In meso in situ In meso in situ serial X‐ray crystallography of soluble and membrane proteins at cryogenic temperatures. Issue 1 (1st January 2016)

• Record Type: Journal Article
• Authors: Huang, Chia-Ying; Olieric, Vincent; Ma, Pikyee; Howe, Nicole; Vogeley, Lutz; Liu, Xiangyu; Warshamanage, Rangana; Weinert, Tobias; Panepucci, Ezequiel; Kobilka, Brian; Diederichs, Kay; Wang, Meitian; Caffrey, Martin
• Is Part Of: Acta crystallographica. Volume 72:Issue 1(2016)
• Page Start: 93
• Page End: 112
• Publication Date: 2016-01-01
• Subjects: AlgE AlgE -- β β2 2-adrenergic receptor ‐adrenergic receptor -- bromine SAD bromine SAD -- cubic phase cubic phase -- DgkA DgkA -- experimental phasing experimental phasing -- GPCR GPCR -- in meso in meso -- in situ in situ -- insulin insulin -- kinase kinase -- lipid cubic phase lipid cubic phase -- membrane protein membrane protein -- mesophase mesophase -- PepT PepTSt St -- serial crystallography serial crystallography -- sponge phase sponge phase -- sulfur SAD sulfur SAD -- transporter transporter; X-ray crystallography -- Periodicals; Crystallography -- Periodicals; Molecular biology -- Periodicals; Molecular structure -- Periodicals; Biomolecules -- Structure -- Periodicals; Cytology -- Periodicals; Biomolecules -- Structure; Crystallography; Cytology; Molecular biology; Molecular structure; X-ray crystallography; Periodicals; 548
• DOI: 10.1107/S2059798315021683
• Languages: English
• ISSNs: 2059-7983

Abstract

Abstract : A method for performing high‐throughput in situ in situ serial X‐ray crystallography with soluble and membrane proteins in the lipid cubic phase at cryogenic temperatures (100 K) is described. It works with nanogram to single‐digit microgram quantities of protein and lipid (and ligand when present), and is compatible with both high‐resolution native data collection and experimental phasing without the need for crystal harvesting. Abstract : Here, a method for presenting crystals of soluble and membrane proteins growing in the lipid cubic or sponge phase for in situ 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 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 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.