title: Direct imaging electron microscopy (EM) methods in modern structural biology: overview and comparison with X-ray crystallography and single-particle cryo-EM reconstruction in the studies of large macromolecules.
abstract: Determining the structure of macromolecules is important for understanding their function. The fine structure of large macromolecules is currently studied primarily by X-ray crystallography and single-particle cryo-electron microscopy (EM) reconstruction. Before the development of these techniques, macromolecular structure was often examined by negative-staining, rotary-shadowing and freeze-etching EM, which are categorised here as 'direct imaging EM methods'. In this review, the results are summarised by each of the above techniques and compared with respect to four macromolecules: the ryanodine receptor, cadherin, rhodopsin and the ribosome-translocon complex (RTC). The results of structural analysis of the ryanodine receptor and cadherin are consistent between each technique. The results obtained for rhodopsin vary to some extent within each technique and between the different techniques. Finally, the results for RTC are inconsistent between direct imaging EM and other analytical techniques, especially with respect to the space within RTC, the reasons for which are discussed. Then, the role of direct imaging EM methods in modern structural biology is discussed. Direct imaging methods should support and verify the results obtained by other analytical methods capable of solving three-dimensional molecular architecture, and they should still be used as a primary tool for studying macromolecule structure in vivo.
authors.affiliation: Shinsapporokeiaikai Hospital, 5-5-35 Ooyachihigashi, Atsubetsuku, Sapporo, 004-0041, Japan.
authors.forename: Katsuyuki
authors.initials: K
authors.lastname: Miyaguchi
mesh_terms: D020285:Cryoelectron Microscopy; D018360:Crystallography, X-Ray; D007091:Image Processing, Computer-Assisted; D046911:Macromolecular Substances / Q000032:analysis / Q000737:chemistry / Q000648:ultrastructure; D008854:Microscopy, Electron / Q000379:methods*; D008958:Models, Molecular
chemical_list: D046911:Macromolecular Substances
keywords: Cadherin; Electron microscopy; Rhodopsin; Ribosome; X-ray crystallography