Structural Biology Using Electron Microscopy
Due to recent developments in cryo-transmission electron microscopy (Cryo-TEM), direct electron counting cameras and reconstruction softwares, cryo-TEM is now widely applied to determine the structure of biological samples including various kinds of proteins and their complexes. Single particle analysis (SPA) using cryo-TEM enables the reconstruction of three-dimensional (3D) structures of proteins from countable numbers of protein particles of usually less than 1 million. Apart from the requirement of crystallization and massive purification, SPA has a great potential to reveal the structures of biologically important proteins, including membrane proteins and macromolecular complexes, which are often hard to crystallize or unsuitable for mass production. Conformational changes of proteins have been implemented in the algorithms and strategies of SPA. Cryo-electron tomography is another approach used to reconstruct 3D structures of frozen samples from a series of images recorded by tilting the specimen in the EM column. It is used for much larger biological structures of higher order, e.g., cells and tissues. Atmospheric scanning electron microscopy (ASEM) directly visualizes two-dimensional (2D) images of aldehyde-fixed cells, tissues, or protein microcrystals in aqueous solution containing radical scavengers (glucose) through electron-transparent SiN membrane at nanometer resolution.
KeywordsCryo-electron microscope Protein Macromolecular complex Single particle analysis Three-dimensional structure Tomography ASEM
This work was supported by a Grant-in-Aid for Scientific Research on Innovative Areas, Sparse modeling (to C.S.), by CREST (to C.S.), by a Grant-in-Aid for Scientific Research from JSPS (15 K14499) (to C.S.), by grants from the Ministry of Education, Culture, Sports, Science, and Technology (MEXT) (to C.S.), by a Grant-in-Aid from CANON (to C.S.), by a Strategic grant to NISP from AIST (to C.S.) and by a Grant-in-Aid from AIST (to C.S).
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