A high-throughput crystallisation facility for protein structure determination
Lead Research Organisation:
Imperial College London
Department Name: Biological Sciences
Abstract
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Technical Summary
The aim of this application is to provide a robotic nanolitre protein crystallisation facility for the Centre for Structural Biology (CSB) at Imperial College London. The CSB is a college centre with affiliated groups from three faculties and provides both expertise and managed core facilities in all major techniques of macromolecular structure determination: X-ray crystallography, nuclear magnetic resonance spectroscopy, and electron microscopy. The research carried out by CSB members (with substantial BBSRC funding) is of the highest international standard and embraces a wide spectrum of topics; membrane proteins; mechanisms of DNA replication, transcription and repair; bacterial gene regulation; enzymes and molecular machines; mechanisms microbial pathogenicity; structural basis for human diseases and drug binding; and method development. X-ray crystallography is used as the major technique for structure determination by 10 research groups and critically contributes to the research of many other groups, thanks to a number of well-established multidisciplinary collaborations. State-of-the-art equipment exists for protein characterisation (funded by BBSRC-REI 2004) and for X-ray diffraction data collection, but the progress of many structural biology research projects is severely hampered by the lack of a robust high-throughput facility for robotic nanolitre protein crystallisation. Therefore, we request the following instrumentation: (1) a robotic liquid handler to fill 96-well plates with commercial or customised crystallisation solutions; (2) a crystallisation robot to set up nanolitre volume crystallisation experiments. These instruments will be managed by the CSB and provide a central crystallisation facility serving over 100 users. This facility will underpin and enhance many BBSRC-supported grants, as well as other research projects. The instruments will complement existing equipment and significantly improve the research infrastructure of a large number of highly productive groups within the CSB. Crystallisation facilities similar to the one envisaged in our application have been operating successfully at several other top UK universities; we strongly believe that the requested instrumentation will be essential for the CSB at Imperial College London to maintain its internationally leading position. We believe that the requested instruments offer good value for money, as they will very significantly reduce the expenses of protein production and crystallisation of a large number of users. Equally important, robotic crystallisation will be essential to make progress in the emerging areas of system-wide structural biology. In summary, we believe that a well-managed crystallisation facility within the CSB will greatly enhance the research infrastructure of a large number of active researchers, complement existing multi-user equipment, and critically contribute to the training of our students and postdocs.
Publications
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(2018)
Structural Basis for the Initiation of Glycosaminoglycan Biosynthesis by Human Xylosyltransferase 1.
in Structure (London, England : 1993)
Carafoli F
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Crystal structure of the LG1-3 region of the laminin alpha2 chain.
in The Journal of biological chemistry
Carafoli F
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Crystallographic insight into collagen recognition by discoidin domain receptor 2.
in Structure (London, England : 1993)
Carafoli F
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An Activating Mutation Reveals a Second Binding Mode of the Integrin a2 I Domain to the GFOGER Motif in Collagens
in PLoS ONE
Carafoli F
(2012)
Crystal structures of the network-forming short-arm tips of the laminin ß1 and ?1 chains.
in PloS one
Carafoli F
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Structure of the tandem fibronectin type 3 domains of neural cell adhesion molecule.
in Journal of molecular biology
Carafoli F
(2012)
Structure of the discoidin domain receptor 1 extracellular region bound to an inhibitory Fab fragment reveals features important for signaling.
in Structure (London, England : 1993)
Chukhutsina VU
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Light activation of Orange Carotenoid Protein reveals bicycle-pedal single-bond isomerization.
in Nature communications
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Serial Femtosecond Crystallography Reveals that Photoactivation in a Fluorescent Protein Proceeds via the Hula Twist Mechanism.
in Journal of the American Chemical Society
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in The Journal of biological chemistry
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Crystal structure and cell surface anchorage sites of laminin alpha1LG4-5.
in The Journal of biological chemistry
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in Proceedings of the National Academy of Sciences of the United States of America
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in EMBO reports
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in RSC medicinal chemistry
Kabasakal BV
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Crystal structure of the [2Fe-2S] protein I (Shethna protein I) from Azotobacter vinelandii.
in Acta crystallographica. Section F, Structural biology communications
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Unraveling the Mechanism of Procollagen C-Proteinase Enhancer
in Structure
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Structural insights into triple-helical collagen cleavage by matrix metalloproteinase 1.
in Proceedings of the National Academy of Sciences of the United States of America
Niu M
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Structure of the transmembrane protein 2 (TMEM2) ectodomain and its apparent lack of hyaluronidase activity.
in Wellcome open research
Niu M
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Structure of the transmembrane protein 2 (TMEM2) ectodomain and its lack of hyaluronidase activity
in Wellcome Open Research
Pathania M
(2021)
Structural basis for the inhibition of the Bacillus subtilis c-di-AMP cyclase CdaA by the phosphoglucomutase GlmM
in Journal of Biological Chemistry
Pulido D
(2018)
Structural Basis for the Acceleration of Procollagen Processing by Procollagen C-Proteinase Enhancer-1.
in Structure (London, England : 1993)
Qin B
(2022)
Acrylamide fragment inhibitors that induce unprecedented conformational distortions in enterovirus 71 3C and SARS-CoV-2 main protease
in Acta Pharmaceutica Sinica B
Raynaud C
(2021)
PilB from Streptococcus sanguinis is a bimodular type IV pilin with a direct role in adhesion.
in Proceedings of the National Academy of Sciences of the United States of America
Sammon D
(2023)
Molecular mechanism of decision-making in glycosaminoglycan biosynthesis.
in Nature communications
Sammon D
(2020)
Two-step release of kinase autoinhibition in discoidin domain receptor 1.
in Proceedings of the National Academy of Sciences of the United States of America
Sellés Vidal L
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Versatile selective evolutionary pressure using synthetic defect in universal metabolism.
in Nature communications
Shahin M
(2023)
Characterization of a glycan-binding complex of minor pilins completes the analysis of Streptococcus sanguinis type 4 pili subunits.
in Proceedings of the National Academy of Sciences of the United States of America
Wu CH
(2021)
Bacillus subtilis YngB contributes to wall teichoic acid glucosylation and glycolipid formation during anaerobic growth.
in The Journal of biological chemistry
Yu Y
(2018)
Xylosyltransferase 1 and the GAG Attachment Site
in Structure