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Title
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Methods for identifying and averaging variable molecular conformations in tomograms of actively contracting insect flight muscle.
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Authors
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S.Wu,
J.Liu,
M.C.Reedy,
H.Winkler,
M.K.Reedy,
K.A.Taylor.
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Ref.
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J Struct Biol, 2009,
168,
485-502.
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PubMed id
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Abstract
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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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