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Entry
Database: PDB / ID: 2w4h
TitleIsometrically contracting insect asynchronous flight muscle quick frozen after a quick release step
Components
  • MYOSIN HEAVY CHAIN, SKELETAL MUSCLE, ADULTMyosin
  • MYOSIN LIGHT CHAIN 3, SKELETAL MUSCLE ISOFORM
  • MYOSIN REGULATORY LIGHT CHAIN 2, SKELETAL MUSCLE ISOFORM
KeywordsCONTRACTILE PROTEIN / METHYLATION / ATP-BINDING / ISOMETRIC CONTRACTION / MICROTOMY / FREEZE SUBSTITUTION / MUSCLE PROTEIN / CALMODULIN-BINDING / MOTOR PROTEIN / ACTIN-BINDING
Function / homology
Function and homology information


contractile fiber / myosin filament / myosin II complex / myosin complex / myofibril / cytoskeletal motor activity / skeletal muscle tissue development / actin filament binding / calmodulin binding / calcium ion binding ...contractile fiber / myosin filament / myosin II complex / myosin complex / myofibril / cytoskeletal motor activity / skeletal muscle tissue development / actin filament binding / calmodulin binding / calcium ion binding / ATP binding / cytoplasm
Similarity search - Function
EF-hand domain / DNA repair protein XRCC4-like, C-terminal / Myosin tail / Myosin tail / Myosin N-terminal SH3-like domain / Myosin S1 fragment, N-terminal / EF-hand domain pair / Myosin, N-terminal, SH3-like / Myosin N-terminal SH3-like domain profile. / Short calmodulin-binding motif containing conserved Ile and Gln residues. ...EF-hand domain / DNA repair protein XRCC4-like, C-terminal / Myosin tail / Myosin tail / Myosin N-terminal SH3-like domain / Myosin S1 fragment, N-terminal / EF-hand domain pair / Myosin, N-terminal, SH3-like / Myosin N-terminal SH3-like domain profile. / Short calmodulin-binding motif containing conserved Ile and Gln residues. / Myosin head, motor domain / Myosin head (motor domain) / Myosin. Large ATPases. / Myosin motor domain profile. / IQ motif profile. / IQ motif, EF-hand binding site / Kinesin motor domain superfamily / EF-hand, calcium binding motif / EF-Hand 1, calcium-binding site / EF-hand calcium-binding domain. / EF-hand calcium-binding domain profile. / EF-hand domain / EF-hand domain pair / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
Myosin light chain 3, skeletal muscle isoform / Myosin regulatory light chain 2, skeletal muscle isoform / Myosin heavy chain, skeletal muscle, adult
Similarity search - Component
Biological speciesGALLUS GALLUS (chicken)
MethodELECTRON MICROSCOPY / helical reconstruction / Resolution: 35 Å
AuthorsWu, S. / Liu, J. / Reedy, M.C. / Tregear, R.T. / Winkler, H. / Franzini-Armstrong, C. / Sasaki, H. / Lucaveche, C. / Goldman, Y.E. / Reedy, M.K. / Taylor, K.A.
Citation
Journal: PLoS One / Year: 2012
Title: Structural changes in isometrically contracting insect flight muscle trapped following a mechanical perturbation.
Authors: Shenping Wu / Jun Liu / Mary C Reedy / Robert J Perz-Edwards / Richard T Tregear / Hanspeter Winkler / Clara Franzini-Armstrong / Hiroyuki Sasaki / Carmen Lucaveche / Yale E Goldman / ...Authors: Shenping Wu / Jun Liu / Mary C Reedy / Robert J Perz-Edwards / Richard T Tregear / Hanspeter Winkler / Clara Franzini-Armstrong / Hiroyuki Sasaki / Carmen Lucaveche / Yale E Goldman / Michael K Reedy / Kenneth A Taylor /
Abstract: The application of rapidly applied length steps to actively contracting muscle is a classic method for synchronizing the response of myosin cross-bridges so that the average response of the ensemble ...The application of rapidly applied length steps to actively contracting muscle is a classic method for synchronizing the response of myosin cross-bridges so that the average response of the ensemble can be measured. Alternatively, electron tomography (ET) is a technique that can report the structure of the individual members of the ensemble. We probed the structure of active myosin motors (cross-bridges) by applying 0.5% changes in length (either a stretch or a release) within 2 ms to isometrically contracting insect flight muscle (IFM) fibers followed after 5-6 ms by rapid freezing against a liquid helium cooled copper mirror. ET of freeze-substituted fibers, embedded and thin-sectioned, provides 3-D cross-bridge images, sorted by multivariate data analysis into ~40 classes, distinct in average structure, population size and lattice distribution. Individual actin subunits are resolved facilitating quasi-atomic modeling of each class average to determine its binding strength (weak or strong) to actin. ~98% of strong-binding acto-myosin attachments present after a length perturbation are confined to "target zones" of only two actin subunits located exactly midway between successive troponin complexes along each long-pitch helical repeat of actin. Significant changes in the types, distribution and structure of actin-myosin attachments occurred in a manner consistent with the mechanical transients. Most dramatic is near disappearance, after either length perturbation, of a class of weak-binding cross-bridges, attached within the target zone, that are highly likely to be precursors of strong-binding cross-bridges. These weak-binding cross-bridges were originally observed in isometrically contracting IFM. Their disappearance following a quick stretch or release can be explained by a recent kinetic model for muscle contraction, as behaviour consistent with their identification as precursors of strong-binding cross-bridges. The results provide a detailed model for contraction in IFM that may be applicable to contraction in other types of muscle.
#1: Journal: J Struct Biol / Year: 2009
Title: Methods for identifying and averaging variable molecular conformations in tomograms of actively contracting insect flight muscle.
Authors: Shenping Wu / Jun Liu / Mary C Reedy / Hanspeter Winkler / Michael K Reedy / Kenneth A Taylor /
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 ...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.
History
DepositionNov 25, 2008Deposition site: PDBE / Processing site: PDBE
Revision 1.0Aug 25, 2010Provider: repository / Type: Initial release
Revision 1.1Jul 18, 2012Group: Database references / Version format compliance
Revision 1.2Apr 19, 2017Group: Other
Revision 1.3Oct 23, 2019Group: Author supporting evidence / Data collection / Other / Category: cell / em_image_scans / em_single_particle_entity / Item: _cell.Z_PDB
Remark 700 SHEET THE SHEET STRUCTURE OF THIS MOLECULE IS BIFURCATED. IN ORDER TO REPRESENT THIS FEATURE IN ... SHEET THE SHEET STRUCTURE OF THIS MOLECULE IS BIFURCATED. IN ORDER TO REPRESENT THIS FEATURE IN THE SHEET RECORDS BELOW, TWO SHEETS ARE DEFINED.

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Assembly

Deposited unit
B: MYOSIN REGULATORY LIGHT CHAIN 2, SKELETAL MUSCLE ISOFORM
C: MYOSIN LIGHT CHAIN 3, SKELETAL MUSCLE ISOFORM
M: MYOSIN HEAVY CHAIN, SKELETAL MUSCLE, ADULT


Theoretical massNumber of molelcules
Total (without water)129,0333
Polymers129,0333
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by author&software
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
MethodPQS
Number of models14

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Components

#1: Protein MYOSIN REGULATORY LIGHT CHAIN 2, SKELETAL MUSCLE ISOFORM / FAST SKELETAL MYOSIN LIGHT CHAIN 2 / MYOSIN S1 / MLC-2 / LC2F / G2 / DTNB


Mass: 16958.186 Da / Num. of mol.: 1 / Fragment: RESIDUES, 16-165 / Source method: isolated from a natural source / Source: (natural) GALLUS GALLUS (chicken) / Tissue: SKELETAL MUSCLE / References: UniProt: P02609
#2: Protein MYOSIN LIGHT CHAIN 3, SKELETAL MUSCLE ISOFORM / / A2 CATALYTIC / ALKALI MYOSIN LIGHT CHAIN 3 / MLC-3 / MYOSIN LIGHT CHAIN 3F / SKELETAL-MUSCLE MYOSIN ...A2 CATALYTIC / ALKALI MYOSIN LIGHT CHAIN 3 / MLC-3 / MYOSIN LIGHT CHAIN 3F / SKELETAL-MUSCLE MYOSIN L-4 LIGHT CHAIN


Mass: 16177.147 Da / Num. of mol.: 1 / Fragment: RESIDUES, 5-149 / Source method: isolated from a natural source / Source: (natural) GALLUS GALLUS (chicken) / Tissue: SKELETAL MUSCLE / References: UniProt: P02605
#3: Protein MYOSIN HEAVY CHAIN, SKELETAL MUSCLE, ADULT / Myosin / MYOSIN S1


Mass: 95898.125 Da / Num. of mol.: 1 / Fragment: RESIDUES, 5-844 / Source method: isolated from a natural source / Source: (natural) GALLUS GALLUS (chicken) / Tissue: SKELETAL MUSCLE / References: UniProt: P13538
Sequence details94 IDENTITY TO P13538 96 IDENTITY TO P02609 97 IDENTITY TO P02605

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: FILAMENT / 3D reconstruction method: helical reconstruction

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Sample preparation

ComponentName: ISOMETRICALLY CONTRACTING ASYNCHRONOUS INSECT FLIGHT MUSCLE FROM THE LARGE WATERBUG LETHOCERUS INDICUS
Type: COMPLEX
Details: TOMOGRAPHIC TILT SERIES COLLECT AROUND TWO MUTUALLY ORTHOGONAL TILT AXES USING THE SAXTON SCHEME FOR DETERMINING THE TILT ANGLES. THE TWO TILT SERIES WERE MERGED USING MARKER FREE ALIGNMENT ...Details: TOMOGRAPHIC TILT SERIES COLLECT AROUND TWO MUTUALLY ORTHOGONAL TILT AXES USING THE SAXTON SCHEME FOR DETERMINING THE TILT ANGLES. THE TWO TILT SERIES WERE MERGED USING MARKER FREE ALIGNMENT AND THE TWO INDEPENDENT TILT SERIES COMBINED USING IMOD.
Buffer solutionName: 20 MM MOPS BUFFER, 5 MM NAN3, AND MGCL2, ATP, CACL2, AND EGTA IN VARYING MILLIMOLAR MILLIMOLAR CONCENTRATIONS
Details: 20 MM MOPS BUFFER, 5 MM NAN3, AND MGCL2, ATP, CACL2, AND EGTA IN VARYING MILLIMOLAR MILLIMOLAR CONCENTRATIONS
SpecimenEmbedding applied: YES / Shadowing applied: NO / Staining applied: NO / Vitrification applied: NO
Specimen supportDetails: CARBON

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Electron microscopy imaging

MicroscopyModel: FEI/PHILIPS CM300FEG/T / Details: NONE
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Cs: 2 mm
Specimen holderTilt angle max: 72 ° / Tilt angle min: -72 °
Image recordingFilm or detector model: TVIPS TEMCAM-F224 (2k x 2k)
Radiation wavelengthRelative weight: 1

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Processing

3D reconstructionDetails: NOTE THAT OUR LOWEST RESOLUTION DATA IS AT INVERSE 1 MICRON. NUMBER OF FOURIER COEFFICIENTS IS ALMOST A HALF MILLION. THESE COORDINATES WERE FITTED TO AVERAGED SUBVOLUMES OBTAINED FROM A ...Details: NOTE THAT OUR LOWEST RESOLUTION DATA IS AT INVERSE 1 MICRON. NUMBER OF FOURIER COEFFICIENTS IS ALMOST A HALF MILLION. THESE COORDINATES WERE FITTED TO AVERAGED SUBVOLUMES OBTAINED FROM A DUAL AXIS TOMOGRAM. THE FITTING WAS DONE MANUALLY USING THE CRYSTALLOGRAPHIC MODEL FITTING PROGRAM O. THERE ARE 14 MODELS.
Symmetry type: HELICAL
RefinementHighest resolution: 35 Å
Refinement stepCycle: LAST / Highest resolution: 35 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms8758 0 0 0 8758

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