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Title: In meso in situ serial X-ray crystallography of soluble and membrane proteins

A method for performing high-throughput in situ serial X-ray crystallography with soluble and membrane proteins in the lipid cubic phase is described. It works with microgram quantities of protein and lipid (and ligand when present) and is compatible with the most demanding sulfur SAD phasing. 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 in meso method. Among the listings are some of the highest profile membrane proteins, including the β{sub 2}-adrenoreceptor–G{sub s} protein complex that figured in the award of the 2012 Nobel Prize in Chemistry to Lefkowitz and Kobilka. The most successful in meso protocol to date uses glass sandwich crystallization plates. Despite their many advantages, glass plates are challenging to harvest crystals from. However, performing in situ 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 in situ measurements. The novel in meso in situ serial crystallography (IMISX) method introduced here has been demonstrated withmore » 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 in situ serial data collection at macromolecular crystallography synchrotron beamlines worldwide. Because of its simplicity and effectiveness, the IMISX approach is likely to supplant existing in meso crystallization protocols. It should prove particularly attractive in the area of ligand screening for drug discovery and development.« less
Authors:
 [1] ;  [2] ;  [1] ;  [2] ;  [3] ;  [2] ;  [1]
  1. Trinity College, Dublin (Ireland)
  2. Paul Scherrer Institute, CH-5232 Villigen (Switzerland)
  3. Universität Konstanz, M647, D-78457 Konstanz (Germany)
Publication Date:
OSTI Identifier:
22389068
Resource Type:
Journal Article
Resource Relation:
Journal Name: Acta Crystallographica. Section D: Biological Crystallography; Journal Volume: 71; Journal Issue: Pt 6; Other Information: PMCID: PMC4461204; PMID: 26057665; PUBLISHER-ID: rr5098; OAI: oai:pubmedcentral.nih.gov:4461204; Copyright (c) Huang et al. 2015; This is an open-access article distributed under the terms of the Creative Commons Attribution Licence, which permits unrestricted use, distribution, and reproduction in any medium, provided the original authors and source are cited.; Country of input: International Atomic Energy Agency (IAEA)
Country of Publication:
Denmark
Language:
English
Subject:
37 INORGANIC, ORGANIC, PHYSICAL AND ANALYTICAL CHEMISTRY; 75 CONDENSED MATTER PHYSICS, SUPERCONDUCTIVITY AND SUPERFLUIDITY; ALGINATES; BROMINE; COMPLEXES; CRYSTALLIZATION; CRYSTALLOGRAPHY; LIGANDS; LIQUID CRYSTALS; LYSOZYME; MEMBRANE PROTEINS; MEMBRANES; PLATES; SCREENING; SULFUR; X-RAY DIFFRACTION