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Research Article

Rhabdovirus Matrix Protein Structures Reveal a Novel Mode of Self-Association

  • Stephen C. Graham,

    Affiliation: Division of Structural Biology and Oxford Protein Production Facility, Wellcome Trust Centre for Human Genetics, University of Oxford, Oxford, United Kingdom

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  • René Assenberg equal contributor,

    equal contributor Contributed equally to this work with: René Assenberg, Olivier Delmas

    Affiliation: Division of Structural Biology and Oxford Protein Production Facility, Wellcome Trust Centre for Human Genetics, University of Oxford, Oxford, United Kingdom

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  • Olivier Delmas equal contributor,

    equal contributor Contributed equally to this work with: René Assenberg, Olivier Delmas

    Affiliation: UPRE Lyssavirus Dynamics and Host Adaptation, WHO Collaborating Centre for Reference and Research on Rabies, Institut Pasteur, Paris, France

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  • Anil Verma,

    Affiliation: Division of Structural Biology and Oxford Protein Production Facility, Wellcome Trust Centre for Human Genetics, University of Oxford, Oxford, United Kingdom

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  • Alireza Gholami,

    Affiliation: UPRE Lyssavirus Dynamics and Host Adaptation, WHO Collaborating Centre for Reference and Research on Rabies, Institut Pasteur, Paris, France

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  • Chiraz Talbi,

    Affiliation: UPRE Lyssavirus Dynamics and Host Adaptation, WHO Collaborating Centre for Reference and Research on Rabies, Institut Pasteur, Paris, France

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  • Raymond J. Owens,

    Affiliation: Division of Structural Biology and Oxford Protein Production Facility, Wellcome Trust Centre for Human Genetics, University of Oxford, Oxford, United Kingdom

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  • David I. Stuart,

    Affiliation: Division of Structural Biology and Oxford Protein Production Facility, Wellcome Trust Centre for Human Genetics, University of Oxford, Oxford, United Kingdom

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  • Jonathan M. Grimes mail,

    jonathan@strubi.ox.ac.uk (JMG); hbourhy@pasteur.fr (HB)

    Affiliation: Division of Structural Biology and Oxford Protein Production Facility, Wellcome Trust Centre for Human Genetics, University of Oxford, Oxford, United Kingdom

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  • Hervé Bourhy mail

    jonathan@strubi.ox.ac.uk (JMG); hbourhy@pasteur.fr (HB)

    Affiliation: UPRE Lyssavirus Dynamics and Host Adaptation, WHO Collaborating Centre for Reference and Research on Rabies, Institut Pasteur, Paris, France

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  • Published: December 26, 2008
  • DOI: 10.1371/journal.ppat.1000251

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VSV M purification

Posted by reneassenberg on 31 Dec 2008 at 02:04 GMT

Cloning, expression, purification, crystallization and data collection
The matrix (M) protein from Lagos bat virus (LBV) was cloned, expressed, purified and diffraction data were collected as described previously [54]. M from VSV serotype New Jersey (VSVNJ) was cloned into pOPINS, encoding an N-terminal His6-SUMO fusion tag, and selenomethionine-labelled (SeMet) protein was expressed and purified as described for LBV M [54]. Purified VSVNJ M was concentrated to 1.2 mg/mL and crystallization trials were attempted at 20.5°C in sitting drops containing 100 nL protein and 100 nL precipitant solution equilibrated against 95 µL reservoirs in 96-well plates. Crystals of SeMet VSVNJ M grew in 20% v/v isopropanol, 20% w/v PEG 4000 and 0.1 M sodium citrate (pH 5.6) and were cryoprotected by a quick pass through reservoir solution supplemented with 20% v/v glycerol before flash cryocooling in a cold (100 K) stream of nitrogen gas. Diffraction data were recorded from a single crystal of SeMet VSVNJ M at a wavelength of 0.9803 Å, to maximize the selenium anomalous signal, on ESRF beamline ID23EH1. Diffraction data were processed using XDS [55] and SCALA [56] as implemented by the xia2 automated data processing package (Winter et al., in preparation).

http://plospathogens.org/article/info:doi/10.1371/journal.ppat.1000251#article1.body1.sec4.sec1.p1

What is not clear from the paper is that for the VSV N-sumo tagged M protein we used only 25ml of a 500ml E. coli culture to purify the VSV M protein. The 25ml E. coli cell pellet was lysed in 35ml of lysis buffer (the same lysis buffer as used for LAG M in the Acta F crystallization paper: Assenberg et al, Acta Cryst. (2008). F64, 258-262) and then sumo cleaved and purified in the same way as the lagos bat virus M protein thereafter. This could be important as this effectively diluted the VSV M protein which may have prevented aggregation.