title: Fabs enable single particle cryoEM studies of small proteins.
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.
authors.affiliation: 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.
authors.forename: Shenping
authors.initials: S
authors.lastname: Wu
authors.forename: Agustin
authors.initials: A
authors.lastname: Avila-Sakar
authors.forename: JungMin
authors.initials: J
authors.lastname: Kim
authors.forename: David S
authors.initials: DS
authors.lastname: Booth
authors.forename: Charles H
authors.initials: CH
authors.lastname: Greenberg
authors.forename: Andrea
authors.initials: A
authors.lastname: Rossi
authors.forename: Maofu
authors.initials: M
authors.lastname: Liao
authors.forename: Xueming
authors.initials: X
authors.lastname: Li
authors.forename: Akram
authors.initials: A
authors.lastname: Alian
authors.forename: Sarah L
authors.initials: SL
authors.lastname: Griner
authors.forename: Narinobu
authors.initials: N
authors.lastname: Juge
authors.forename: Yadong
authors.initials: Y
authors.lastname: Yu
authors.forename: Claudia M
authors.initials: CM
authors.lastname: Mergel
authors.forename: Javier
authors.initials: J
authors.lastname: Chaparro-Riggers
authors.forename: Pavel
authors.initials: P
authors.lastname: Strop
authors.forename: Robert
authors.initials: R
authors.lastname: Tampé
authors.forename: Robert H
authors.initials: RH
authors.lastname: Edwards
authors.forename: Robert M
authors.initials: RM
authors.lastname: Stroud
authors.forename: Charles S
authors.initials: CS
authors.lastname: Craik
authors.forename: Yifan
authors.initials: Y
authors.lastname: Cheng
mesh_terms: D020285:Cryoelectron Microscopy / Q000379:methods; D004926:Escherichia coli; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006801:Humans; D007091:Image Processing, Computer-Assisted; D007140:Immunoglobulin Fab Fragments / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008958:Models, Molecular; D008970:Molecular Weight; D019151:Peptide Library; D000071449:Proprotein Convertase 9; D043484:Proprotein Convertases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012697:Serine Endopeptidases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D050496:Vesicular Glutamate Transport Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
chemical_list: D029968:Escherichia coli Proteins; D007140:Immunoglobulin Fab Fragments; D019151:Peptide Library; D011994:Recombinant Proteins; D050496:Vesicular Glutamate Transport Proteins; C472125:PCSK9 protein, human; D000071449:Proprotein Convertase 9; D043484:Proprotein Convertases; D012697:Serine Endopeptidases