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Animals; Chromatography, Affinity, methods; Cryoelectron Microscopy, methods; Freezing; Humans; Membrane Proteins, metabolism; Negative Staining, methods; Protein Conformation; Recombinant Proteins, metabolism; Single Molecule Imaging, methods; Specimen Handling, methods; Time Factors
Abstract
The ongoing development of single-particle cryo-electron microscopy (cryo-EM) is leading to fast data acquisition, data processing, and protein structure elucidation. Quick and reliable methods to go from protein purification and optimization to grid preparation will significantly improve the reach and power of cryo-EM. Such methods would particularly constitute a tremendous advantage in structural biology of membrane proteins, whose published structures stay still far behind the number of soluble protein structures. Here we describe a fast, low-cost, and user-friendly method for the purification and cryo-EM analysis of a recombinant membrane protein. This method minimizes the amount of starting material and manipulation steps needed to go from purification to grid preparation, and could potentially be expanded to other membrane protein purification systems for its direct application in structure determination by single-particle cryo-EM.
