine Metabolism. Sci Rep. (2016) 6:19501.
Project Title: Structural characterization of the evolutionary relationship between the orthologues enzymes DHS and HSS
Discipline: Molecular and cellular biology

Project summary
Using the sample preparation and characterization (SPC) facilities at EMBL Hamburg, the group of Axel Scheidig obtained crystals of the bacterial orthologue of homospermidine synthase (HSS).
This is a key enzyme of the polyamine metabolism of many proteobacteria including pathogenic strains such as Legionella pneumophila and Pseudomonas aeruginosa. The crystal structure revealed differences in the substrate binding site between prokaryotic and eukaryotic HSS, which can be exploited to find specific inhibitors. These inhibitors could potentially be used as a new line of antibiotics.
Reproduced from Krossa et al Scientific Reports (2016) 6:19501. Figure 4. On the left, a view of the substrate pocket (in red) of the bacterial HSS crystal structure. On the right, a schematic drawing of the different moieties of the substrate binding pocket, including the “Side pocket” that is only observed in bacterial HSS.

Joint Research Activities (JRA)

The four transnational access workpackages in BioStruct-X were critically enhanced by four targeted Joint Research Activities established with the essential goal of improving the quality and output of the respective TNA. The overall objectives of the JRA activities were to develop: a) new data processing tools, to the benefit of access to applications in macromolecular X-ray crystallography; b) an integrated on-line sample characterisation system, to the benefit of the access to small angle X-ray scattering, c) correlated fluorescence light microscopy components that will enhance X-ray imaging applications offered for access; d) a toolbox for mammalian cell line expression, to the direct benefit of the access to protein production facilities.

WP6 - Data Integration and Analysis for Synchrotron and FEL crystallography

Work package 6 has delivered two new substantial software packages for macromolecular crystallography, DIALS and dViewer. In addition, significant progress in the development of a high-speed analysis framework for the processing of forthcoming serial femtosecond crystallography (SFX) data from the European X-ray Free Electron Laser (Eu-XFEL) has been delivered allowing highly parallel analysis and triaging of snapshot crystal diffraction images.
DIALS is the first substantially new software package for analysing rotation data in two decades. Due to the open-source, collaborative approach to DIALS developments we have managed to bring a number of other groups into the DIALS collaboration, including the group of Nick Sauter at the Lawrence Berkeley National Laboratory, CA, USA., Gerard Bricogne from Global Phasing, Cambridge, UK. The Sauter group in particular have actively contributed to various aspects of DIALS development and are particularly interested in the implementation of XFEL SFX data analysis within DIALS.

This is all made possible by the unique modular and extensible framework underpinning DIALS that allows multiple approaches to particular data processing steps to coexist easily within the same software package. Equally this permits parallel development of these approaches by different groups and makes DIALS a very powerful development environment.

In the four and a half years of BioStruct-X the DIALS development team have delivered a development framework (Waterman et al., 2016, Gildea et al., 2014, Parkhurst et al., 2014) and a competitive data analysis suite that is already outperforming existing software packages in some important areas. One important area of development during the BioStruct-X project was on improving the analysis of data comprising very tightly overlapping diffraction spots resulting from large unit cell dimensions and improved handling of very weak mosaic crystal diffraction.

During our developments it became clear that underlying these developments was a key need to improve existing methods of accurately determining the X-ray background underneath diffraction spots. The presence of residual systematic error in the determination of background, especially where both diffraction spot and background counts are very low, has an impact of data quality at high resolution. Some users of DIALS (R. Lewis, P. Salgado, Uni. Newcastle; K.S. Wilson, Uni York; M. Isupov, Uni Exeter; R. Bunker, Friedrich Miescher Institute; personal communications) have reported significant gains in resolution, improved electron density maps and improved refinement statistics due the new DIALS background handling algorithms.
We are now in a strong position to progress the development of the integration of closely overlapping spots in such a way as to introduce less systematic error and allow the decoupling of potential residual twinning-like effects caused by the over estimation of very weak reflections. However, our new algorithms in DIALS are already having a significant impact on, and specifically address, problems caused by weakly diffracting, highly mosaic data such as membrane protein crystals.
The treatment of weak diffraction data (e.g. by profile fitting) is dependent on the accuracy with which we can predict the positions of diffraction spots. This is determined by how well we can model our experiment in terms of source, crystal and detector properties. In DIALS we have taken a new approach to this modelling that permits a global approach to parameterizing the experiment. Multi-panel detectors such as the Pilatus and Eiger detectors from Dectris or the CS-PAD detectors at the LCLS can be modelled as many separate detectors. This allows the detailed metrology to be optimised (Hattne et al., 2014).

Further DIALS allows joint refinement of detector parameters using many crystals creating opportunities for brand new approaches to the analysis of serial crystallography data. The new refinement algorithms are implemented with the dials.refine (Waterman et al., 2016) module and this has already been applied to the analysis of serial crystallography data from the LCLS.
Key to the development of DIALS has been our collaboration with Dectris and the Paul Scherrer Institute. The ability within DIALS to isolate computer code that is specifically tailored to the performance and physics of a given detector provide opportunities to work very closely with detector manufacturers. Dectris, producers of the Pilatus 6M detectors prevalent at many synchrotron MX beamlines have developed the ability to apply customized count rate corrections to the diffraction data recorded with their detectors (Trueb et al., 2012) and work is now on-going to implement these advances in DIALS.
The development of dViewer as part of the BioStruct-X project has provided the MX user community in Europe with access to a state-of-the-art tool for diffraction image inspection. The software has so far been in use at ESRF MX beamlines with users and staff providing critical feedback to the developers at EMBL Grenoble. The software has now matured to version 1.6.0 and includes a number of very useful features, shown in Figure 5) for visualising data from Pixel Array Detectors (PADs). The software has been developed within the DAWN framework (Basham et al., 2015) and is available from http://www.dawnsci.org/.

Figure 5. Diffraction image visualization features offered by dViewer version 1.6.0. Top: dViewer can display images in groups and for each data set permits the display of multiple merged images to improve visualization where very fine sliced data has been recorded. An image slider allows the full data set to be conveniently navigated by the user while assessing diffraction quality and the presence of any pathology. Middle: the independence of PAD detector pixels can give rise to very small diffraction spots that are hard to pick out visually when manually inspecting an image. The spot highlighter tool applies a blurring function to the image display to make the reciprocal lattice more easily visible. Bottom: Navigation into the detail of an image has been improved through the provision of Google-style zooming capability.

Karabo, the European-XFELs software framework for managing the immense data measurement and analysis challenges raised by XFELs, provides device servers (send and receive data) and empty devices that are programmable by the user. A simulated Serial Femtosecond Crystallography data processing pipeline using Karabo devices has been set up as part of the BioStruct-X project to investigate the detector → crystal indexing → hkl merging pipeline.
The detector device sends simulated diffraction patterns to the indexing devices. The indexing devices can run on different machines at different locations and Karabo ensures that the fast CPUs see more of the data to balance the processing load. Merging device merges all the hkl information into a small stream file as output.

Figure 6. Indexing using libcrystfel: Karabo provides the device servers and empty devices for distributed computing. A simulated SFX data processing pipeline was set up and crystal diffraction pattern indexing was implemented in the compute method using libcrystfel functions.
Figure 7. Essentially linear speed up in image indexing from the use of up to 80 CPU cores.
The scalability of the SFX data processing pipeline was tested on a computer cluster (up to 80 CPU cores) and is shown in Figure 3. An essentially linear speed up with the number of cores was observed. Assuming linear speed up, we would need about 3500 CPU cores to index crystals at 5000 images/sec (given that indexing one diffraction pattern takes 2.3 seconds and only 30% of the frames contains crystal diffraction).
This first simulation provides confidence that serial crystallographic data analysis is feasible within Karabo at the European XFEL, and gives an initial estimate of the scale of computing resources required as a function of data load.

Modelling work as a basis for more complete modelling of serial crystallography experiments has been performed at the European XFEL by Chun Hong Yoon, who was employed within the BioStruct-X project at both CFEL, DESY, Hamburg and European XFEL. A complete XFEL experiment that includes the modelling of source properties, propagation through realistic X-ray optics, the interaction of intense, XFEL pulses with a non-crystalline bio-sample (including the associated time-dependent radiation damage) and analysis of the modelled signal has been performed for different source parameters of the European XFEL exploiting the SPB/SFX instrument. The software modules developed therein provide a first framework for future modelling of more general XFEL experiments, including structure determination of crystalline samples. This first tool-chain is publicly available (xfel.eu/simS2E) and its associated description is presently under review (C.H. Yoon, et al, submitted, 2016).

Achieved Deliverables:
D6.1 Framework delivered - Prototype (P)
D6.2 Algorithms delivered - Prototype (P)
D6.3 Simulation and visualization tools developed - Prototype (P)
D6.4 Prototype pipeline delivered - Prototype (P)
D6.5 Delivery of integration suite - (Report (R))

WP7 - Integrated Beamline Environments for Biological Sample Characterisation and Optimisation

In Workpackage 7, the combination of different biophysical characterization techniques has provided a powerful new method to determine concentration-dependent oligomerization of biological samples. The complementarity between static light scattering and SAXS has been applied for the first time as a pipeline at a synchrotron, through a collaboration with Malvern Instruments. Software was developed to provide high-throughput analysis of scattering curves, allowing non-experts to analyze SAXS experiments on the fly. The distribution of knowledge on SAXS analysis is thus facilitated through robust and informative software interfaces that are easily portable to other facilities and can interface through xml with other software packages. New technology for sample delivery in SAXS and macromolecular crystallography has been developed and this will likely become the standard technology at synchrotron beamlines around Europe. Patents were filed for a SAXS sample changer and a new storage Dewar for macromolecular crystals that prevent ice formation. BioStruct-X also provided the means to engage the different synchrotron sites in discussions to develop these technologies, and to distribute and test them so they become ultimately available to the structure biology community.

The advance of highly brilliant and focused X-ray beams has caused a revolution in X-ray based structure determination, both in the field of small angle X-ray scattering (SAXS) and macromolecular crystallography (MX). A large user community can collect tremendous amounts of high-quality X-ray data in a small time frame. However, these high-throughput capabilities require new technologies in sample handling and challenge users to make intelligent and comprehensive decisions in data collection. Work package 7 has provided novel developments in sample delivery, characterization and on-the-fly data evaluation that have begun to impact the user community on a large scale.

SAXS is a powerful technique to gauge the structural properties of biological macromolecules in solution. For the proper determination of these properties, it is important that the sample solution is pure and monodisperse. A modular multi-method purification and characterization set-up was developed at EMBL Hamburg together with Malvern Instruments to purify and characterize the macromolecular sample at the SAXS beamline, so that the sample is measured by SAXS immediately after purification. The biophysical characterization included right angle light scattering to determine the molecular weight of the purified sample, and the determination of sample concentration using both UV-Vis spectroscopy and a refractive index detector. The most optimal set-up was achieved when the biophysical characterization was done in parallel with the SAXS measurement (Graewert et al 2015). A similar system was integrated at the ESRF/EMBL beamline BM29 in Grenoble, where special modifications of the BioSAXS sample changer were made to ensure easy and secure exchange between the SEC-SAXS and batch data collection mode. The system has been in user operation since February 2013. Approximately, 15% of the users request the set-up during the beam line proposal process at EMBL Hamburg. Importantly, the configurability of the tested hardware and software components will allow for a seamless integration of the set-up at other synchrotron facilities.

An automatic sample changer for the measurement of SAXS using sample batches loaded into a 96-well format has been incorporated at BioSAXS beamlines on the ESRF, EMBL Hamburg, Diamond and MAXIV synchrotrons. This system now makes full use of the state-of-the-art beamline properties, requiring small amounts (10-50 uL) of sample in a high-throughput mode (Blanchet et al. 2015).

Applications such as on-line purification of samples and high-throughput batch measurements require a data management system that is comprehensive and flexible. The highly successful Information System for Protein Crystallography Beamlines (ISPyB) was adapted to produce a prototype data base for biological SAXS. The new data base named ISPyBB (ISPyB for BioSAXS) enables users to manage SAXS experiments in a high-throughput manner (De Maria Antolinos et al. 2015). Feedback from the user community has been very positive, emphasizing that ISPyBB provides users with greater independence during experiments and provides confidence that all data required for further analysis have been collected within the limits of sample availability. Since its installation at the ESRF it is now also installed at the partner institutes DIAMOND and EMBL@PETRAIII and is being installed at 2 additional sites (SOLEIL and MAXIV).
The tremendous growth in the use of SAXS as a complementary technique for structural studies has led to an influx of users with little experience in performing SAXS experiments. To facilitate these users to perform high-quality SAXS experiments, a set of protocols for automated data reduction and analysis has been implemented into the SASFLOW data analysis pipeline. The pipeline provides the user with an immediate feedback about the sample characteristics and enables the users to modify the sample preparation conditions on the fly to optimize the SAXS experiment. The pipeline includes steps for primary data processing, automated merging of data collected from different solute concentrations, analysis of scattering curves and ab inito shape reconstruction. Standarization of the ATSAS programs’ command-line interfaces and of the reading and writing of data files has facilitated the addition of new components to the piepeline. Based on the collected SAXS data, structural neighbours already available at the Protein Data Bank are suggested on-the-fly by means of a module linked to the reimplementation of DARA (Kikhney et al. 2016). If the user provided a priori high-resolution models, their fit against the SAXS data being collected is calculated and flexible refinement is also available through the SREFLEX program recently developed at EMBL Hamburg (Panjkovich A. and Svergun DI 2016a). All the information generated by SASFLOW is summarized using XML format to be conveniently presented with a Web browser and, if the ISPyBB database is used, to represent and compare the obtained results with previous acquisitions. Additionally, a PyMOL plugin that provides users with a point-and-click interface to build and refine models was implemented as part of the combined effort (Panjkovich A. and Svergun DI 2016b).

Macromolecular crystallography has also become a highly automated discipline, but it has not yet fully developed sample delivery to exploit the latest capabilities in brilliance and high-throughput provided by the synchrotron beamlines. A new high-density system was developed to store and mount pin-containing crystals. After several rounds of design were tested at EMBL Grenoble, a MiniSPINE standard was agreed upon by synchrotron sites in Europe, the USA and Japan. The MiniSPINE allows a storage density of 36 samples per puck (288 per shipping Dewar (Taylor Warton CX100)). This is substantially more than the traditional SPINE system, but still ensures that both systems can be mounted on goniometers around the world. A complete kit to harvest, store and mount crystals using the MiniSPINE system was produced and tested at several synchrotron sites. On the whole, the sample storage and transporting cost will be reduced with MiniSPINE and Dewar and puck handling efforts will be reduced at high throughput beamlines. Finally, improved crystal positioning will increase the speed of crystal alignment and benefit from emerging automated harvesting systems anticipated to provide crystal position information.

Publications:
Blanchet CE, Spilotros A, Schwemmer F, Graewert MA, Kikhney A, Jeffries CM, Franke D, Mark D, Zengerle R, Cipriani F, Fiedler S, Roessle M, Svergun DI. (2015). Versatile sample environments and automation for biological solution X-ray scattering experiments at the P12 beamline (PETRA III, DESY). Journal of Applied Crystallography (2015). 48, 431–443.

A. De Maria Antolinos, P. Pernot, M. E. Brennich, J. Kieffer, M. W. Bowler, S. Delageniere, S. Ohlsson, S. Malbet Monaco, A. Ashton, D. Franke, D. Svergun, S. McSweeney, E. Gordon and A. Round. (2015). ISPyB for BioSAXS, the gateway to user autonomy in solution scattering experiments. Acta Cryst. D71, 76-85.

Graewert MA, Franke D, Jeffries CM, Blanchet CE, Ruskule D, Kuhle K, Flieger A, Schäfer B, Tartsch B, Meijers R, Svergun DI. (2015). Automated pipeline for purification, biophysical and x-ray analysis of biomacromolecular solutions. Sci Rep 5 doi:10.1038/srep10734.

Kikhney AG, Panjkovich A, Sokolova AV and Svergun DI (2016) DARA: a web server for rapid search of structural neighbours using solution small angle X-ray scattering data.Bioinformatics 32, 616-8.

Panjkovich A. and Svergun D.I.(2016a) Deciphering conformational transitions of proteins by small angle X-ray scattering and normal mode analysis. Phys. Chem. Chem. Phys. 18, 5707-19.

Panjkovich A. and Svergun D.I.(2016b) SASpy: A PyMOL plugin for manipulation and refinement of hybrid models against small angle X-ray scattering data. Bioinformatics 28 Feb. doi: 10.1093/bioinformatics/btw071

Achieved Deliverables:
D7.1 Multi-method characterization prototype - Prototype (P)
D7.2 Extended prototype ISPyB database - Report (R)
D7.3 Flexible sample charger - Prototype (P)
D7.4 Unified SAXS pipeline - Report (R)
D7.5 Multi-method analysis - Report (R)
D7.6 Database intercommunication protocols - Report (R)
D7.7 Development and testing of NewPin - Report (R)

WP8 – 3D Tomographic X-ray Imaging of Biological Cells

The aim of BioStruct-X WP8 was to further the development of X-ray microscopy. At the start of the funding period, X-ray microscopy had just evolved to the point where the first 3D high resolution images of cells had been obtained at one synchrotron X-ray microscope. The BioStruct-X funding has been essential to firmly establish this approach and also greatly expand its capabilities. Thus over the period of BioStruct-X funding X-ray microscopy approaches have been exported to a number of different synchrotron facilities, and these X-ray microscopes are now being used by numerous scientists from around the world. Furthermore, the funding has led to the development and implementation of correlative X-ray fluorescence imaging and the development of improved methods for image reconstruction designed specifically for X-ray microscopy.

A major advance catalyzed by BioStruct-X funding was the development of correlative X-ray and fluorescence microscopy. This marriage combines the molecular specificity of fluorescence with the high resolution 3D imaging of X-ray microscopy. In particular, the ability of the X-ray microscope to obtain 3D images of cells at ~50 nm lateral resolution without chemical fixation, staining or sectioning makes it a powerful approach to visualize cellular ultrastructure in its near native state subject only to cryo-preservation. The overlay of fluorescence light microscope images then offers the ability to translate this molecular labelling onto the underlying ultrastructure detected by the X-ray microscope.
This correlative approach is accomplished by incorporating both microscope modalities into one instrument such that the same cell can be examined first by the fluorescence microscope, and then without any movement of the specimen examined next by X-ray microscopy. Shown below is one of many examples enabled by the BioStruct-X funding in which this correlative technique has been applied to important cell biological problems.

Figure 8. X-ray microscopy images at three different focal planes (235, 204 and 199) from a 3D stack of images obtained from an Hek293 cell. Correlative fluorescence image (2) labeled with two different markers for autophagosomes, namely RFP-Atg9 (red) and GFP-LC3 (green). The labeled structures can be found in the X-ray slices enabling a 3D rendering of the different organelles (schematic at right). The green organelles tagged by LC3 reflect organelles known as omegasomes, and the X-ray microscopy data suggest that multiple omegasomes can arise from the same endoplasmic reticulum subdomain. (from Duke et al. Ultramicroscopy 143:77 (2014).

The expansion of the X-ray microscopy approach to multiple synchrotrons and its entry into a mainstream technique has provided a number of new biological insights. One striking example was the use of X-ray imaging to help define the structure and function of the nuclear export complex. This is a system involved in the release of virus particles from the nucleus that may also reflect a general, hitherto under-appreciated mechanism for vesicular export of larger cargos from the nucleus to the cytoplasm.

Figure 9. X-ray microscopy images of nuclear membrane budding structures (yellow box in A, D’’’) plus schematic model of the budding process. From Hagen et al. Cell 163:1692 (2015).
Finally, the BioStruct-X funding has also enabled the development of custom approaches for image restoration in X-ray microscopy. Prior to this funding, the tomographic tilt series acquired in X-ray microscopy was reconstructed using software designed for data from cryo-electron microscopy (cryo-EM). This produced good reconstructions, but the procedure did not account for the finite depth of field in X-ray microscopy that spans only about 1/3 of the thickness of a typical mammalian cell. BioStruct-X funding has enabled the development of new reconstruction procedures that account for this finite depth of field by acquiring tilt series at several different focal planes and then merging these data to produce a more accurate reconstruction of the specimen. Clear improvements in the images have resulted, as shown below.
Figure 10. Cytoplasm from a mammalian cell processed by the conventional tomography approach (d), and by the new approach accounting for the depth of focus (l).

n summary, BioStruct-X has been vital for significant improvements in X-ray microscopy that has enabled this approach to enter into the mainstream of tools for biological imaging.

Achieved deliverables
D8.1 Report on the implementation of a dedicated reconstruction algorithm for X-ray tomography - Report (R)
D8.2 Report on the developments to the HZB TXM - Report (R)
D8.3 Report on the transfer of developments to the beamlines - Report (R)

WP9 – Mammalian Cell Toolbox for Structural Biology

Most of the current available automated cell culture systems are developed in collaboration with pharmaceutical industries. Those systems are mainly configured for screen assays with 96 or 384 well plates. Those assays require a very limited number of cells in comparison to the number of cells used for protein production. On implementing the CompacSelecT system for large scale mammalian expression, the most challenging configuration was how to use the completely novel type of 10-layer HYPERflasks to fulfill our requirement of a larger number of cells for high yield protein expression.
Sartorus Stedim (previously known as The Automation Partnership, TAP) together with Corning developed a new set of robot movement modules especially for dealing with HyperFlask handling. UOXF are the first to use HYPERFlasks in the automated cell culture system for large scale protein production and as a consequence, have tackled several problems to implement the use of HyperFlasks in the CompactSelecT mammalian system. The HyperFlask has a slightly bigger footprint and requires medium to be filled to the neck brim. This leads the gripper sensor to receive a false signal from the reflecting surface of the HYPERFlask (Fig 11a).

Figure 11. Implementation of HyperFlask in SelecT Compact system. a. reflection onto the gripper sensor;
b. Flask fill to neck brim;c. Flask fill to neck brim; c. Media leakage from HyperFlasks; d. HyperFlask in operation in Robot
Together with TAP engineers, we investigated a number of potential solutions for this and finally adjusted the sensor to a special angle where no false signals were detected while still handling all flasks perfectly.
The HYPERFlask requires very precise filling of flasks (Fig.11b). When filling the HYPERFlask in manual mode, at near full level, it requires a pipette to remove serum foam at the top. As the robot cannot do that, the filling is likely either too little or media comes out of the flask because of foaming at the neck. We solved the problem by precisely calibrating the media tubing each time before use and we also introduced a new specific gentle robot movement before final 10 ml of media was delivered into HYPERFlask. This rectified the flask filling problems. The worst problem we encountered when implementing the use of HYPERFlasks in the robot was that the early generation of HYPERFlasks leaked at the junction of layers on one seam of the flask (Fig.11c). This was due to design/manufacturing problems of the flask. After a long investigation and further development with advice and testing at UOXF, Corning finally redesigned the product, strengthening the junction between the cell surface membrane and the plastic support. We further developed the transient transfection protocol for HYPERFlasks that allows the robot to do automated transfection on introduction of the DNA-PEI mixture. Previously, this step had only been possible manually. The HYPERFlask system for large scale mammalian expression within the automated cell culture system is now routinely in use (Fig.11d). Furthermore, UOXF has developed the stable cell line protocols, and stable cells can routinely maintained at CompactSelecT robot. TNA users have benefited from the system.

Methods for genetic engineering of constructs to improve activity, folding, solubility and crystallization of proteins are important in many research fields. Classically, modifications to the gene sequence are guided by comparison with similar related proteins, or using structural knowledge. However, many interesting targets bear little resemblance to known proteins and, in such cases, random library approaches may help. Mutation procedures and phenotypic screens or selections can be used to obtain new variants with the desired properties. The requirement for handling large numbers of clonally distinct library members generally limits these strategies to in vitro systems or bacterial hosts that are limited in their abilities to express complex post-translationally modified or multidomain proteins. In WP9, we have generated a system MamESPRIT (to be published as EMPEX), a system that combines an E. coli-based life-or-death selection for in-frame constructs from incremental gene truncation libraries with a multiwell protein expression screening step in mammalian cell lines.

To benchmark EMPEX, we screened human p85 alpha (p85α) resulting in the successful isolation of single and tandem domains when expressed cytoplasmically. We then applied a protein secretion version of the EMPEX system to the extracellular region of the human receptor protein tyrosine phosphatase sigma (RPTPs) resulting in the isolation of stable and soluble tandem and triple domain constructs. The method requires little-to-no automation, and should therefore facilitate construct screening in challenging projects, notably those on eukaryotic membrane and cytosolic proteins.
A very large number of transduction experiments were carried out with the novel MultiBacMam reagents (Fig.12). Applications included bi-molecular fluorescence complementation (BiFC) for small molecular ligand screening targeting protein protein interactions PPIs evidencing the superior transduction property of MultiBacMam as compared to conventional transduction. Further applications included multigene transduction of primary cells (HUVEC, REF,...) and even iPS cells. Reprogramming of mouse embryonic fibroblasts (MEFs) to neurons was achieved by transducing transcription factors. MultiBacMam was applied to zebrafish cells successfully, broadening the scope of application of our new tool-kit. Notably, CRISPR/Cas9 based genome engineering with a single MultiBacMam reagent comprising all factors required (Cas9, guide RNAs, DNA containing homology arms). The results of these studies, jointly performed by partners EMBL and PSI, are under revision in Nature Communications. A manuscript describing the MultiBacMam-based BiFC tool-kit has been submitted.

Figure 12. (A) MultiBacMam system is shown schematically. MulitBacbMam follows the logic of the MultiBac system for multigene insertion into a baculoviral genome which has been customized for efficient transduction of mammalian cells. Composite baculoirus is generated and amplified in insect cells and then applied to established or primary mammalian cells (right, top). (B) The striking gain of transduction efficacy by MultiBacMam (right) is shown as compared to conventional plasmid transfection (left).

GPCRs are integral membrane proteins that play fundamental roles in many physiological and pathological processes. Because GPCRs are the target of ~30% of all medical drugs, they have attracted huge interest and commercial investment as drug targets and for the high-throughput screening of drugs.
Most GPCRs are glycosylated, which often poses a problem for crystallization as a result of the heterogeneity of the protein sample. To overcome this hurdle, we used a genetically engineered cell line, HEKS293 GnTI-, which is defective in N-acetylglucosaminyltransferase I and therefore unable to synthesize complex glycans. Furthermore, we also used a tetracycline-inducible expression system for the production of target proteins. Using this approach, we established a robust method for the identification, stable expression and purification of homogeneously-glycosylated membrane proteins from suspension cultures that are suitable for structural work.
The novel fusion approach developed in this study provided very promising results that were generally better than those obtained for the established fusion proteins T4L and bRIL that are successfully used to determine high-resolution GPCR crystal structures. Using this novel strategy, we recently obtained crystals of two different GPCRs.

Achieved deliverables
D9.1 TAP Compact SelecT Robot protocols for mammalian cell expression - Report (R)
D9.2 MamESPRIT construct library screening in mammalian cells - Report (R)
D9.3 MultiBacMam system construction and evaluation - Report (R)
D9.4 Limited glycosylation system - Report (R)

WP10 – Integrated Networking Between TNA Providers, JRA Providers and the TNA User Community

This workpackage, devoted to networking activities, was the largest and most diverse of all BioStruct-X workpackages and actively engaged all 19 partners of the Consortium from the very onset of the project. The overall goal was to foster a culture of cooperation between the BioStruct-X participants and the scientific communities benefiting from the research infrastructures with the aim of aiding in the development of an efficient and attractive European Research Area.

Five different objectives divided into eight specific tasks in this workpackage ensured a high level of training activities, exchange of information between the TNA/JRA partners and the targeted research user community, and dissemination of knowledge, methods and technologies to both the BioStruct-X user community as well as to other relevant research organisations and the industry.
Furthermore, through its networking activities, BioStruct-X promoted the dissemination of good practices, clustering and coordinated actions with related projects and international related initiatives (such as Calipso, BioNMR and INSTRUCT) as well as supported the deployment of global and sustainable approaches in the field and supported training of new users.

The four Training and Dissemination (TID) centres from Finland, Hungary, Portugal and Israel played an instrumental role in ensuring the widest possible level of networking, including remote EU member and associated states and states that do not own competitive synchrotron radiation facilities for TNA applications.
During the BioStruct-X project 36 scientific events have taken place across Europe involving participants from many European and also overseas countries. In addition to the scientific value and networking possibilities one would highlight the special events on gender balance, which took place at the Industrial meeting in Hamburg (2015) and during the final BioStruct-X meeting in Lisbon (2016). Both events were highly successful and prompted lively discussions among the participants.

Achieved deliverables
D10.1 Summary report on networking activities - Report (R)
D10.2 Summary documentation on the pilot application form and data exchange protocols - Report (R)
D10.3 Report on the pilot version of Single sign on (SSO) authentication - Report (R)

Potential Impact:
Structural biology has provided the means to achieve three-dimensional atomic resolution images of biological macromolecules that underpin basic biological processes. Many of these molecules are potential targets for the biotechnology and pharmaceutical industries, and their structural biology underpins enormous economic and health benefits. The elucidation of the structural details of molecular complexes can provide important information on functional interactions, regulatory mechanisms and sub-cellular dynamics that are relevant to new therapies, and to our fundamental understanding of cell biology.

In order to address some of the most challenging scientific questions, the BioStruct-X consortium was designed to combine scientific excellence, multidisciplinarity, complementarity and experience, by exploiting longstanding interactions and joint collaboration records between its partners. It has brought together top-notch research organisations with a leading capacity to build a broad platform of infrastructures that is able to address all stages of biological structure determination: from protein production of sufficient quantity and quality for structure analysis, to sample production and data collection by a variety of X-ray methods. With its successful implementation, the BioStruct-X consortium pioneered the integrated European infrastructure provision allowing diverse structural biology synchrotron activities to be organised by structural biologists for the scientific community. Through its website, BioStruct-X developed and deployed project tools that enabled the offer of a unified portal for simultaneous multi-site user project applications and their evaluation by a centralized project review committee - a key instrument for integration of all BioStruct-X activities.

The BioStruct-X TNA activity supported more than 1900 users that performed more than 1100 experiments. This activity has been particularly instrumental for users from countries without a national synchrotron facility. Furthermore, it is of importance to mention that 48% of the total number of BioStruct-X supported users were young scientist below the age of 35. Through BioStruct-X TNA support those scientist were given the opportunity to carry out their experiments and acquire specific expertise at an early stages of their career for the overall benefit of their future research activities.

Moreover, BioStruct-X provided the means to engage different synchrotron sites in discussions to develop new technologies, and to distribute and test them so they become ultimately available to the structural biology community. The commitment of BioStruct-X project to the development of new technologies and methods resulted in a number of technological advances in the field.

The four targeted Joint Research Activities were established with the essential goal of improving the quality and output of the respective TNA. The overall objectives of the JRA activities were to develop: a) new data processing tools, to the benefit of access to applications in macromolecular X-ray crystallography; b) an integrated on-line sample characterisation system, to the benefit of the access to small angle X-ray scattering, c) correlated fluorescence light microscopy components that will enhance X-ray imaging applications offered for access; d) a toolbox for mammalian cell line expression, to the direct benefit of the access to protein production facilities.
WP6 has created a new modular and extensible data integration package, DIALS, for synchrotron and XFEL crystallography plus the dViewer diffraction visualisation software. The WP6 researchers designed and implemented the programming framework for DIALS and populated it with both existing and novel algorithms for the full analysis of macromolecular crystallography data, both recorded from single crystal and serial crystallography data. Key algorithms from DIALS have had a significant impact in the XFEL field in enabling high quality metrology on X-ray detectors to be performed thereby increasing the effective resolution of XFEL diffraction data.

The JRA WP7 has delivered advances in sample handling and analysis for SAXS and MX that have been adopted at many laboratories around Europe. The development of complementary biophysical characterization of the sample in line with a SAXS experiment has provided a new method to deliver pure sample that is at the same time validated for its homogeneity. An expert package for SAXS data collection and interpretation called SASFLOW was developed, in conjunction with the laboratory information management system ISPyBB. Together, these software packages provide synchrotron sites with the tools to allow users to collect meaningful data, and help to disseminate the expert knowledge how to perform a proper SAXS experiment.

The purpose of the JRA for X-ray microscopy was to disseminate new developments in this field as quickly and as widely as possible. The funding enabled advances in instrumentation for X-ray microscopy, including correlative X-ray microscopy and fluorescence light microscopy, as wells as new developments in image processing for X-ray microscopy, namely deconvolution approaches tailored to the specific features of X-ray microscopy. These developments were quickly exported to other synchrotron facilities around the world, and therefore enabled many new biological investigations using X-ray microscopy. Furthermore, some of the techniques developed in BioStruct-X were adopted by Zeiss and are now being sold commercially, generating a larger economic impact. In sum, BioStruct-X funding provided critical support to enable X-ray microscopy to establish itself as a valuable and essential technique in structural biology

As part of the JRA WP9 activities, the University of Oxford implemented robotic tissue culture system and advanced the mammalian expression system for protein production for structural studies. The mammalian expression techniques have been disseminated across EU laboratories. Structural determination of human proteins or human pathogens is fundamentally important for understanding mechanisms of human diseases and for potential drug development. However, quite some important target proteins could only expressed well in mammalian system. For example, a TNA user from Greece determined the human antigen processing aminopeptidase, which is only could expressed with mammalian stable cell system. The structure of insulin regulated aminopeptidase advanced our knowledge of human immune response (Mpakali et al, PMID:26259583). Similarly, a French TNA user determined the structure of human chitotriosidase, may help to understand the human allergic action (Fadel et al., PMID 26143917). We determined structure of NPC1, an Ebola virus receptor (Zhao et al., PMID26846330) and used mammalian expressed Ebola virus glycoprotein for drug screen, which may lead to important advances for anti-Ebola drug development (Zhao et al., manuscript close to submission).

Main dissemination activities
The main project dissemination activities included the development of the public website, raising public awareness and reaching out to the new user communities. Furthermore, as an integral part of the Networking workpackage activities, a series of training events, scientific workshops and courses was organised and successfully carried out.

The dissemination of scientific results resulting from transnational access activities and joint research activities was primarily carried out via publications in peer reviewed journals, presentations at meetings, workshops and international conferences as well as the annual project meetings and user meetings in individual synchrotron facilities. In order to attract new user communities, a detailed project flyer describing the opportunities for access was designed, regularly updated and distributed among consortium partners, at meetings, workshops and conferences.

Exploitation of results
The full release of DIALS to the community as an open-source package will now allow it to be exploited both as a data analysis package by users worldwide and as a development toolkit to explore new ideas for data analysis. The team and Diamond and CCP4 in particular will actively be developing DIALS further in coming years to meet new challenges arising from the development of new X-ray sources, the use of electrons for 3D crystallography, new detectors, neutron crystallography and nano crystallography to name a few.
Making macromolecular crystallography more efficient will benefit structural biology research in general and therefore contributes to the advance of projects that aim at understanding and treating human diseases and fundamental biological processes. Within the WP7 JRA a new system to store and mount crystals was developed, and the BioStruct-X project was ideal to bring together most of the European synchrotrons. Similarly, companies working in the field of consumables and robotics for macromolecular crystallography were integrated in the project (IRELEC-Alcen, MiTeGen LLC, Molecular Dimensions Ltd, NatX-Ray). The new system is now in its implementation phase, providing the user community with an efficient and affordable means to handle their precious macromolecular crystals.

List of Websites:
BioStruct-X website: http://www.biostructx.eu/

Contact: Dr. Dmitri Svergun (Coordinator), svergun@embl-hamburg.de
Dr. Ivana Custic (Project Manager), custic@embl-hamburg.de
Ivanka Araujo (Project Manager Assistant), araujo@embl-hamburg.de

Related information

Reported by

EUROPEAN MOLECULAR BIOLOGY LABORATORY
Germany

Subjects

Other Technology
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