title: Electron counting and beam-induced motion correction enable near-atomic-resolution single-particle cryo-EM.
abstract: In recent work with large high-symmetry viruses, single-particle electron cryomicroscopy (cryo-EM) has achieved the determination of near-atomic-resolution structures by allowing direct fitting of atomic models into experimental density maps. However, achieving this goal with smaller particles of lower symmetry remains challenging. Using a newly developed single electron-counting detector, we confirmed that electron beam-induced motion substantially degrades resolution, and we showed that the combination of rapid readout and nearly noiseless electron counting allow image blurring to be corrected to subpixel accuracy, restoring intrinsic image information to high resolution (Thon rings visible to ∼3 Å). Using this approach, we determined a 3.3-Å-resolution structure of an ∼700-kDa protein with D7 symmetry, the Thermoplasma acidophilum 20S proteasome, showing clear side-chain density. Our method greatly enhances image quality and data acquisition efficiency-key bottlenecks in applying near-atomic-resolution cryo-EM to a broad range of protein samples.
authors: The Keck Advanced Microscopy Laboratory, Department of Biochemistry and Biophysics, University of California, San Francisco (UCSF), San Francisco, California, USA.
authors: Xueming
authors: X
authors: Li
authors: Paul
authors: P
authors: Mooney
authors: Shawn
authors: S
authors: Zheng
authors: Christopher R
authors: CR
authors: Booth
authors: Michael B
authors: MB
authors: Braunfeld
authors: Sander
authors: S
authors: Gubbens
authors: David A
authors: DA
authors: Agard
authors: Yifan
authors: Y
authors: Cheng
mesh_terms: D020285:Cryoelectron Microscopy / Q000379:methods*; D004583:Electrons; D021621:Imaging, Three-Dimensional / Q000379:methods; D009038:Motion; D046988:Proteasome Endopeptidase Complex / Q000648:ultrastructure*; D013822:Thermoplasma / Q000201:enzymology*
chemical_list: D046988:Proteasome Endopeptidase Complex
references: J Comput Chem. 2004 Oct;25(13):1605-12
references: 15264254
references: Science. 1995 Apr 28;268(5210):533-9
references: 7725097
references: Q Rev Biophys. 1995 May;28(2):171-93
references: 7568675
references: Proc Natl Acad Sci U S A. 2009 Jun 30;106(26):10644-8
references: 19487668
references: J Struct Biol. 2010 Dec;172(3):407-12
references: 20558298
references: J Struct Biol. 2011 Dec;176(3):404-8
references: 21933715
references: J Mol Biol. 2003 Oct 31;333(4):721-45
references: 14568533
references: J Struct Biol. 2006 Dec;156(3):556-63
references: 17067819
references: Elife. 2013 Feb 19;2:e00461
references: 23427024
references: Nat Protoc. 2008;3(12):1941-74
references: 19180078
references: Ultramicroscopy. 2000 Sep;85(1):9-13
references: 10981735
references: Methods Cell Biol. 2007;79:661-719
references: 17327180
references: Nat Methods. 2012 Sep;9(9):853-4
references: 22842542
references: Proc Natl Acad Sci U S A. 2010 Apr 6;107(14):6298-303
references: 20308582
references: J Struct Biol. 2012 Mar;177(3):630-7
references: 22366277
references: Ultramicroscopy. 2007 Feb-Mar;107(2-3):106-15
references: 16905258
references: Structure. 2012 Nov 7;20(11):1823-8
references: 23022349
references: J Struct Biol. 2001 Jan;133(1):32-42
references: 11356062
references: Ultramicroscopy. 2009 Nov;109(12):1411-6
references: 19647366
references: Ultramicroscopy. 2009 Aug;109(9):1144-7
references: 19541421
references: J Struct Biol. 2004 Jan-Feb;145(1-2):91-9
references: 15065677
references: Cell. 2010 Apr 30;141(3):472-82
references: 20398923
references: Nature. 2010 Jan 21;463(7279):379-83
references: 20090755
references: Ultramicroscopy. 2009 Aug;109(9):1126-43
references: 19497671
references: Mol Cell. 2008 May 9;30(3):360-8
references: 18471981
references: J Struct Biol. 2012 Mar;177(3):589-601
references: 22285189
references: J Struct Biol. 2001 Feb-Mar;133(2-3):143-55
references: 11472086
references: EMBO J. 2010 Feb 3;29(3):692-702
references: 20019667
references: Structure. 2008 May;16(5):673-83
references: 18462672
references: J Struct Biol. 2003 Jun;142(3):334-47
references: 12781660
references: Ultramicroscopy. 2011 Jan;111(2):90-100
references: 21185452
references: Proc Natl Acad Sci U S A. 2008 Feb 12;105(6):1867-72
references: 18238898
references: J Mol Biol. 1999 May 14;288(4):765-86
references: 10329178
references: EMBO J. 2011 Aug 09;30(18):3854-63
references: 21829169