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- PDB-2w49: ISOMETRICALLY CONTRACTING INSECT ASYNCHRONOUS FLIGHT MUSCLE -

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Basic information

Entry
Database: PDB / ID: 2w49
TitleISOMETRICALLY CONTRACTING INSECT ASYNCHRONOUS FLIGHT MUSCLE
Components
  • ACTIN, ALPHA SKELETAL MUSCLE
  • TROPOMYOSIN ALPHA-1 CHAIN
  • TROPONIN C, SKELETAL MUSCLE
  • TROPONIN I, FAST SKELETAL MUSCLE
  • TROPONIN T, FAST SKELETAL MUSCLE ISOFORMS
KeywordsCONTRACTILE PROTEIN / ISOMETRIC CONTRACTION
Function / homology
Function and homology information


troponin C binding / troponin complex / Striated Muscle Contraction / regulation of muscle contraction / sarcomere organization / cytoskeletal motor activator activity / tropomyosin binding / myosin heavy chain binding / mesenchyme migration / troponin I binding ...troponin C binding / troponin complex / Striated Muscle Contraction / regulation of muscle contraction / sarcomere organization / cytoskeletal motor activator activity / tropomyosin binding / myosin heavy chain binding / mesenchyme migration / troponin I binding / actin filament bundle / filamentous actin / actin filament bundle assembly / skeletal muscle thin filament assembly / striated muscle thin filament / skeletal muscle myofibril / skeletal muscle contraction / actin monomer binding / skeletal muscle fiber development / stress fiber / titin binding / cardiac muscle contraction / actin filament polymerization / filopodium / actin filament / Hydrolases; Acting on acid anhydrides; Acting on acid anhydrides to facilitate cellular and subcellular movement / calcium-dependent protein binding / actin filament binding / actin cytoskeleton / lamellipodium / cell body / actin binding / hydrolase activity / protein heterodimerization activity / protein domain specific binding / calcium ion binding / positive regulation of gene expression / magnesium ion binding / protein homodimerization activity / ATP binding / identical protein binding / cytoplasm
Similarity search - Function
Troponin T / Tropomyosins signature. / Tropomyosin / Troponin / Troponin domain superfamily / Tropomyosin / Troponin / EF hand / EF-hand domain pair / Actins signature 1. ...Troponin T / Tropomyosins signature. / Tropomyosin / Troponin / Troponin domain superfamily / Tropomyosin / Troponin / EF hand / EF-hand domain pair / Actins signature 1. / Actin, conserved site / Actins signature 2. / Actin/actin-like conserved site / Actins and actin-related proteins signature. / Actin / Actin family / Actin / EF-hand domain pair / EF-hand, calcium binding motif / ATPase, nucleotide binding domain / EF-Hand 1, calcium-binding site / EF-hand calcium-binding domain. / EF-hand calcium-binding domain profile. / EF-hand domain / EF-hand domain pair
Similarity search - Domain/homology
Troponin C, skeletal muscle / Troponin T, fast skeletal muscle isoforms / Tropomyosin alpha-1 chain / Actin, alpha skeletal muscle / Troponin I, fast skeletal muscle
Similarity search - Component
Biological speciesGALLUS GALLUS (chicken)
ORYCTOLAGUS CUNICULUS (rabbit)
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 24, 2008Deposition site: PDBE / Processing site: PDBE
Revision 1.0May 5, 2010Provider: repository / Type: Initial release
Revision 1.1May 8, 2011Group: Version format compliance
Revision 1.2Jul 13, 2011Group: Version format compliance
Revision 1.3Aug 23, 2017Group: Data collection / Category: em_image_scans / em_software / Item: _em_software.image_processing_id
Revision 1.4Oct 23, 2019Group: Data collection / Other / Category: cell / Item: _cell.Z_PDB

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Structure visualization

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Assembly

Deposited unit
0: TROPONIN C, SKELETAL MUSCLE
1: TROPONIN T, FAST SKELETAL MUSCLE ISOFORMS
2: TROPONIN I, FAST SKELETAL MUSCLE
3: TROPONIN C, SKELETAL MUSCLE
4: TROPONIN T, FAST SKELETAL MUSCLE ISOFORMS
5: TROPONIN I, FAST SKELETAL MUSCLE
6: TROPONIN C, SKELETAL MUSCLE
7: TROPONIN T, FAST SKELETAL MUSCLE ISOFORMS
8: TROPONIN I, FAST SKELETAL MUSCLE
9: TROPONIN C, SKELETAL MUSCLE
A: TROPOMYOSIN ALPHA-1 CHAIN
B: TROPOMYOSIN ALPHA-1 CHAIN
C: TROPOMYOSIN ALPHA-1 CHAIN
D: ACTIN, ALPHA SKELETAL MUSCLE
E: ACTIN, ALPHA SKELETAL MUSCLE
F: ACTIN, ALPHA SKELETAL MUSCLE
G: ACTIN, ALPHA SKELETAL MUSCLE
H: ACTIN, ALPHA SKELETAL MUSCLE
I: ACTIN, ALPHA SKELETAL MUSCLE
J: ACTIN, ALPHA SKELETAL MUSCLE
K: ACTIN, ALPHA SKELETAL MUSCLE
L: ACTIN, ALPHA SKELETAL MUSCLE
M: ACTIN, ALPHA SKELETAL MUSCLE
N: ACTIN, ALPHA SKELETAL MUSCLE
O: ACTIN, ALPHA SKELETAL MUSCLE
P: ACTIN, ALPHA SKELETAL MUSCLE
Q: ACTIN, ALPHA SKELETAL MUSCLE
R: ACTIN, ALPHA SKELETAL MUSCLE
S: ACTIN, ALPHA SKELETAL MUSCLE
T: TROPOMYOSIN ALPHA-1 CHAIN
U: TROPOMYOSIN ALPHA-1 CHAIN
V: TROPOMYOSIN ALPHA-1 CHAIN
W: TROPOMYOSIN ALPHA-1 CHAIN
X: TROPOMYOSIN ALPHA-1 CHAIN
Y: TROPONIN T, FAST SKELETAL MUSCLE ISOFORMS
Z: TROPONIN I, FAST SKELETAL MUSCLE
hetero molecules


Theoretical massNumber of molelcules
Total (without water)1,100,72552
Polymers1,100,08436
Non-polymers64116
Water0
1


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

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Components

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Protein , 5 types, 36 molecules 0369147Y258ZABCTUVWXDEFGHIJKLM...

#1: Protein
TROPONIN C, SKELETAL MUSCLE / / TNNC2


Mass: 17971.836 Da / Num. of mol.: 4 / Fragment: RESIDUES 5-163 / Source method: isolated from a natural source / Source: (natural) GALLUS GALLUS (chicken) / Tissue: SKELETAL MUSCLE / References: UniProt: P02588
#2: Protein
TROPONIN T, FAST SKELETAL MUSCLE ISOFORMS / / TNNT3


Mass: 10976.568 Da / Num. of mol.: 4 / Fragment: RESIDUES 148-237 / Source method: isolated from a natural source / Source: (natural) GALLUS GALLUS (chicken) / Tissue: SKELETAL MUSCLE / References: UniProt: P12620
#3: Protein
TROPONIN I, FAST SKELETAL MUSCLE / / TROPONIN I / FAST-TWITCH ISOFORM / TNNI2


Mass: 16304.978 Da / Num. of mol.: 4 / Fragment: RESIDUES 4-144 / Source method: isolated from a natural source / Source: (natural) GALLUS GALLUS (chicken) / Tissue: SKELETAL MUSCLE / References: UniProt: P68246
#4: Protein
TROPOMYOSIN ALPHA-1 CHAIN / TROPOMYOSIN-1 / TPM1 / TPMA / ALPHA-TROPOMYOSIN


Mass: 31917.555 Da / Num. of mol.: 8 / Fragment: RESIDUES 8-284 / Source method: isolated from a natural source / Source: (natural) ORYCTOLAGUS CUNICULUS (rabbit) / Tissue: SKELETAL MUSCLE / References: UniProt: P58772
#5: Protein
ACTIN, ALPHA SKELETAL MUSCLE / / ALPHA-ACTIN-1 / ACTA1 / ACTA


Mass: 41483.117 Da / Num. of mol.: 16 / Fragment: RESIDUES 3-374 / Source method: isolated from a natural source / Source: (natural) ORYCTOLAGUS CUNICULUS (rabbit) / Tissue: SKELETAL MUSCLE / References: UniProt: P68135

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Non-polymers , 1 types, 16 molecules

#6: Chemical
ChemComp-CA / CALCIUM ION


Mass: 40.078 Da / Num. of mol.: 16 / Source method: obtained synthetically / Formula: Ca

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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 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

EM software
IDNameCategory
1IMOD3D reconstruction
2PROTOMO3D reconstruction
3D reconstructionMethod: MARKER FREE ALIGNMENT / Resolution 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 FIT TO AVERAGED SUBVOLUMES OBTAINED FROM A DUAL ...Details: NOTE THAT OUR LOWEST RESOLUTION DATA IS AT INVERSE 1 MICRON. NUMBER OF FOURIER COEFFICIENTS IS ALMOST A HALF MILLION. THESE COORDINATES WERE FIT TO AVERAGED SUBVOLUMES OBTAINED FROM A DUAL AXIS TOMOGRAM. THE ACTIN FILAMENT WAS GENERATED TO HAVE THE HELICAL SYMMETRY OF 28 SUBUNITS IN 13 TURNS OF THE 5.9 NM HELIX. 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. atoms69360 0 16 0 69376

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