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Yorodumi- PDB-2w4u: Isometrically contracting insect asynchronous flight muscle quick... -
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-Basic information
Entry | Database: PDB / ID: 2w4u | ||||||
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Title | Isometrically contracting insect asynchronous flight muscle quick frozen after a length step | ||||||
Components |
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Keywords | CONTRACTILE PROTEIN / METHYLATION / ATP-BINDING / ISOMETRIC CONTRACTION / MICROTOMY / FREEZE SUBSTITUTION / MUSCLE PROTEIN / CALMODULIN-BINDING / MOTOR PROTEIN / ACTIN-BINDING | ||||||
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 | ||||||
Biological species | GALLUS GALLUS (chicken) ORYCTOLAGUS CUNICULUS (rabbit) | ||||||
Method | ELECTRON MICROSCOPY / helical reconstruction / Resolution: 35 Å | ||||||
Authors | Wu, 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 |
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-Structure visualization
Movie |
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Structure viewer | Molecule: MolmilJmol/JSmol |
-Downloads & links
-Download
PDBx/mmCIF format | 2w4u.cif.gz | 1.6 MB | Display | PDBx/mmCIF format |
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PDB format | pdb2w4u.ent.gz | 1.3 MB | Display | PDB format |
PDBx/mmJSON format | 2w4u.json.gz | Tree view | PDBx/mmJSON format | |
Others | Other downloads |
-Validation report
Arichive directory | https://data.pdbj.org/pub/pdb/validation_reports/w4/2w4u ftp://data.pdbj.org/pub/pdb/validation_reports/w4/2w4u | HTTPS FTP |
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-Related structure data
Related structure data | 1584MC 1585MC 2w4hC 2w4vC 2w4wC M: map data used to model this data C: citing same article (ref.) |
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Similar structure data |
-Links
-Assembly
Deposited unit |
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-Components
#1: Protein | Mass: 17971.836 Da / Num. of mol.: 4 / Fragment: RESIDUES 5-163 / Source method: isolated from a natural source / Source: (natural) GALLUS GALLUS (chicken) / References: UniProt: P02588 #2: Protein | Mass: 10976.568 Da / Num. of mol.: 4 / Fragment: RESIDUES 148-237 / Source method: isolated from a natural source / Source: (natural) GALLUS GALLUS (chicken) / References: UniProt: P12620 #3: Protein | Mass: 16304.978 Da / Num. of mol.: 4 / Fragment: RESIDUES 4-144 / Source method: isolated from a natural source / Source: (natural) GALLUS GALLUS (chicken) / References: UniProt: P68246 #4: Protein | Mass: 31917.555 Da / Num. of mol.: 8 / Fragment: RESIDUES 8-284 / Source method: isolated from a natural source / Source: (natural) ORYCTOLAGUS CUNICULUS (rabbit) / References: UniProt: P58772 #5: Protein | 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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-Experimental details
-Experiment
Experiment | Method: ELECTRON MICROSCOPY |
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EM experiment | Aggregation state: FILAMENT / 3D reconstruction method: helical reconstruction |
-Sample preparation
Component | Name: 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. |
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Buffer solution | Name: 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 |
Specimen | Embedding applied: YES / Shadowing applied: NO / Staining applied: NO / Vitrification applied: NO |
Specimen support | Details: CARBON |
Vitrification | Instrument: HOMEMADE PLUNGER / Cryogen name: HELIUM Details: SMASH AGAINST A LIQUID HELIUM COOLED GOLD COATED COPPER MIRROR |
-Electron microscopy imaging
Microscopy | Model: FEI/PHILIPS CM300FEG/T / Details: NONE |
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Electron gun | Electron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM |
Electron lens | Mode: BRIGHT FIELDBright-field microscopy / Cs: 2 mm |
Specimen holder | Tilt angle max: 72 ° / Tilt angle min: -72 ° |
Image recording | Film or detector model: TVIPS TEMCAM-F224 (2k x 2k) |
Radiation wavelength | Relative weight: 1 |
-Processing
3D reconstruction | 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 | ||||||||||||
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Refinement | Highest resolution: 35 Å | ||||||||||||
Refinement step | Cycle: LAST / Highest resolution: 35 Å
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