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J Struct Biol. 2014 Mar;185(3):295-302. doi: 10.1016/j.jsb.2014.01.004. Epub 2014 Jan 24.

Automatic cryo-EM particle selection for membrane proteins in spherical liposomes.

Author information

1
Department of Cellular and Molecular Physiology, Yale University School of Medicine, New Haven, CT 06520, USA; School of Computer and Information Technology, Beijng Jiaotong University, Beijing 100044, China.
2
Department of Cellular and Molecular Physiology, Yale University School of Medicine, New Haven, CT 06520, USA. Electronic address: Fred.Sigworth@yale.edu.

Abstract

Random spherically constrained (RSC) single particle reconstruction is a method to obtain structures of membrane proteins embedded in lipid vesicles (liposomes). As in all single-particle cryo-EM methods, structure determination is greatly aided by reliable detection of protein "particles" in micrographs. After fitting and subtraction of the membrane density from a micrograph, normalized cross-correlation (NCC) and estimates of the particle signal amplitude are used to detect particles, using as references the projections of a 3D model. At each pixel position, the NCC is computed with only those references that are allowed by the geometric constraint of the particle's embedding in the spherical vesicle membrane. We describe an efficient algorithm for computing this position-dependent correlation, and demonstrate its application to selection of membrane-protein particles, GluA2 glutamate receptors, which present very different views from different projection directions.

KEYWORDS:

Cross-correlation; Glutamate receptor; Particle picker; RSC reconstruction; Template matching

PMID:
24468290
PMCID:
PMC3978669
DOI:
10.1016/j.jsb.2014.01.004
[Indexed for MEDLINE]
Free PMC Article
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