Electron cryomicroscopy of membrane proteins: specimen preparation for two-dimensional crystals and single particles.
Membrane protein structure and function can be studied by two powerful and highly complementary electron cryomicroscopy (cryo-EM) methods: electron crystallography of two-dimensional (2D) crystals and single particle analysis of detergent-solubilized protein complexes. To obtain the highest-possible resolution data from membrane proteins, whether prepared as 2D crystals or single particles, cryo-EM samples must be vitrified with great care. Grid preparation for cryo-EM of 2D crystals is possible by back-injection, the carbon sandwich technique, drying in sugars before cooling in the electron microscope, or plunge-freezing. Specimen grids for single particle cryo-EM studies of membrane proteins are usually produced by plunge-freezing protein solutions, supported either by perforated or a continuous carbon film substrate. This review outlines the different techniques available and the suitability of each method for particular samples and studies. Experimental considerations in sample preparation and preservation include the protein itself and the presence of lipid or detergent. The appearance of cryo-EM samples in different conditions is also discussed.
Schmidt-Krey
Ingeborg
I
Georgia Institute of Technology, School of Biology, School of Chemistry and Biochemistry, 310 Ferst Drive, Rm. A118, Atlanta, GA 30332-0230, USA. Ingeborg.Schmidt-Krey@biology.gatech.edu
Rubinstein
John L
JL
D020285:Cryoelectron Microscopy / Q000379:methods* / Q000639:trends; D003461:Crystallography / Q000379:methods; D008565:Membrane Proteins / Q000648:ultrastructure*; D013048:Specimen Handling / Q000379:methods*
D008565:Membrane Proteins