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P-CUBE Newsletter No. 2

March 2, 2011

One word from the CoordinatorMarkus Grütter talks about the first 18 months of P-CUBE

P-CUBE is the first project within the Seventh Framework Program that supports cutting edge infrastructures critical for the efficient production and crystallization of proteins and protein complexes in

contemporary structural biology. The infrastructures comprise technology platforms for (i) high-throughput cloning and expression of proteins in prokaryotic and eukaryotic cells, (ii) high-throughput crystallization, (iii) high throughput selection technologies and (iv) advanced light microscopy. The program on the one hand offers access to its infrastructures free of charge for scientists all across Europe. The program in addition supports activities to further improve and automate the methods within each infrastructure. European experts in the field from the University of Zurich, the University of Oxford and the European Molecular Biology Laboratory share expertise, equipment and their know-how.

The first 18 months of P-CUBE have been reviewed in the user meeting held in September 2010 at the EMBL in Grenoble. At the same time a satellite workshop on high-throughput protein expression and crystallization was held for

future potential users of the P-CUBE infrastructures (see P-CUBE Training p. 5).

This meeting nicely illustrated the excellent start of P-CUBE as judged by the large number of applications from scientists throughout Europe as well as by the number of projects already carried out in the different platforms.

Most surprising was the outstanding quality of the research that could critically profit from the cutting edge infrastructures which is already manifested in publications in high-ranked journals.

Besides the successful transnational access to infrastructures aspect of the program a number of technology improvements in different infrastructure platforms were reported.

The most important general conclusion from the review meeting is the successful and fast way the technology know-how of only a few specialized research units has been disseminated throughout the wider European scientific community and the high quality of projects that could profit through the P-CUBE program.

Dissemination of know-how will remain a central goal of the program which is also documented by the various training activities already carried out and planned in the future.

Contents

Transnational Access 2

Scientific Highlights 3

P-CUBE Training 5

Feature on:

Advanced Microscopy Platform 7

News from the platforms 9

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One big pillar of P-CUBE besides research is Transnational Access (TNA). P-CUBE aims at offering and granting access to all currently available and essential technologies in structural biology such as cloning, expression, protein characterization and crystallization. It provides facilities as well as the expertise and knowhow of experts in the field to European researchers. Scientists can apply for TNA via the P-CUBE web page. Upon positive review of the P-CUBE external expert board, the proposed project will be implemented at the corresponding platform.

Transnational AccessP-CUBE strives to enable access to cutting-edge technologies in structural biology.

Jutta TatzelProgram Manager(University Zurich)

During the first 18 months of P-CUBE, a large number of projects have already been conducted at the different platforms, ranging from time- and labour-intensive projects at the DARPin selection facility at the University of Zurich (UZH) to crystallization experiments at the High-throughput crystallization (HTX) platforms.

The University of Oxford (UOXF) implemented one project at the bacterial and two projects at the mammalian expression platform, respectively. In addition, 3 projects were conducted at their HTX facility corresponding to 139 crystallization plates. The UZH carried out four projects at the DARPin selection facility. At EMBL Grenoble (GRB), the ESPRIT platform conducted 4 projects, whereas the MultiBac facility completed 18 projects. At the HTX platform in GRB, 24 projects were carried out, corresponding to 445 crystallization

plates. The HTX platform at EMBL Hamburg (HAM) performed 16 projects on 313 crystallization plates. At EMBL Heidelberg (HD), four projects were executed at the Advanced Microscopy platform (also see Feature p. 7).

Researchers applying and conducting the projects at the P-CUBE platforms have different scientific backgrounds. Experienced scientists (EXP) represent the largest group, followed by Post-Docs (PDOC), Post-Graduates (PGR) and Technicians (TEC).

Successful applications have been received from 17 different European countries. The majority of users came from institutions residing in Germany and France (16 and 19 projects, respectively). However, it was particularly nice to receive applications from several Eastern European countries, such as Hungary, Poland, Romania and Slovakia.

Due to extensive advertisement through the P-CUBE partners at conferences and meetings, TNA is now widely known and well accepted within the European community.

1!

2!

3!

4!

4!

18!

24!

16!

4!

Bacterial Expression!

Mammalian Expression!

HTX-OXF!

DARPin!

ESPRIT!

MultiBac!

HTX-GBR!

HTX-HAM!

Advanced Microscopy!

UO

XF!

UZ

H!

EMBL

GR

B!EM

BL

HA

M!

EMBL

H

D!

Projects per platform!

AT (3)!BE (2)!

CH (2)!

DE (16)!

DK (3)!

ES (7)!

FI (5)!

FR (19)!

GB (5)!

HU (1)!IT (1)!LU (1)!

PL (1)!

PT (7)!

RO (3)!SI (2)!

SK (1)!

Location of users' institutions!

40!

17!

4!

15!

Researcher Status!

EXP! PDOC! TEC! PGR!

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Scientific HighlightsThe switch that activates the defence against drought in plants

Detail of the plant hormone Abscisic Acid (center) bound to its cellular receptor PYR1.

Abscisic Acid (ABA) is an hormone that controls many aspects of plant development, such as seed dormancy, germination and vegetative development. At the same time it plays a key role in activating the defence mechanisms against adverse environmental conditions, like drought or cold. During extended dry periods, ABA levels in the plant can rise up to 40-fold, triggering a specific signalling cascade that will ultimately result in the activation of a number of metabolic responses to avoid and adapt to the loss of water. It’s importance in plant development, but also it’s obvious biotechnological potential has made of the ABA signalling pathway one of the most intensively studied in plants.

The ABA signalling pathway requires the activity of two types of proteins with opposed actions, the protein phosphatases 2C (PP2Cs) and Serine-Threonine protein kinases, of the SnRK family. In the absence of ABA, the protein kinases are “held-hostage”, in an inactive state, by the PP2Cs, however in the presence of ABA the PP2Cs become inactive, releasing the SnRKs which leads in turn to the transcriptional activation of stress responsive genes. However, since ABA neither binds nor inhibits the activity of PP2Cs directly there had to be a receptor that conveyed the signal. The search for the ABA receptors has been long and intense, with a false start and some highly disputed candidates… until recently!

In May 2009, a number of research groups reported in the journal Science the discovery of a family of proteins named PYR/PYL/RCAR, with the capacity to bind ABA specifically and to inhibit the activity of the PP2Cs. The crystal structure of the ABA receptor PYR1 has now been obtained

by our groups and shows how the hormone is bound in a large cavity inside the PYR1 protein. The loops surrounding the entry to this cavity act as “gates”, closing over the hormone once it enters the cavity. Interestingly amino acids in these same loops are also important for the interaction between PYR1 and the phosphatases. Binding of ABA to the receptor and closure of the gating loops thus generates an optimal surface for and promotes binding of the PP2Cs. This leads to the release of the protein kinases of the SnRK family which turn on the stress response.

Simplified scheme of the ABA signalling pathway.

This work has been the result of a collaboration between the group of Dr. Pedro Luis Rodriguez of the Instituto de Biologia Molecular de Plantas (Valencia, Spain) and the High Throughput Crystallization Laboratory at EMBL Grenoble. The group of Dr. Rodriguez participated in the initial discovery of the receptors and wanted to pursue structural studies. The EC-funded PCUBE program allowed this group to benefit from access to the high throughput crystallization screening services offered by the HTX lab, which was the beginning of a successful collaboration between the two groups. The access to the MX beamlines at the ESRF, the recently inaugurated BioSAX station and the biophysical characterization capabilities of the CISB (Centre for Integrated Structural Biology) in Grenoble, were also critical.

This work does not only represent the definitive confirmation of the PYR/PYL/RCAR protein family as ABA receptors, but

it also provides insights into the basic mechanisms through which proteins are capable of perceiving a chemical signal, the concentration of an hormone in this case, and transform it into a biological signal, inactivation of a protein activity. Moreover, the PYR1 structure provides a very detailed view on the interactions that stabilize the hormone-receptor complex, which paves the way for the design of small molecules able to bind to the ABA receptor and activate the stress signalling pathway. These molecules should be easier to synthesise and more stable than ABA itself and could potentially be used to improve the tolerance of crops to drought and other type of environmental stress.

José A. Marquez(EMBL Grenoble)

Pedro Rodriguez (IBMCP-CSIC, Valencia, Spain)

Julia Santiago (IBMCP-CSIC, Valencia, Spain)

J. Santiago et al., Nature. 2009 Dec 3;462(7273):665-8.

The crystal structure of the receptor for the plant-hormone Abscisic Acid illuminates one of the key steps in the mechanisms of defence of the plants against adverse environmental conditions and opens the door to the synthesis of chemicals to improve the tolerance of cultivated plants to drought and cold.

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Two recent papers describe important advances in multiprotein expression in mammalian and insect cells.

A key method in contemporary biology involves the integration of foreign genetic material into cells. This can serve many purposes. Structural biologists clone genes into expression plasmids which they then introduce for example in E.coli cells, for producing proteins they then analyze by EM, NMR or X-ray crystallography.

Many essential proteins of eukaryotic origin, however, cannot be produced efficiently in prokaryotic hosts. This can be due to post-translational modifications or eukaryotic folding systems which are absent in prokaryotes. Eukaryotic proteins may require additional binding partners to exert their activity. In fact, such protein machines with 10 and more components are at the core of many vital processes in the cell. As a consequence, protein expression in eukaryotic host organisms has become increasingly important for many applications including structural biology, for producing eukaryotic proteins and notably, protein complexes.

One of the technologies for producing eukaryotic complexes in the quantity and quality required for high-resolution structural and functional analyses uses recombinant baculovirus, into which a gene of interest has been introduced. The resulting baculovirus is then used to infect insect cell cultures to produce the encoded protein. Among the most powerful baculovirus/insect cell expression technologies to date is the MultiBac system (below), conceived by Imre Berger and colleagues, and implemented at EMBL Grenoble’s Eukaryotic Expression Facility (EEF).

The MultiBac system is particularly tailored for rapidly assembling recombinant baculoviruses containing many genes, by using a technique called tandem recombineering (TR) which the scientists at Grenoble have developed. TR relies on a specifically designed array of so-called

Donor and Acceptor DNA modules, which are rapidly loaded with recombinant genes and contamerized in a robust process that can be automated. The multigene fusions are then integrated into a baculovirus genome which is produced as a bacterial artificial chromosome (BAC) in special E.coli cells. The resulting composite baculovirus is then used to infect insect cell cultures.

Many large proteins and protein complexes have now been produced by using MultiBac, often for the first time. P-CUBE has made it possible to provide transnational access to researchers from all over Europe to the MultiBac platform, which is constantly being further developed by Berger and colleagues, for achieving even higher efficacy and superior performance. Excellent research has been catalyzed by the training scientists received through the P-CUBE program. In a recent invited paper published as part of a special issue of Journal of Structural Biology devoted to advances in protein expression technologies, Berger and colleagues summarize in detail the state-of-the-art of the MultiBac baculovirus/insect cell expression platform at EMBL Grenoble and provide hands-on advice and information to facilitate setting-up and successfully implementing MultiBac in many laboratories that are interested in eukaryotic protein complex production.

The tandem recombineering method, however, is not limited to baculovirus/insect cell expression. In fact, introducing foreign genes into mammalian cells, is still more of a challenge to date, especially if many genes are to be introduced simultaneously. Genetic engineering of mammalian cells with transgenes is an essential need in contemporary biology. Reprogramming of somatic cells into stem cells by coexpressing an array of specific transcription factors is a prominent example. Efficient simultaneous monitoring of many parameters in living cells with fluorescent-protein sensors is an essential prerequisite for cell biology experiments, for example if multi-component pathways are to be followed precisely. Achieving robust results in such experiments has been a considerable challenge, requiring specialist knowledge and highly trained personnel. Existing approaches were hampered by many impediments, rendering it virtually impossible to generate mammalian cell populations in which every cell simultaneously expresses all desired genes.

In a recent publication in Nature Communications, Imre Berger and Philipp Berger from the Paul Scherrer Institute

(PSI) in Villigen, Switzerland, now teamed up to adapt the tandem recombineering concept to introducing multiple transgenes into mammalian cells. The resulting technology, called MultiLabel, overcomes previous impediments by using a single multigene plasmid which is rapidly built from custom-designed, tiny pro-genitor DNA molecules. In the article, the scientists demonstrate highly efficient co-expression of currently up to five genes in transfected cells from a pig's cardiovascular system. Different components of the cells are labelled simultaneously by genetically encoded tags, using the new MultiLabel technology (below, still image from video courtesy of P. Berger/PSI).

MultiLabel also enables the generation of stable multigene expresser cell lines due to specific elements that can be readily introduced by the TR method. The viability of the transfected cells is not adversely affected, as they show the expected behaviour for example when stimulated with growth factors.

A broad range of applications will benefit from this methodology. In the present study, fluorescent markers where used to genetically tag specific proteins to study cellular processes. Protein affinity purification tags could be introduced instead, thus benefitting structural biologists who wish to produce their protein complexes in mammalian cells as an expression host.

Imre Berger(EMBL

Grenoble)

S. Trowitzsch et al., J. Struct. Biol. 2010, 172(1):45-54.A. Kriz et al., Nat. Commun. 2010, 1, DOI: 10.1038/ncomms1120

Scientific HighlightsLight it up – Multiprotein expression in eukaryotes takes center stage

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It is of great interest of P-CUBE to ensure efficient training in the state-of-the-art technologies that are available at the transnational access infrastructures, Therefore, P-CUBE training activities primarily target users who are granted transnational access to the P-CUBE platforms.

This is achieved by offering or contributing to focused training courses in specific techniques that are used at the platforms. During the first 18 months of the project, P-CUBE has already participated in several successful training events (read more about them in this section of the newsletter) covering techniques such as production, purification and characterization of protein complexes as well as crystallization methods. We hope to intensify this in the future and also cover topics such as library technologies, HT-expression methodologies and protein-labeling techniques for advanced microscopy. We are confident that we will offer P-CUBE users access to exciting training courses that will deepen their knowledge in protein production and characterization techniques.

Another emphasis lies on our yearly user meetings for platform users and operators. In this setting, former users are given the chance

to present their scientific projects and to provide feedback about running the platforms and input on how to improve the services and training. Future users can get detailed information on the various platforms. They are able to learn from the experiences of former users and get in contact with the platform operators to discuss their projects. The platform operators will inform the participants on the latest technological developments of their platforms, present the scientific services that they offer and also give some insight on successful user projects that have been performed already. Our first user meeting took place in September 2010 in Grenoble. It was exciting and delightful to experience the quality of the scientific talks

and projects by the platform users and the fruitful discussions with the platform operators. We are looking forward to another excellent user meeting in 2011.

Petra Lindemann(EMBL Heidelberg)

P-CUBE TrainingThe ambition to enable dissemination of cutting-edge methodologies.

Workshop on “Advanced Protein Expression and Crystallization Methods” and 1st P-CUBE user meeting

A one-day workshop on “Advanced Protein Expression and Crystallization Methods” took place on 8 September 2010 in Grenoble, followed by the 1st P-CUBE user meeting on 9 September 2010. The workshop focused on the newest developments in MultiBac, ESPRIT and HT-X technologies. Tutors included Imre Berger, Darren Hart and Jose A. Marquez from EMBL Grenoble.

It was a bit uncertain whether the workshop would be affected by a general strike in France that was scheduled for the arrival day of most participants coming into Grenoble from all over Europe. But despite some difficulties, nearly everyone eventually managed to find their way to Grenoble. The participants were rewarded by a workshop that gave them the opportunity to closely interact with the tutors and to directly discuss their research projects with the experts in small workgroups. Another highlight was the tour through the labs and demonstration of the robots.

Approximately 50 scientists including P-CUBE partners as well as former and prospective users attended the 1st P-CUBE user meeting.

The meeting started with a warm welcome by the coordinator, Markus Grütter, who also introduced the Keynote Speaker Gunter Schneider from the Karolinska Institute. He gave a fascinating talk about the challenges of developing new antibiotics and the role of structural biology in this quest.

The platform operators presented their scientific services including the latest technological developments of their platforms to inform prospective P-CUBE users about the Transnational Access opportunities of the project. In addition, they gave some insight into successful user projects that had already been performed at the different platforms.

Importantly, a number of former users were given the chance to present their exciting research projects and could provide feedback about their experiences in using the platforms and input on how to improve the services and training. The user presentations showed the broad range of different projects that are supported by the PCUBE project, but also impressively demonstrated the high quality of the supported research. User presentations were supplemented with an interesting poster session that evoked fruitful discussions amongst the attendees.

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EMBO course in GrenobleP-CUBE co-sponsored the EMBO Practical Course “Structural Characterization of Macromolecular Complexes” which took place in

Grenoble from the 30th of May to the 5th of June 2010. Students and postdocs from 13 different countries (UK, France, Germany, Austria, Spain, Belgium, Israel, India, Italy,

Switzerland, Sweden, Hungary and Australia) participated in the course, which covered a range of topics on how to produce, purify and characterize multi-subunit protein and protein/nucleic acid complexes for structural analysis.

EMBO course in HamburgThe EMBO Practical Course “Protein Expression, Purification and Crystallisation” which took place in Hamburg from 23rd – 31st August 2010 was co-sponsored by P-CUBE. Students and Postdocs from 15 different countries (Germany, Denmark, Belgium, UK, France, Austria, Italy, Turkey, Latvia, Croatia, Poland, Ireland, Netherlands,

USA and South Africa) were trained in topics covering over-expression of proteins in prokaryotic and eukaryotic systems, protein purification

and characterization, crystallization techniques and determination of crystal quality.

Workshop on “Advanced Protein Expression and Crystallization Methods” and 1st P-CUBE user meeting

A picture’s worth a thousand words

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Feature on: Advanced Microscopy PlatformThe facility at EMBL Heidelberg offers expertise and support to use modern fluorescent light microscopy techniques.

Which scientific services do we offer?

We offer the possibility to perform state-of-the-art microscopy experiments using the adequate equipment. Users and staff will plan the experiments and organise the required reagents prior to the visit. EMBL Heidelberg can provide expression vectors for different fluorescent proteins, fluorescence labels, and chemical reagents. At the facility users will receive support for the production of fluorescently labeled samples (e.g. protein expression in different cell lines for in vivo analysis, recombinant protein production for in vitro studies, chemical labeling of protein etc.). Subsequently, they will use the appropriate microscopes to collect imaging data, process and evaluate them using optimal equipment and software.

Which techniques can be used?

A wide range of microscopy techniques can be applied from the determination of protein localization in cells or tissue, analysis of protein interactions and mobility through fluorescence recovery after photobleaching (FRAP), fluorescence energy transfer (FRET)

to the tracking of dynamic processes in live environment. The ALMF offers a great number of diverse microscopes suited for all kind of imaging experiments. In addition all the microscopes are fitted for live imaging with an environmental chamber capable of regulating temperature and CO2 concentration.

What kind of projects have we had already?

We have had a variety of challenging projects ranging from analysis of in vitro actin dynamics to live imaging of proteins during neuronal growth in culture of drosophila brain cells or the localization of extracellular enzymes in bacterial flocs. We’re looking forward to some exciting projects in the future dealing with mechanisms of protein activation and interactions.

Which countries can our users come from?

To be eligible for Transnational Access, the main proposer and the majority of the participants must work in an institution established in an EU Member State or Associated State. So far, our visitors have come from laboratories in Luxembourg,

France, Romania, Czech Republic, Israel, Italy and Germany.

What do potential new users need to do?

Applications can be done via the P-CUBE website (www.p-cube.eu). For further inquiries please feel free to contact Wolfgang Hübner (huebner@embl.de) or Petra Lindemann (petra.lindemann@embl.de) directly.

What logistic support are we offering?

An average user will stay around 4 weeks to prepare samples, do the light microscopy analysis and process data. Users will be provided with accommodation at the EMBL Guesthouse or the ISG hotel which are located in close proximity to the EMBL Heidelberg building. Travel costs will be reimbursed after submission of an experimental report. More specific information can be found here: http://www.embl.de/research/units/scb/mueller_christoph/projects/index.html.

Many structural biologists would like to study their favourite molecule or protein complex not only in vitro, but also in the natural environment of a living cell. In vivo light microscopy studies provide important information about the quantity, sub-cellular localization and dynamics of proteins and protein complexes and about the functional relevance of the molecular complexes that are often complementary to the results obtained in vitro. To facilitate access for non-expert users the Advanced Microscopy Platform offers support from experts in fluorescently labeling of the complexes and in performing light microscopy experiments.

The platform in detail:EMBL Heidelberg provides expression vectors for different fluorescent proteins, fluorescence labels and chemical reagents to produce fluorescently labelled biomolecules for light microscopy analysis in vitro and in vivo.

Users can perform experiments in the cell culture facilities for mammalian and insect cells. They can collect fluorescence data, process and evaluate them using optimal equipment and software under the guidance of highly experienced light microscopists.

The combination of protein expression (PEPCORE) and advanced light microscopy facilities (ALMF) as a platform within P-CUBE enables e.g. structural biologists to study their proteins of interest in the natural environment in living cells or tissues.

The people behind the platform:

Hüseyin BesirHead of PEPCORE

Rainer PepperkokHead of ALMF

Stefan Terjung, Christian Tischer, Yury Belyaev Scientific officers ALMF

Christoph MüllerHead of the Structural and Computational Biology Unit and EMBL P-CUBE coordinator

Wolfgang HübnerScientific officer

Petra LindemannScientific Administrator

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How did you learn about P-CUBE?

I have learnt about P- CUBE through personal communication with Wolfgang Hübner.Do you consider the access modalities adequate/appropriate for your purpose?

Yes. However, I was not aware about P-CUBE before talking to Wolfgang Hübner, so maybe more advertisement would be even better!What was your goal (scientific project and/or acquisition of technology/expertise)?

The lab of Florence Besse in the Institute of Developmental Biology and Cancer (IBDC) in Nice studies the mechanisms of RNA transport in a group of neurons involved in olfactory memory in the central nervous system of the fly (mushroom body neurons).

Drosophila mushroom body (MB) neurons.

My first goal in this project was to define axon growth properties, such as speed, directionality and branching in wild type conditions. Moreover, I wanted to compare the characteristics observed in wild type brains, with those obtained from brains in which the function of the mRNA transport factor Imp has been impaired. Indeed, strong axon growth/guidance defects are

observed in fixed brains when Imp function is disrupted. Real time observations are however required to precisely determine the origin of these defects. In parallel, my second objective was to develop an assay to visualise in real time the subcellular

distribution of Imp and its mRNA targets within axons. Axonal growth within drosophila MB.

Did your visit meet your expectations?

To image developing axons in real-time, I have then used up-to-date microscopes (spinning disc and scanning laser confocal) available at EMBL Heidelberg. These microscopes are ideally suited for live-imaging of deep tissues, but not yet available in my institute. For the first time, I have been able to follow axon growth for about 15 hours within an intact fly brain, and to image this process with a very high spatial and temporal resolution. Using various genetic tools, I have followed the growth of single axons either wild-type, or mutant for imp.What did the results mean for your project? This innovative project had provided novel information on the role of mRNA transport during axon growth. Having the possibility to work at EMBL has allowed me to further improve my in vivo cell biology/imaging

experience, and to open new research avenues in the laboratory, as we have until now been relying on analysis of fixed samples. Moreover, thanks to my stay at EMBL, I will soon be able to submit an article containing these results.How was the scientific support in the lab?

I received a lot of help and valuable support from Wolfgang Hübner, the ALMF and the laboratories of Anne Ephrussi and Alexander Aulehla at EMBL Heidelberg.Do you consider collaborating in the future with the laboratory that hosted your P-CUBE visit?

That could be an option, precisely to improve my knowledge on the structural aspect of Imp, the RNA binding protein I am studying.How do you consider the P-CUBE administrative support?

Very good, especially I have to thank a lot Petra Lindemann for her help/support.Would you recommend the infrastructures of P-CUBE and especially the one you used to anyone else?

Of course! I already recommended them to the people of my institute in Nice, France.

Interview with Caroline Medioni - user of the Advanced Microscopy Platform working on RNA localisation during axon growth in Drosophila

Workshop on Mammalian Expression Technologies

The P-CUBE Mammalian Expression platform at the University of Oxford is now accepting applications for the practical workshop on mammalian expression to be held in Oxford, UK on 3rd - 8th April, 2011.

The course is designed primarily for structural biologists who have prokaryotic expression experience and would like to use the mammalian expression system for more challenging targets. The goals of the course are to allow participants to have hands-on experience of mammalian cell culture, small and large scale transient gene expression including the use of automated systems, protein purification and crystallization. The course will be structured so as to interleave lectures and practical sessions. The participants will have the opportunity to closely interact with the tutors and to directly discuss their research projects with the experts in small workgroups.

Applications can be submitted until 15th March through the P-CUBE website: www.p-cube.eu

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T h e P - C U B E O x f o r d M a m m a l i a n Expression platform has successfully implemented a CompacT SelecT cell culture robot for large-scale expression of multiple target proteins in a transient format. Successful protocols for automated cell culture and transient transfection of human embryonic kidney (HEK), 293T and 293S GnTI- cells in various flask formats have been designed. The ability of the robot to handle 10 layers of HYPERFlasks (High Yield PERformance Flask, surface area 1720 cm2) further enhanced the capacity for high throughput protein production. Protein yields obtained by this method were similar to those produced manually, with the added benefit of reproducibility. Automating cell

maintenance and transient transfection allows the expression of high quality recombinant protein in a completely sterile environment with limited support from a cell culture scientist. This automated method for large scale transient transfection is offered as a Europe-wide service via the P-CUBE initiative.

The CompacT SelecT robot consists of two compartments (Fig 1a). The first is a humidified 5% CO2, 37°C incubator/flask hotel unit (Fig 1b) with space for 40 new input flasks and 90 production flasks for cell maintenance and transfection. The second is a laminar flow compartment containing a Stäubli robotic arm (Stäubli Robotics, Faverges, France) (Fig 1c), a pipette head (Fig 1d), a waste receptacle and cocktail bar (Fig 1e). The laminar flow compartment also has a barcode reader to track flasks and a flask de-capper (Fig 1f). The unit also incorporates a Cedex automated cell counting module (Fig 1g). Cells are

automatically maintained by regular harvesting and seeding of T175 flasks, while production is performed in Triple flasks and HYPERFlasks, following the flowchart described in Fig 1h.

Da ta f rom HYPERFlask t r ans i en t transfections performed on the CompacT SelecT robot over a period of four months, using both HEK 293T and HEK 293S GnTI- cells and using only constructs confirmed to be secreted in small-scale experiments, revealed a broad range of pure, crystallization grade, protein yields (Fig. 2). An overall trend of higher expression levels in HEK 293T (Fig. 2a) versus HEK 293S GnTI- cells (Fig. 2b) was observed, but in all cases the protein quantities obtained w e r e s u f f i c i e n t t o s e t e x t e n s i v e crystallization trials using nanolitre scale technologies.

News from the platformsImplementation of robot at Oxford Mammalian Expression Platform

Figure 2 Figure 1

Advanced high throughput capillary plate for protein crystallizationThe University of Zurich has designed and developed a new crystallization plate, the CrystalHarp. The CrystalHarp plate is designed for crystallization based on capillary diffusion and can be used for crystallization screening and optimization. Capillary diffusion achieves a much broader screening of variables in one single experiment. The CrystalHarp plate contains 48 capillaries in total and is in an ANSI/SBS 1-2004-standard format to facilitate handling and imaging. Addition of cryoprotectants or derivatives for phasing studies can be easily added after crystal appearance. The SBS format enables the usage of the plate directly on beam line robots or alternatively single capillaries can be easily mounted to standard magnetic base, enabling in-situ diffraction analysis. The unique capillary material allows data collection at room temperature. Flash-freezing in a liquid nitrogen stream (with or without the use of cryoprotectants) is also feasible. The formation of ice-rings is kept to a manageable minimum.

You can view the new advanced Counter Diffusion Crystallisation plate system user video online at:http://www.youtube.com/watch?v=houcn_1Vnws

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AnnouncementsThe P-CUBE midterm review meeting will be on May 2-3, 2011. Our scientific advisory board (Gunter Schneider, Karolinska Institute, Brian Matthews, University of Oregon and Anthony Kossjakoff, University of Chicago) will join us at the meeting. Further information regarding the location etc. will follow in due time.

The following training events are planned for 2011:

• Mammalian Expression Technologies Workshop, Oxford, UK, 3rd - 8th April 2011 (see p.8)

• Protein Crystallization Workshop and 2nd P-CUBE User Meeting, Zurich, CH, Autumn 2011

P-CUBE is a EU-funded project that offers free access to infrastructures for the European science community in structural biology

P-CUBE is the first project within the Seventh Framework Program that brings together

research, networking and service activities. It offers exciting cutting edge technologies in contemporary structural biology and provides access to high-throughput cloning and expression technologies in prokaryotic and eukaryotic cells, high-throughput crystallization facilities, to the DARPin-selection methodology, the ESPRIT technology and advanced light microscopy. Service and access to these infrastructures are free of charge for scientists all across Europe from now on until March 2013.

ContactsCoordinator: Markus Grütter (Uni Zurich), email: gruetter@bioc.uzh.ch

Newsletter editors: Petra Lindemann (P-CUBE Training, EMBL Heidelberg) and Jutta Tatzel (Program Manager, Uni Zurich), emails: petra.lindemann@embl.de, j.tatzel@bioc.uzh.ch

Link: http://www.p-cube.eu/index.php?option=com_content&view=category&layout=blog&id=67&Itemid=96

The identification of initial crystallization conditions has been facilitated by the introduction of automated laboratory equipment and nanovolume crystallization robots. However, initial crystallization conditions often produce crystals with poor x-ray diffraction properties and have to be optimized. Different strategies have been developed to assist in this process in a high throughput scale, however the traditional approach consisting in the elaboration of multi-well optimization screens where the concentration of one or several components of the crystallization solution are varied systematically remains a common approach. Commercial liquid-handling robots can be used to generate multi-well customized optimisation screens from a limited number of component stock solutions and this type of equipment is often present in crystallization laboratories. However the varied nature of the optimization screens results in the need to reprogram the liquid-handling robot for each specific experiment, which requires a significant amount of time and manpower. A commonly used approach to overcome this problem is to introduce constrains in the design of the optimisation screens so that a limited number of programs can be used to satisfy many conditions.

Software to assist crystallographers in the design and reporting of optimisation screens, such as OptiCrys developed as part of the E.C.-funded BioxHit project by the Institut de Biologie Structurale Jean-Pierre Ebel (IBS, Grenoble, France) with the support of CEA/DSV (Céline Charavay, GIPSE, Grenoble) exist. OptiCrys provides a graphical user interface for the generation of crystallization screens without any limitation in any plate format and provides convenient tools like, for example, the automatic calculation of component gradients. OptiCrys outputs the volumes of reagents to be delivered at each well position as well as a table of individual component concentrations to facilitate reporting. OptiCrys, is freely available for academic researchers and is in use in many crystallography laboratories. However, its use in a high throughput context was limited by the lack of an output format that could be directly read by

liquid-handling robots. In order to address this problem the GIPSE group of the IBS in collaboration with the High Throughput Crystallization Laboratory (HTX lab) of the EMBL Grenoble outstation has developed OptiLink; a new software module that takes the output from OptiCrys and generates files that can be directly read by a Tecan Robot, ridding of the need to reprogram the liquid-handling system. OptiLink can be adapted to a variety of robot configurations through a simple configuration file and allows to specify different liquid classes. The combined use of OptiCrys and OptiLink can contribute to save time and manpower facilitating significantly the optimisation of crystal diffraction properties. Unlike other existing programs OptiCyrs and OptiLink are freely available to academic users under a LGPL license. For further information visit the IBS (http://www.ibs.fr) or HTX lab web sites (https://embl.fr/htxlab).

Above: The OptiCrys software for the design of optimization experiments (J. appl Crys 39, 446-453.).

Delphine Blot (IBS) and José A. Márquez (EMBL Grenoble)

Speeding up crystallization refinement experiments with the OptiLink software module