In meso in situ serial X-ray crystallography of soluble and membrane proteins at cryogenic temperatures

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• 作者：Chia-Ying Huang ; Vincent Olieric ; Pikyee Ma ; Nicole Howe ; Lutz Vogeley ; Xiangyu Liu ; Rangana Warshamanage ; Tobias Weinert ; Ezequiel Panepucci ; Brian Kobilka ; Kay Diederichs ; Meitian Wang and Martin Caffrey
• 关键词：AlgE ; &beta ; 2-adrenergic receptor ; bromine SAD ; cubic phase ; DgkA ; experimental phasing ; GPCR ; in meso ; in situ ; insulin ; kinase ; lipid cubic phase ; membrane protein ; mesophase ; PepTSt ; serial crystallography ; sponge phase ; sulfur SAD ; transporter
• 刊名：Acta Crystallographica Section D
• 出版年：2016
• 出版时间：January 2016
• 年：2016
• 卷：72
• 期：1
• 页码：93-112
• 全文大小：2273K
• ISSN：1399-0047

文摘

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.