Structural analysis of membrane protein complexes by single particle electron microscopy.
Single particle electron microscopy (EM) is an increasingly important tool for the structural analysis of macromolecular complexes. The main advantage of the technique over other methods is that it is not necessary to precede the analysis with the growth of crystals of the sample. This advantage is particularly important for membrane proteins and large protein complexes where generating crystals is often the main barrier to structure determination. Therefore, single particle EM can be employed with great utility in the study of large membrane protein complexes. Although the construction of atomic resolution models by single particle EM is possible in theory, currently the highest resolution maps are still limited to approximately 7-10A resolution and 15-30 A resolution is more typical. However, by combining single particle EM maps with high-resolution models of subunits or subcomplexes from X-ray crystallography and NMR spectroscopy it is possible to build up an atomic model of a macromolecular assembly. Image analysis procedures are almost identical for micrographs of soluble protein complexes and detergent solubilized membrane protein complexes. However, electron microscopists attempting to prepare specimens of a membrane protein complex for imaging may find that these complexes require different handling than soluble protein complexes. This paper seeks to explain how high-quality specimen grids of membrane protein complexes may be prepared to allow for the determination of their structure by EM and image analysis.
Rubinstein
John L
JL
Molecular Structure and Function Program, Research Institute, The Hospital for Sick Children, 555 University Avenue, Toronto, Ont., Canada M5G 1X8. john.rubinstein@utoronto.ca
D002244:Carbon; D020285:Cryoelectron Microscopy / Q000295:instrumentation* / Q000379:methods*; D003902:Detergents; D007053:Ice; D046911:Macromolecular Substances / Q000737:chemistry*; D008565:Membrane Proteins / Q000737:chemistry*; D012995:Solubility
D003902:Detergents; D007053:Ice; D046911:Macromolecular Substances; D008565:Membrane Proteins; D002244:Carbon