title: Methods for identifying and averaging variable molecular conformations in tomograms of actively contracting insect flight muscle.
abstract: During active muscle contraction, tension is generated through many simultaneous, independent interactions between the molecular motor myosin and the actin filaments. The ensemble of myosin motors displays heterogeneous conformations reflecting different mechanochemical steps of the ATPase pathway. We used electron tomography of actively contracting insect flight muscle fast-frozen, freeze substituted, Araldite embedded, thin-sectioned and stained, to obtain 3D snapshots of the multiplicity of actin-attached myosin structures. We describe procedures for alignment of the repeating lattice of sub-volumes (38.7 nm cross-bridge repeats bounded by troponin) and multivariate data analysis to identify self-similar repeats for computing class averages. Improvements in alignment and classification of repeat sub-volumes reveals (for the first time in active muscle images) the helix of actin subunits in the thin filament and the troponin density with sufficient clarity that a quasiatomic model of the thin filament can be built into the class averages independent of the myosin cross-bridges. We show how quasiatomic model building can identify both strong and weak myosin attachments to actin. We evaluate the accuracy of image classification to enumerate the different types of actin-myosin attachments.
authors.affiliation: Institute of Molecular Biophysics, Florida State University, Tallahassee, FL 32306-4380, USA.
authors.forename: Shenping
authors.initials: S
authors.lastname: Wu
authors.forename: Jun
authors.initials: J
authors.lastname: Liu
authors.forename: Mary C
authors.initials: MC
authors.lastname: Reedy
authors.forename: Hanspeter
authors.initials: H
authors.lastname: Winkler
authors.forename: Michael K
authors.initials: MK
authors.lastname: Reedy
authors.forename: Kenneth A
authors.initials: KA
authors.lastname: Taylor
mesh_terms: D000818:Animals; D055032:Electron Microscope Tomography / Q000379:methods*; D007313:Insecta / Q000648:ultrastructure*; D009119:Muscle Contraction / Q000502:physiology*; D009132:Muscles / Q000473:pathology
references.citation: Proc Natl Acad Sci U S A. 2000 Oct 10;97(21):11238-43
references.pmid: 11016966
references.citation: J Mol Biol. 1996 Nov 29;264(2):302-22
references.pmid: 8951378
references.citation: J Cell Biol. 1981 Aug;90(2):459-66
references.pmid: 6793598
references.citation: J Cell Biol. 1989 Sep;109(3):1103-23
references.pmid: 2768335
references.citation: J Cell Biol. 1978 Dec;79(3):846-52
references.pmid: 569662
references.citation: Nature. 1982 Jul 8;298(5870):131-5
references.pmid: 7201078
references.citation: Nat Struct Biol. 2000 Sep;7(9):711-4
references.pmid: 10966635
references.citation: J Cell Biol. 1978 Jun;77(3):837-52
references.pmid: 28332
references.citation: J Mol Biol. 1993 Sep 5;233(1):86-108
references.pmid: 8377196
references.citation: Biophys J. 2004 May;86(5):3009-19
references.pmid: 15111415
references.citation: J Cell Biol. 1989 Sep;109(3):1085-102
references.pmid: 2768334
references.citation: J Biol Chem. 2001 Oct 26;276(43):39812-8
references.pmid: 11517216
references.citation: Biophys J. 1993 Jul;65(1):397-408
references.pmid: 8369445
references.citation: J Cell Biol. 1994 Nov;127(3):763-78
references.pmid: 7962058
references.citation: Biophys J. 1995 Apr;68(4 Suppl):194S-199S; discussion 199S-201S
references.pmid: 7787067
references.citation: J Cell Biol. 1991 Nov;115(3):689-703
references.pmid: 1918159
references.citation: Biophys J. 2003 Aug;85(2):1063-79
references.pmid: 12885653
references.citation: Biophys J. 1994 Oct;67(4):1620-33
references.pmid: 7819494
references.citation: Biophys J. 1986 Jan;49(1):353-64
references.pmid: 3955176
references.citation: Mol Cell. 2005 Sep 2;19(5):595-605
references.pmid: 16137617
references.citation: Ultramicroscopy. 1992 Oct;46(1-4):207-27
references.pmid: 1481272
references.citation: Biophys J. 1992 Nov;63(5):1299-305
references.pmid: 1477281
references.citation: J Cell Biol. 1997 Nov 3;139(3):695-707
references.pmid: 9348286
references.citation: J Mol Biol. 1985 Sep 5;185(1):145-76
references.pmid: 4046036
references.citation: J Struct Biol. 2004 Sep;147(3):315-26
references.pmid: 15450300
references.citation: J Mol Biol. 1972 Sep 14;70(1):85-104
references.pmid: 5073353
references.citation: J Struct Biol. 2004 Sep;147(3):247-58
references.pmid: 15450294
references.citation: J Mol Biol. 2006 Mar 31;357(3):707-17
references.pmid: 16469331
references.citation: Acta Crystallogr A. 1991 Mar 1;47 ( Pt 2):110-9
references.pmid: 2025413
references.citation: Nature. 1984 Jul 26-Aug 1;310(5975):285-91
references.pmid: 6540369
references.citation: Proc Natl Acad Sci U S A. 2008 Jul 29;105(30):10372-7
references.pmid: 18658238
references.citation: Cell. 1999 Nov 12;99(4):421-31
references.pmid: 10571184
references.citation: Annu Rev Biochem. 2005;74:833-65
references.pmid: 15952904
references.citation: Nature. 1965 Sep 18;207(5003):1276-80
references.pmid: 5884645
references.citation: Nature. 2005 Jan 20;433(7023):330-4
references.pmid: 15662427
references.citation: Curr Opin Struct Biol. 1996 Oct;6(5):585-94
references.pmid: 8913679
references.citation: J Mol Biol. 2006 Sep 29;362(4):844-60
references.pmid: 16949613
references.citation: J Struct Biol. 2004 Sep;147(3):268-82
references.pmid: 15450296
references.citation: J Mol Biol. 1996 Nov 29;264(2):279-301
references.pmid: 8951377
references.citation: Proc Natl Acad Sci U S A. 2005 Nov 1;102(44):15889-94
references.pmid: 16246999
references.citation: Philos Trans R Soc Lond B Biol Sci. 2000 Apr 29;355(1396):419-31
references.pmid: 10836495
references.citation: J Mol Biol. 1968 Jan 28;31(2):155-76
references.pmid: 5635532
references.citation: Biophys J. 1996 Nov;71(5):2289-306
references.pmid: 8913571
references.citation: J Struct Biol. 2002 Apr-May;138(1-2):92-104
references.pmid: 12160705
references.citation: Acta Crystallogr D Biol Crystallogr. 2000 Oct;56(Pt 10):1341-9
references.pmid: 10998631
references.citation: Ultramicroscopy. 2006 Feb;106(3):240-54
references.pmid: 16137829
references.citation: J Muscle Res Cell Motil. 1983 Feb;4(1):25-53
references.pmid: 6841591
references.citation: Ultramicroscopy. 1984;13(1-2):57-70
references.pmid: 6382732
references.citation: Nature. 2003 Sep 25;425(6956):423-7
references.pmid: 14508495
references.citation: J Struct Biol. 2006 Aug;155(2):273-84
references.pmid: 16793285
references.citation: J Struct Biol. 2007 Jan;157(1):126-37
references.pmid: 16973379
references.citation: J Struct Biol. 1997 Dec;120(3):343-52
references.pmid: 9441937