In meso in situ serial X‐ray crystallography of soluble and membrane proteins. (1st June 2015)

• Record Type: Journal Article
• Authors: Huang, Chia‐Ying; Olieric, Vincent; Ma, Pikyee; Panepucci, Ezequiel; Diederichs, Kay; Wang, Meitian; Caffrey, Martin
• Is Part Of: Acta crystallographica. Volume 71:Part 6(2015:Jun.)
• Page Start: 1238
• Page End: 1256
• Publication Date: 2015-06-01
• Subjects: Biomolecules -- Structure -- Periodicals; Physical biochemistry -- Periodicals; X-ray crystallography -- Periodicals; Crystallography -- Periodicals; 572
• DOI: 10.1107/S1399004715005210
• Languages: English
• ISSNs: 0907-4449

Abstract

<abstract abstract-type="main" xml:lang="en"> <title> <x xml:space="preserve">Abstract</x> </title> <p>The lipid cubic phase (LCP) continues to grow in popularity as a medium in which to generate crystals of membrane (and soluble) proteins for high‐resolution X‐ray crystallographic structure determination. To date, the PDB includes 227 records attributed to the LCP or <italic>in meso</italic> method. Among the listings are some of the highest profile membrane proteins, including the β<sub>2</sub>‐adrenoreceptor–G<sub>s</sub> protein complex that figured in the award of the 2012 Nobel Prize in Chemistry to Lefkowitz and Kobilka. The most successful <italic>in meso</italic> protocol to date uses glass sandwich crystallization plates. Despite their many advantages, glass plates are challenging to harvest crystals from. However, performing <italic>in situ</italic> X‐ray diffraction measurements with these plates is not practical. Here, an alternative approach is described that provides many of the advantages of glass plates and is compatible with high‐throughput <italic>in situ</italic> measurements. The novel <italic>in meso in situ</italic> serial crystallography (IMISX) method introduced here has been demonstrated with AlgE and PepT (alginate and peptide transporters, respectively) as model integral membrane proteins and with lysozyme as a test soluble protein. Structures were solved by molecular replacement and by experimental phasing using bromine SAD and native sulfur SAD methods to resolutions ranging from 1.8 to 2.8 Å using single‐digit microgram quantities of protein. That sulfur SAD phasing worked is testament to the exceptional quality of the IMISX diffraction data. The IMISX method is compatible with readily available, inexpensive materials and equipment, is simple to implement and is compatible with high‐throughput <italic>in situ</italic> serial data collection at macromolecular crystallography synchrotron beamlines worldwide. Because of its simplicity and effectiveness, the IMISX approach is likely to supplant existing <italic>in meso</italic> crystallization protocols. It should prove particularly attractive in the area of ligand screening for drug discovery and development.</p> </abstract>