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J Struct Biol. 2014 May;186(2):265-72. doi: 10.1016/j.jsb.2014.04.003. Epub 2014 Apr 12.

A method for 3D-reconstruction of a muscle thick filament using the tilt series images of a single filament electron tomogram.

Author information

1
Centro de Biología Estructural, Instituto Venezolano de Investigaciones Científicas (IVIC), Apdo. 20632, Caracas 1020A, Venezuela. Electronic address: eltavogus@gmail.com.
2
Centro de Biología Estructural, Instituto Venezolano de Investigaciones Científicas (IVIC), Apdo. 20632, Caracas 1020A, Venezuela. Electronic address: pinto.misle@gmail.com.
3
Centro de Biología Estructural, Instituto Venezolano de Investigaciones Científicas (IVIC), Apdo. 20632, Caracas 1020A, Venezuela. Electronic address: alamo@ivic.gob.ve.
4
Institute of Molecular Biophysics, Florida State University, 91 Chieftan way, Tallahassee, FL 32306-4380, USA. Electronic address: baumann@sb.fsu.edu.
5
Institute of Molecular Biophysics, Florida State University, 91 Chieftan way, Tallahassee, FL 32306-4380, USA. Electronic address: feye@ucsd.edu.
6
Institute of Molecular Biophysics, Florida State University, 91 Chieftan way, Tallahassee, FL 32306-4380, USA. Electronic address: winkler@sb.fsu.edu.
7
Institute of Molecular Biophysics, Florida State University, 91 Chieftan way, Tallahassee, FL 32306-4380, USA. Electronic address: taylor@bio.fsu.edu.
8
Centro de Biología Estructural, Instituto Venezolano de Investigaciones Científicas (IVIC), Apdo. 20632, Caracas 1020A, Venezuela. Electronic address: raul.padron@gmail.com.

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.

KEYWORDS:

3D-reconstruction; Electron microscopy; Electron tomography; Muscle thick filament; Myosin interacting-heads (MIH) motif; Negative staining

PMID:
24727133
PMCID:
PMC4102135
DOI:
10.1016/j.jsb.2014.04.003
[Indexed for MEDLINE]
Free PMC Article

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