title: Structure determination of an integral membrane protein at room temperature from crystals in situ.
abstract: The structure determination of an integral membrane protein using synchrotron X-ray diffraction data collected at room temperature directly in vapour-diffusion crystallization plates (in situ) is demonstrated. Exposing the crystals in situ eliminates manual sample handling and, since it is performed at room temperature, removes the complication of cryoprotection and potential structural anomalies induced by sample cryocooling. Essential to the method is the ability to limit radiation damage by recording a small amount of data per sample from many samples and subsequently assembling the resulting data sets using specialized software. The validity of this procedure is established by the structure determination of Haemophilus influenza TehA at 2.3 Å resolution. The method presented offers an effective protocol for the fast and efficient determination of membrane-protein structures at room temperature using third-generation synchrotron beamlines.
authors.affiliation: Diamond Light Source, Harwell Science and Innovation Campus, Oxfordshire OX11 0DE, England.
authors.forename: Danny
authors.initials: D
authors.lastname: Axford
authors.affiliation: Membrane Protein Laboratory, Diamond Light Source, Harwell Science and Innovation Campus, Oxfordshire OX11 0DE, England.
authors.forename: James
authors.initials: J
authors.lastname: Foadi
authors.affiliation: Membrane Protein Laboratory, Diamond Light Source, Harwell Science and Innovation Campus, Oxfordshire OX11 0DE, England.
authors.forename: Nien Jen
authors.initials: NJ
authors.lastname: Hu
authors.affiliation: Membrane Protein Laboratory, Diamond Light Source, Harwell Science and Innovation Campus, Oxfordshire OX11 0DE, England.
authors.forename: Hassanul Ghani
authors.initials: HG
authors.lastname: Choudhury
authors.affiliation: Diamond Light Source, Harwell Science and Innovation Campus, Oxfordshire OX11 0DE, England.
authors.forename: So
authors.initials: S
authors.lastname: Iwata
authors.affiliation: Membrane Protein Laboratory, Diamond Light Source, Harwell Science and Innovation Campus, Oxfordshire OX11 0DE, England.
authors.forename: Konstantinos
authors.initials: K
authors.lastname: Beis
authors.affiliation: Diamond Light Source, Harwell Science and Innovation Campus, Oxfordshire OX11 0DE, England.
authors.forename: Gwyndaf
authors.initials: G
authors.lastname: Evans
authors.affiliation: Membrane Protein Laboratory, Diamond Light Source, Harwell Science and Innovation Campus, Oxfordshire OX11 0DE, England.
authors.forename: Yilmaz
authors.initials: Y
authors.lastname: Alguel
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry; D018360:Crystallography, X-Ray / Q000379:methods*; D006193:Haemophilus influenzae / Q000737:chemistry; D008565:Membrane Proteins / Q000737:chemistry*; D008958:Models, Molecular; D011487:Protein Conformation; D013696:Temperature
chemical_list: D001426:Bacterial Proteins; D008565:Membrane Proteins
keywords: in situ data collection; membrane protein; multiple data sets; synchrotron beamline