TY - JOUR
T1 - Fabs enable single particle cryoEM studies of small proteins
AU - Wu, Shenping
AU - Avila-Sakar, Agustin
AU - Kim, Jungmin
AU - Booth, David S.
AU - Greenberg, Charles H.
AU - Rossi, Andrea
AU - Liao, Maofu
AU - Li, Xueming
AU - Alian, Akram
AU - Griner, Sarah L.
AU - Juge, Narinobu
AU - Yu, Yadong
AU - Mergel, Claudia M.
AU - Chaparro-Riggers, Javier
AU - Strop, Pavel
AU - Tampé, Robert
AU - Edwards, Robert H.
AU - Stroud, Robert M.
AU - Craik, Charles S.
AU - Cheng, Yifan
N1 - Funding Information:
This work is partly supported by NIH Grants 5R01GM098672 and 1S10RR026814-01 (to Y.C.), P50GM082250 (HARC Center to A. Frankel), and 1P50 GM073210 (Membrane Protein Expression Center to R.M.S.), the UCSF Program for Breakthrough Biomedical Research (Opportunity Award in Basic Science and New Technology Award to Y.C.), and The German Research Foundation (SFB 807 Transport and Communication across Biological Membranes and TA157/7 to R.T.). D.S.B. is supported by an NIGMS-IMSD Fellowship. The authors thank Jaume Pons, Arvind Rajpal, Dave Shelton, and Hong Liang for their advice and insightful discussion in generating PCSK9 and Fab J16 samples.
PY - 2012/4/4
Y1 - 2012/4/4
N2 - 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.
AB - 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.
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U2 - 10.1016/j.str.2012.02.017
DO - 10.1016/j.str.2012.02.017
M3 - Article
C2 - 22483106
AN - SCOPUS:84859405812
SN - 0969-2126
VL - 20
SP - 582
EP - 592
JO - Structure with Folding & design
JF - Structure with Folding & design
IS - 4
ER -