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- PDB-2w4g: ISOMETRICALLY CONTRACTING INSECT ASYNCHRONOUS FLIGHT MUSCLE QUICK... -

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Basic information

Entry
Database: PDB / ID: 2w4g
TitleISOMETRICALLY CONTRACTING INSECT ASYNCHRONOUS FLIGHT MUSCLE QUICK FROZEN AFTER A QUICK STRETCH STEP
Components
  • MYOSIN HEAVY CHAIN, SKELETAL MUSCLE, ADULT
  • 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 muscle fiber / Striated Muscle Contraction / myosin filament / myosin II complex / myosin complex / microfilament motor activity / myofibril / skeletal muscle tissue development / muscle contraction / actin filament binding ...contractile muscle fiber / Striated Muscle Contraction / myosin filament / myosin II complex / myosin complex / microfilament motor activity / myofibril / skeletal muscle tissue development / muscle contraction / 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 ...: / 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. / IQ motif, EF-hand binding site / Myosin head, motor domain / Myosin head (motor domain) / Myosin motor domain profile. / Myosin. Large ATPases. / IQ motif profile. / 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 11 / 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: 2010
Title: Electron tomography of cryofixed, isometrically contracting insect flight muscle reveals novel actin-myosin interactions.
Authors: Shenping Wu / Jun Liu / Mary C Reedy / Richard T Tregear / Hanspeter Winkler / Clara Franzini-Armstrong / Hiroyuki Sasaki / Carmen Lucaveche / Yale E Goldman / Michael K Reedy / Kenneth A Taylor /
Abstract: BACKGROUND: Isometric muscle contraction, where force is generated without muscle shortening, is a molecular traffic jam in which the number of actin-attached motors is maximized and all states of ...BACKGROUND: Isometric muscle contraction, where force is generated without muscle shortening, is a molecular traffic jam in which the number of actin-attached motors is maximized and all states of motor action are trapped with consequently high heterogeneity. This heterogeneity is a major limitation to deciphering myosin conformational changes in situ.
METHODOLOGY: We used multivariate data analysis to group repeat segments in electron tomograms of isometrically contracting insect flight muscle, mechanically monitored, rapidly frozen, freeze ...METHODOLOGY: We used multivariate data analysis to group repeat segments in electron tomograms of isometrically contracting insect flight muscle, mechanically monitored, rapidly frozen, freeze substituted, and thin sectioned. Improved resolution reveals the helical arrangement of F-actin subunits in the thin filament enabling an atomic model to be built into the thin filament density independent of the myosin. Actin-myosin attachments can now be assigned as weak or strong by their motor domain orientation relative to actin. Myosin attachments were quantified everywhere along the thin filament including troponin. Strong binding myosin attachments are found on only four F-actin subunits, the "target zone", situated exactly midway between successive troponin complexes. They show an axial lever arm range of 77°/12.9 nm. The lever arm azimuthal range of strong binding attachments has a highly skewed, 127° range compared with X-ray crystallographic structures. Two types of weak actin attachments are described. One type, found exclusively in the target zone, appears to represent pre-working-stroke intermediates. The other, which contacts tropomyosin rather than actin, is positioned M-ward of the target zone, i.e. the position toward which thin filaments slide during shortening.
CONCLUSION: We present a model for the weak to strong transition in the myosin ATPase cycle that incorporates azimuthal movements of the motor domain on actin. Stress/strain in the S2 domain may ...CONCLUSION: We present a model for the weak to strong transition in the myosin ATPase cycle that incorporates azimuthal movements of the motor domain on actin. Stress/strain in the S2 domain may explain azimuthal lever arm changes in the strong binding attachments. The results support previous conclusions that the weak attachments preceding force generation are very different from strong binding attachments.
#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 1.1May 8, 2011Group: Version format compliance
Revision 1.2Jul 13, 2011Group: Version format compliance
Revision 1.3Apr 19, 2017Group: Other
Revision 1.4Oct 23, 2019Group: Author supporting evidence / Data collection / Other / Category: cell / em_image_scans / em_single_particle_entity / Item: _cell.Z_PDB
Revision 1.5May 8, 2024Group: Data collection / Database references / Category: chem_comp_atom / chem_comp_bond / database_2
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession
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
Water00
1


  • Idetical with deposited unit
  • defined by author&software
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
MethodPISA
Number of models20

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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 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: INSECT FIBRILLAR FLIGHT MUSCLE / Type: COMPLEX
Details: THIS SPECIMEN IS OBTAINED FROM A QUICK FROZEN, ISOMETRICALLY CONTRACTING ASYNCHRONOUS INSECT FLIGHT MUSCLE THAT HAS BEEN FREEZE SUBSTITUTED, PLASTIC EMBEDDED, AND THIN SECTIONED. THE FIBERS ...Details: THIS SPECIMEN IS OBTAINED FROM A QUICK FROZEN, ISOMETRICALLY CONTRACTING ASYNCHRONOUS INSECT FLIGHT MUSCLE THAT HAS BEEN FREEZE SUBSTITUTED, PLASTIC EMBEDDED, AND THIN SECTIONED. THE FIBERS FOR THIS STUDY WERE SUBJECTED TO MECHANICAL TRANSIENTS. FOR THE QUICK STRETCH, THE FIBER WAS STRETCHED 6 NM PER HALF-SARCOMERE IN 2 MS AND LENGTH WAS HELD CONSTANT AFTER THE LENGTH STEP. THE FREEZING IMPACT OCCURRED 6-7 MS LATER.
Buffer solutionName: 20 MM MOPS BUFFER, 5 MM NAN3, AND MGCL2, ATP, CACL2, AND EGTA IN VARYING MILLIMOLAR CONCENTRATIONS
Details: 20 MM MOPS BUFFER, 5 MM NAN3, AND MGCL2, ATP, CACL2, AND EGTA IN VARYING MILLIMOLAR CONCENTRATIONS
SpecimenEmbedding applied: YES / Shadowing applied: NO / Staining applied: NO / Vitrification applied: NO
Specimen supportDetails: CARBON
VitrificationInstrument: HOMEMADE PLUNGER / Cryogen name: HELIUM
Details: SMASH AGAINST A LIQUID HELIUM COOLED GOLD COATED COPPER MIRROR

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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 FIELD / 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 reconstructionResolution method: FSC 0.5 CUT-OFF
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 ...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.
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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