title: In meso in situ serial X-ray crystallography of soluble and membrane proteins at cryogenic temperatures.
abstract: Here, a method for presenting crystals of soluble and membrane proteins growing in the lipid cubic or sponge phase for in situ diffraction data collection at cryogenic temperatures is introduced. The method dispenses with the need for the technically demanding and inefficient crystal-harvesting step that is an integral part of the lipid cubic phase or in meso method of growing crystals. Crystals are dispersed in a bolus of mesophase sandwiched between thin plastic windows. The bolus contains tens to hundreds of crystals, visible with an in-line microscope at macromolecular crystallography synchrotron beamlines and suitably disposed for conventional or serial crystallographic data collection. Wells containing the crystal-laden boluses are removed individually from hermetically sealed glass plates in which crystallization occurs, affixed to pins on goniometer bases and excess precipitant is removed from around the mesophase. The wells are snap-cooled in liquid nitrogen, stored and shipped in Dewars, and manually or robotically mounted on a goniometer in a cryostream for diffraction data collection at 100 K, as is performed routinely with standard, loop-harvested crystals. The method is a variant on the recently introduced in meso in situ serial crystallography (IMISX) method that enables crystallographic measurements at cryogenic temperatures where crystal lifetimes are enormously enhanced whilst reducing protein consumption dramatically. The new approach has been used to generate high-resolution crystal structures of a G-protein-coupled receptor, α-helical and β-barrel transporters and an enzyme as model integral membrane proteins. Insulin and lysozyme were used as test soluble proteins. The quality of the data that can be generated by this method was attested to by performing sulfur and bromine SAD phasing with two of the test proteins.
authors.affiliation: Membrane Structural and Functional Biology Group, School of Medicine and School of Biochemistry and Immunology, Trinity College, Dublin 2, D02 R590, Ireland.
authors.forename: Chia Ying
authors.initials: CY
authors.lastname: Huang
authors.affiliation: Swiss Light Source, Paul Scherrer Institute, CH-5232 Villigen, Switzerland.
authors.forename: Vincent
authors.initials: V
authors.lastname: Olieric
authors.affiliation: Membrane Structural and Functional Biology Group, School of Medicine and School of Biochemistry and Immunology, Trinity College, Dublin 2, D02 R590, Ireland.
authors.forename: Pikyee
authors.initials: P
authors.lastname: Ma
authors.affiliation: Membrane Structural and Functional Biology Group, School of Medicine and School of Biochemistry and Immunology, Trinity College, Dublin 2, D02 R590, Ireland.
authors.forename: Nicole
authors.initials: N
authors.lastname: Howe
authors.affiliation: Membrane Structural and Functional Biology Group, School of Medicine and School of Biochemistry and Immunology, Trinity College, Dublin 2, D02 R590, Ireland.
authors.forename: Lutz
authors.initials: L
authors.lastname: Vogeley
authors.affiliation: School of Medicine, Tsinghua University, Beijing 100084, People's Republic of China.
authors.forename: Xiangyu
authors.initials: X
authors.lastname: Liu
authors.affiliation: Swiss Light Source, Paul Scherrer Institute, CH-5232 Villigen, Switzerland.
authors.forename: Rangana
authors.initials: R
authors.lastname: Warshamanage
authors.affiliation: Swiss Light Source, Paul Scherrer Institute, CH-5232 Villigen, Switzerland.
authors.forename: Tobias
authors.initials: T
authors.lastname: Weinert
authors.affiliation: Swiss Light Source, Paul Scherrer Institute, CH-5232 Villigen, Switzerland.
authors.forename: Ezequiel
authors.initials: E
authors.lastname: Panepucci
authors.affiliation: School of Medicine, Tsinghua University, Beijing 100084, People's Republic of China.
authors.forename: Brian
authors.initials: B
authors.lastname: Kobilka
authors.affiliation: Fachbereich Biologie, Universität Konstanz, Box 647, D-78457 Konstanz, Germany.
authors.forename: Kay
authors.initials: K
authors.lastname: Diederichs
authors.affiliation: Swiss Light Source, Paul Scherrer Institute, CH-5232 Villigen, Switzerland.
authors.forename: Meitian
authors.initials: M
authors.lastname: Wang
authors.affiliation: Membrane Structural and Functional Biology Group, School of Medicine and School of Biochemistry and Immunology, Trinity College, Dublin 2, D02 R590, Ireland.
authors.forename: Martin
authors.initials: M
authors.lastname: Caffrey
mesh_terms: D000818:Animals; D001419:Bacteria / Q000737:chemistry; D001426:Bacterial Proteins / Q000737:chemistry; D002645:Chickens; D003080:Cold Temperature; D003460:Crystallization / Q000379:methods; D018360:Crystallography, X-Ray / Q000379:methods; D007328:Insulin / Q000737:chemistry*; D008565:Membrane Proteins / Q000737:chemistry*; D008958:Models, Molecular; D009113:Muramidase / Q000737:chemistry*; D044367:Phase Transition; D012995:Solubility; D013552:Swine
chemical_list: D001426:Bacterial Proteins; D007328:Insulin; D008565:Membrane Proteins; D009113:Muramidase
keywords: AlgE; DgkA; GPCR; PepTSt; bromine SAD; cubic phase; experimental phasing; in meso; in situ; insulin; kinase; lipid cubic phase; membrane protein; mesophase; serial crystallography; sponge phase; sulfur SAD; transporter; β2-adrenergic receptor