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- PDB-2w4t: ISOMETRICALLY CONTRACTING INSECT ASYNCHRONOUS FLIGHT MUSCLE -

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Entry
Database: PDB / ID: 2w4t
TitleISOMETRICALLY CONTRACTING INSECT ASYNCHRONOUS FLIGHT MUSCLE
Components
  • MYOSIN ESSENTIAL LIGHT CHAIN, STRIATED ADDUCTOR MUSCLE
  • MYOSIN HEAVY CHAIN, STRIATED MUSCLEMyosin
  • MYOSIN REGULATORY LIGHT CHAIN, STRIATED ADDUCTOR MUSCLE
KeywordsCONTRACTILE PROTEIN / NUCLEOTIDE-BINDING / TROPOMYOSIN / LIGHT CHAINS / ACTIN- BINDING / FREEZE SUBSTITUTION / ISOMETRIC CONTRACTION / FREEZING / MICROTOMY / ATP-BINDING / THIN FILAMENT / MOTOR PROTEIN / THICK FILAMENT / MUSCLE PROTEIN / IMAGE PROCESSING / CALMODULIN-BINDING / ACTIN / INSECT / MYOSIN / MUSCLE / TROPONIN / MULTIVARIATE DATA ANALYSIS
Function / homology
Function and homology information


myosin filament / myosin complex / myofibril / cytoskeletal motor activity / actin filament binding / calmodulin binding / calcium ion binding / ATP binding
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 / 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 ...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 / 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 motor domain profile. / Myosin. Large ATPases. / IQ motif profile. / IQ motif, EF-hand binding site / Kinesin motor domain superfamily / EF-hand domain pair / 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 essential light chain, striated adductor muscle / Myosin regulatory light chain, striated adductor muscle / Myosin heavy chain, striated muscle
Similarity search - Component
Biological speciesARGOPECTEN IRRADIANS (bay scallop)
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
DepositionDec 2, 2008Deposition site: PDBE / Processing site: PDBE
Revision 1.0Jan 19, 2010Provider: repository / Type: Initial release
Revision 1.1May 8, 2011Group: Version format compliance
Revision 1.2Jul 13, 2011Group: Version format compliance
Revision 1.3Oct 23, 2019Group: Author supporting evidence / Data collection / Other / Category: cell / em_image_scans / em_single_particle_entity / Item: _cell.Z_PDB

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Assembly

Deposited unit
C: MYOSIN HEAVY CHAIN, STRIATED MUSCLE
Y: MYOSIN REGULATORY LIGHT CHAIN, STRIATED ADDUCTOR MUSCLE
Z: MYOSIN ESSENTIAL LIGHT CHAIN, STRIATED ADDUCTOR MUSCLE


Theoretical massNumber of molelcules
Total (without water)127,4713
Polymers127,4713
Non-polymers00
Water0
1
C: MYOSIN HEAVY CHAIN, STRIATED MUSCLE


Theoretical massNumber of molelcules
Total (without water)94,8441
Polymers94,8441
Non-polymers00
Water0
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
MethodPISA
2
Y: MYOSIN REGULATORY LIGHT CHAIN, STRIATED ADDUCTOR MUSCLE


Theoretical massNumber of molelcules
Total (without water)15,5761
Polymers15,5761
Non-polymers00
Water0
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
MethodPISA
3
Z: MYOSIN ESSENTIAL LIGHT CHAIN, STRIATED ADDUCTOR MUSCLE


Theoretical massNumber of molelcules
Total (without water)17,0521
Polymers17,0521
Non-polymers00
Water0
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
MethodPISA
Number of models43

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Components

#1: Protein MYOSIN HEAVY CHAIN, STRIATED MUSCLE / Myosin / MYOSIN S1


Mass: 94843.883 Da / Num. of mol.: 1 / Fragment: RESIDUES 5-835 / Source method: isolated from a natural source / Details: MYOSIN S1 HEAVY CHAIN / Source: (natural) ARGOPECTEN IRRADIANS (bay scallop) / References: UniProt: P24733
#2: Protein MYOSIN REGULATORY LIGHT CHAIN, STRIATED ADDUCTOR MUSCLE / MYOSIN S1 / R-LC


Mass: 15575.700 Da / Num. of mol.: 1 / Fragment: RESIDUES 16-151 / Source method: isolated from a natural source
Details: MYOSIN REGULATORY LIGHT CHAIN, RLC, STRIATED ADDUCTOR MUSCLE
Source: (natural) ARGOPECTEN IRRADIANS (bay scallop) / References: UniProt: P13543
#3: Protein MYOSIN ESSENTIAL LIGHT CHAIN, STRIATED ADDUCTOR MUSCLE / MYOSIN S1 / E-LC


Mass: 17051.912 Da / Num. of mol.: 1 / Fragment: RESIDUES 5-155 / Source method: isolated from a natural source
Details: MYOSIN ESSENTIAL LIGHT CHAIN, ELC, STRIATED ADDUCTOR MUSCLE
Source: (natural) ARGOPECTEN IRRADIANS (bay scallop) / References: UniProt: P07291

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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 COLLECTED AROUND TWO MUTUALLY ORTHOGONAL TILT AXES USING THE SAXTON SCHEME FOR DETERMINING THE TILT ANGLES. THE TWO TILT SERIES WERE MERGED USING MARKER FREE ...Details: TOMOGRAPHIC TILT SERIES COLLECTED 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
VitrificationInstrument: HOMEMADE PLUNGER / Cryogen name: HELIUM

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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 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. THERE ARE 43 MODELS.
Symmetry type: HELICAL
RefinementHighest resolution: 35 Å
Refinement stepCycle: LAST / Highest resolution: 35 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms8501 0 0 0 8501

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