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- PDB-3j4k: Cryo-EM structures of the actin:tropomyosin filament reveal the m... -

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Basic information

Entry
Database: PDB / ID: 3j4k
TitleCryo-EM structures of the actin:tropomyosin filament reveal the mechanism for the transition from C- to M-state
Components
  • Actin, alpha skeletal muscle
  • tropomyosin
KeywordsSTRUCTURAL PROTEIN / actin / tropomyosin / coiled-coil C-state
Function / homology
Function and homology information


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 ...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 / actin monomer binding / skeletal muscle fiber development / stress fiber / titin binding / 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 / lamellipodium / cell body / hydrolase activity / protein domain specific binding / calcium ion binding / positive regulation of gene expression / magnesium ion binding / ATP binding / identical protein binding / cytoplasm
Similarity search - Function
Actins signature 1. / Actin, conserved site / Actins signature 2. / Actin/actin-like conserved site / Actins and actin-related proteins signature. / Actin / Actin family / Actin / ATPase, nucleotide binding domain
Similarity search - Domain/homology
ADENOSINE-5'-DIPHOSPHATE / Actin, alpha skeletal muscle
Similarity search - Component
Biological speciesOryctolagus cuniculus (rabbit)
Gallus gallus (chicken)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 8 Å
AuthorsSousa, D.R. / Stagg, S.M. / Stroupe, M.E.
CitationJournal: J Mol Biol / Year: 2013
Title: Cryo-EM structures of the actin:tropomyosin filament reveal the mechanism for the transition from C- to M-state.
Authors: Duncan R Sousa / Scott M Stagg / M Elizabeth Stroupe /
Abstract: Tropomyosin (Tm) is a key factor in the molecular mechanisms that regulate the binding of myosin motors to actin filaments (F-Actins) in most eukaryotic cells. This regulation is achieved by the ...Tropomyosin (Tm) is a key factor in the molecular mechanisms that regulate the binding of myosin motors to actin filaments (F-Actins) in most eukaryotic cells. This regulation is achieved by the azimuthal repositioning of Tm along the actin (Ac):Tm:troponin (Tn) thin filament to block or expose myosin binding sites on Ac. In striated muscle, including involuntary cardiac muscle, Tm regulates muscle contraction by coupling Ca(2+) binding to Tn with myosin binding to the thin filament. In smooth muscle, the switch is the posttranslational modification of the myosin. Depending on the activation state of Tn and the binding state of myosin, Tm can occupy the blocked, closed, or open position on Ac. Using native cryogenic 3DEM (three-dimensional electron microscopy), we have directly resolved and visualized cardiac and gizzard muscle Tm on filamentous Ac in the position that corresponds to the closed state. From the 8-Å-resolution structure of the reconstituted Ac:Tm filament formed with gizzard-derived Tm, we discuss two possible mechanisms for the transition from closed to open state and describe the role Tm plays in blocking myosin tight binding in the closed-state position.
History
DepositionAug 26, 2013Deposition site: RCSB / Processing site: RCSB
Revision 1.0Sep 25, 2013Provider: repository / Type: Initial release
Revision 1.1Nov 13, 2013Group: Database references
Revision 1.2Jul 18, 2018Group: Author supporting evidence / Data collection
Category: em_image_scans / em_single_particle_entity / em_software
Item: _em_software.image_processing_id
Revision 1.3Feb 21, 2024Group: Data collection / Database references ...Data collection / Database references / Derived calculations / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / em_3d_fitting_list / pdbx_initial_refinement_model / struct_site
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id / _em_3d_fitting_list.source_name / _em_3d_fitting_list.type / _struct_site.pdbx_auth_asym_id / _struct_site.pdbx_auth_comp_id / _struct_site.pdbx_auth_seq_id

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Structure visualization

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  • Deposited structure unit
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  • Simplified surface model + fitted atomic model
  • EMDB-5751
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  • Superimposition on EM map
  • EMDB-5751
  • Imaged by UCSF Chimera
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Structure viewerMolecule:
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Assembly

Deposited unit
A: Actin, alpha skeletal muscle
B: Actin, alpha skeletal muscle
C: Actin, alpha skeletal muscle
D: Actin, alpha skeletal muscle
E: Actin, alpha skeletal muscle
F: tropomyosin
G: tropomyosin
hetero molecules


Theoretical massNumber of molelcules
Total (without water)234,63412
Polymers232,4987
Non-polymers2,1365
Water0
1


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

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Components

#1: Protein
Actin, alpha skeletal muscle / / Alpha-actin-1


Mass: 41862.613 Da / Num. of mol.: 5 / Source method: isolated from a natural source / Source: (natural) Oryctolagus cuniculus (rabbit) / References: UniProt: P68135
#2: Protein tropomyosin /


Mass: 11592.281 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Source: (natural) Gallus gallus (chicken) / Organ: gizzard
#3: Chemical
ChemComp-ADP / ADENOSINE-5'-DIPHOSPHATE / Adenosine diphosphate


Mass: 427.201 Da / Num. of mol.: 5 / Source method: obtained synthetically / Formula: C10H15N5O10P2 / Comment: ADP, energy-carrying molecule*YM

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Experimental details

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Experiment

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

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Sample preparation

ComponentName: F-actin/tropomyosin / Type: COMPLEX
Buffer solutionName: 70 mM NaCl, 3 mM MgCl2, 0.2 mM EGTA, 5 mM NaH2PO4, 5 mM PIPES buffer
pH: 7.5
Details: 70 mM NaCl, 3 mM MgCl2, 0.2 mM EGTA, 5 mM NaH2PO4, 5 mM PIPES buffer
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: glow discharged Quantifoil 2/2 perforated carbon copper grids
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 293 K
Details: 3 second blot before plunging into liquid ethane (FEI Vitrobot Mark IV)
Method: 3 second blot

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS / Date: Mar 2, 2010
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 59000 X / Calibrated magnification: 101555 X / Nominal defocus max: 4000 nm / Nominal defocus min: 1500 nm / Cs: 2 mm
Specimen holderTemperature: 90 K / Tilt angle max: 0 ° / Tilt angle min: 0 °
Image recordingElectron dose: 30 e/Å2 / Film or detector model: GATAN ULTRASCAN 4000 (4k x 4k)

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Processing

EM software
IDNameCategoryDetails
1UCSF Chimeramodel fittingfit-in-map
2EMAN3D reconstruction
3MATLAB3D reconstruction
4SPIDER3D reconstruction
CTF correctionDetails: individual images
Helical symmertyAngular rotation/subunit: 167 ° / Axial rise/subunit: 28 Å / Axial symmetry: C1
3D reconstructionMethod: IHRSR / Resolution: 8 Å / Resolution method: FSC 0.5 CUT-OFF / Num. of particles: 224337 / Nominal pixel size: 1.5 Å / Actual pixel size: 1.477 Å / Magnification calibration: helical diffraction / Symmetry type: HELICAL
Atomic model buildingProtocol: RIGID BODY FIT / Space: REAL / Target criteria: Cross correlation
Details: REFINEMENT PROTOCOL--RIGID BODY DETAILS--The F-actin and tropomyosin were fitted separately.
Atomic model building

3D fitting-ID: 1 / Accession code: 4A7F / Initial refinement model-ID: 1 / PDB-ID: 4A7F

4a7f

/ Source name: PDB / Type: experimental model

IDPdb chain-ID
1A
2B
3D
4E
5F
6H
7I
Refinement stepCycle: LAST
ProteinNucleic acidLigandSolventTotal
Num. atoms16025 0 135 0 16160

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