title: 3D imaging and quantitative analysis of small solubilized membrane proteins and their complexes by transmission electron microscopy.
abstract: Inherently unstable, detergent-solubilized membrane protein complexes can often not be crystallized. For complexes that have a mass of >300 kDa, cryo-electron microscopy (EM) allows their three-dimensional (3D) structure to be assessed to a resolution that makes secondary structure elements visible in the best case. However, many interesting complexes exist whose mass is below 300 kDa and thus need alternative approaches. Two methods are reviewed: (i) Mass measurement in a scanning transmission electron microscope, which has provided important information on the stoichiometry of membrane protein complexes. This technique is applicable to particulate, filamentous and sheet-like structures. (ii) 3D-EM of negatively stained samples, which determines the molecular envelope of small membrane protein complexes. Staining and dehydration artifacts may corrupt the quality of the 3D map. Staining conditions thus need to be optimized. 3D maps of plant aquaporin SoPIP2;1 tetramers solubilized in different detergents illustrate that the flattening artifact can be partially prevented and that the detergent itself contributes significantly. Another example discussed is the complex of G protein-coupled receptor rhodopsin with its cognate G protein transducin.
authors.affiliation: Department of Pharmacology, School of Medicine, Case Western Reserve University, 10900 Euclid Ave, Cleveland, OH 44106-4965, USA.
authors.forename: Ardeschir
authors.initials: A
authors.lastname: Vahedi-Faridi
authors.forename: Beata
authors.initials: B
authors.lastname: Jastrzebska
authors.forename: Krzysztof
authors.initials: K
authors.lastname: Palczewski
authors.forename: Andreas
authors.initials: A
authors.lastname: Engel
mesh_terms: D020346:Aquaporins / Q000737:chemistry / Q000648:ultrastructure; D020285:Cryoelectron Microscopy / Q000379:methods; D003902:Detergents / Q000032:analysis; D005069:Evaluation Studies as Topic; D015966:Gene Expression Regulation, Fungal; D021621:Imaging, Three-Dimensional / Q000379:methods*; D008565:Membrane Proteins / Q000737:chemistry / Q000648:ultrastructure*; D046529:Microscopy, Electron, Transmission / Q000379:methods*; D016624:Negative Staining / Q000379:methods; D010843:Pichia / Q000235:genetics*; D017433:Protein Structure, Secondary; D012243:Rhodopsin / Q000737:chemistry / Q000648:ultrastructure; D015293:Transducin / Q000737:chemistry / Q000648:ultrastructure
chemical_list: D020346:Aquaporins; D003902:Detergents; D008565:Membrane Proteins; D012243:Rhodopsin; D015293:Transducin