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Structure. 2012 Apr 4;20(4):582-92. doi: 10.1016/j.str.2012.02.017. Epub 2012 Apr 3.

Fabs enable single particle cryoEM studies of small proteins.

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1
The W.M. Keck Advanced Microscopy Laboratory, Department of Biochemistry and Biophysics, University of California San Francisco, 600 16th Street, San Francisco, CA 94158, USA.

Abstract

In spite of its recent achievements, the technique of single particle electron cryomicroscopy (cryoEM) has not been widely used to study proteins smaller than 100 kDa, although it is a highly desirable application of this technique. One fundamental limitation is that images of small proteins embedded in vitreous ice do not contain adequate features for accurate image alignment. We describe a general strategy to overcome this limitation by selecting a fragment antigen binding (Fab) to form a stable and rigid complex with a target protein, thus providing a defined feature for accurate image alignment. Using this approach, we determined a three-dimensional structure of an ∼65 kDa protein by single particle cryoEM. Because Fabs can be readily generated against a wide range of proteins by phage display, this approach is generally applicable to study many small proteins by single particle cryoEM.

PMID:
22483106
PMCID:
PMC3322386
DOI:
10.1016/j.str.2012.02.017
[Indexed for MEDLINE]
Free PMC Article

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