Article

Methods for identifying and averaging variable molecular conformations in tomograms of actively contracting insect flight muscle.

Institute of Molecular Biophysics, Florida State University, Tallahassee, FL 32306-4380, USA.
Journal of Structural Biology (impact factor: 3.41). 09/2009; 168(3):485-502. DOI:10.1016/j.jsb.2009.08.007 pp.485-502
Source: PubMed

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.

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Keywords

38.7 nm cross-bridge
 
actin filaments
 
actin-attached myosin structures
 
active muscle contraction
 
active muscle images
 
alignment
 
class averages
 
class averages independent
 
contracting insect flight muscle fast-frozen
 
different mechanochemical steps
 
different types
 
electron tomography
 
molecular motor myosin
 
multivariate data analysis
 
myosin motors displays heterogeneous conformations
 
quasiatomic model building
 
repeat sub-volumes
 
repeating lattice
 
troponin density
 
weak myosin attachments