title: A method for 3D-reconstruction of a muscle thick filament using the tilt series images of a single filament electron tomogram.
abstract: Myosin interacting-heads (MIH) motifs are visualized in 3D-reconstructions of thick filaments from striated muscle. These reconstructions are calculated by averaging methods using images from electron micrographs of grids prepared using numerous filament preparations. Here we propose an alternative method to calculate the 3D-reconstruction of a single thick filament using only a tilt series images recorded by electron tomography. Relaxed thick filaments, prepared from tarantula leg muscle homogenates, were negatively stained. Single-axis tilt series of single isolated thick filaments were obtained with the electron microscope at a low electron dose, and recorded on a CCD camera by electron tomography. An IHRSR 3D-recontruction was calculated from the tilt series images of a single thick filament. The reconstruction was enhanced by including in the search stage dual tilt image segments while only single tilt along the filament axis is usually used, as well as applying a band pass filter just before the back projection. The reconstruction from a single filament has a 40 Å resolution and clearly shows the presence of MIH motifs. In contrast, the electron tomogram 3D-reconstruction of the same thick filament - calculated without any image averaging and/or imposition of helical symmetry - only reveals MIH motifs infrequently. This is - to our knowledge - the first application of the IHRSR method to calculate a 3D reconstruction from tilt series images. This single filament IHRSR reconstruction method (SF-IHRSR) should provide a new tool to assess structural differences between well-ordered thick (or thin) filaments in a grid by recording separately their electron tomograms.
authors.affiliation: Centro de Biología Estructural, Instituto Venezolano de Investigaciones Científicas (IVIC), Apdo. 20632, Caracas 1020A, Venezuela. Electronic address: eltavogus@gmail.com.
authors.forename: G
authors.initials: G
authors.lastname: Márquez
authors.affiliation: Centro de Biología Estructural, Instituto Venezolano de Investigaciones Científicas (IVIC), Apdo. 20632, Caracas 1020A, Venezuela. Electronic address: pinto.misle@gmail.com.
authors.forename: A
authors.initials: A
authors.lastname: Pinto
authors.affiliation: Centro de Biología Estructural, Instituto Venezolano de Investigaciones Científicas (IVIC), Apdo. 20632, Caracas 1020A, Venezuela. Electronic address: alamo@ivic.gob.ve.
authors.forename: L
authors.initials: L
authors.lastname: Alamo
authors.affiliation: Institute of Molecular Biophysics, Florida State University, 91 Chieftan way, Tallahassee, FL 32306-4380, USA. Electronic address: baumann@sb.fsu.edu.
authors.forename: B
authors.initials: B
authors.lastname: Baumann
authors.affiliation: Institute of Molecular Biophysics, Florida State University, 91 Chieftan way, Tallahassee, FL 32306-4380, USA. Electronic address: feye@ucsd.edu.
authors.forename: F
authors.initials: F
authors.lastname: Ye
authors.affiliation: Institute of Molecular Biophysics, Florida State University, 91 Chieftan way, Tallahassee, FL 32306-4380, USA. Electronic address: winkler@sb.fsu.edu.
authors.forename: H
authors.initials: H
authors.lastname: Winkler
authors.affiliation: Institute of Molecular Biophysics, Florida State University, 91 Chieftan way, Tallahassee, FL 32306-4380, USA. Electronic address: taylor@bio.fsu.edu.
authors.forename: K
authors.initials: K
authors.lastname: Taylor
authors.affiliation: Centro de Biología Estructural, Instituto Venezolano de Investigaciones Científicas (IVIC), Apdo. 20632, Caracas 1020A, Venezuela. Electronic address: raul.padron@gmail.com.
authors.forename: R
authors.initials: R
authors.lastname: Padrón
mesh_terms: D000818:Animals; D055032:Electron Microscope Tomography; D005121:Extremities / Q000033:anatomy & histology*; D021621:Imaging, Three-Dimensional; D008854:Microscopy, Electron; D008958:Models, Molecular; D054792:Muscle, Striated / Q000648:ultrastructure*; D009218:Myosins / Q000648:ultrastructure*; D013112:Spiders / Q000033:anatomy & histology*
chemical_list: D009218:Myosins
keywords: 3D-reconstruction; Electron microscopy; Electron tomography; Muscle thick filament; Myosin interacting-heads (MIH) motif; Negative staining
references.citation: J Struct Biol. 2007 Jan;157(1):83-94
references.pmid: 16919474
references.citation: J Mol Biol. 2011 Nov 18;414(1):44-61
references.pmid: 21959262
references.citation: Science. 1969 Jun 20;164(3886):1356-65
references.pmid: 4181952
references.citation: J Mol Biol. 1981 Dec 15;153(3):781-90
references.pmid: 7338924
references.citation: J Mol Biol. 2008 Dec 26;384(4):780-97
references.pmid: 18951904
references.citation: J Struct Biol. 1996 Jan-Feb;116(1):190-9
references.pmid: 8742743
references.citation: Proc Natl Acad Sci U S A. 2013 May 21;110(21):8561-6
references.pmid: 23650385
references.citation: J Struct Biol. 2007 Jan;157(1):126-37
references.pmid: 16973379
references.citation: Biophys J. 2001 Nov;81(5):2817-26
references.pmid: 11606293
references.citation: BMC Genomics. 2009 Mar 19;10:117
references.pmid: 19298669
references.citation: J Cell Biol. 1985 Aug;101(2):395-401
references.pmid: 2410429
references.citation: Proc Natl Acad Sci U S A. 2013 Jan 2;110(1):318-23
references.pmid: 23251030
references.citation: J Mol Biol. 1967 Dec 14;30(2):383-434
references.pmid: 5586931
references.citation: J Struct Biol. 2003 Jan;141(1):43-52
references.pmid: 12576019
references.citation: J Struct Biol. 2012 Dec;180(3):469-78
references.pmid: 22982253
references.citation: J Comput Chem. 2004 Oct;25(13):1605-12
references.pmid: 15264254
references.citation: J Struct Biol. 2003 Oct-Nov;144(1-2):162-71
references.pmid: 14643219
references.citation: Proc Natl Acad Sci U S A. 2001 Apr 10;98(8):4361-6
references.pmid: 11287639
references.citation: Proc Natl Acad Sci U S A. 2008 Feb 19;105(7):2386-90
references.pmid: 18252826
references.citation: J Cell Biol. 1999 Dec 27;147(7):1385-90
references.pmid: 10613897
references.citation: Biophys J. 2013 Nov 5;105(9):2114-22
references.pmid: 24209856
references.citation: J Mol Biol. 1963 Sep;7:281-308
references.pmid: 14064165
references.citation: Ultramicroscopy. 2000 Dec;85(4):225-34
references.pmid: 11125866
references.citation: Nature. 2005 Aug 25;436(7054):1195-9
references.pmid: 16121187
references.citation: J Mol Biol. 1985 Aug 5;184(3):429-39
references.pmid: 4046022
references.citation: J Mol Biol. 2009 Jan 16;385(2):423-31
references.pmid: 18976661
references.citation: J Mol Biol. 2003 Jun 20;329(5):963-72
references.pmid: 12798686
references.citation: J Cell Biol. 1987 Sep;105(3):1319-27
references.pmid: 2958483
references.citation: J Struct Biol. 1996 Jan-Feb;116(1):71-6
references.pmid: 8742726
references.citation: Ultramicroscopy. 2006 Feb;106(3):240-54
references.pmid: 16137829
references.citation: Ultramicroscopy. 1984;13(1-2):57-70
references.pmid: 6382732