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- PDB-6x5z: Bovine Cardiac Myosin in Complex with Chicken Skeletal Actin and ... -

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Basic information

Entry
Database: PDB / ID: 6x5z
TitleBovine Cardiac Myosin in Complex with Chicken Skeletal Actin and Human Cardiac Tropomyosin in the Rigor State
Components
  • Actin, alpha skeletal muscle
  • Myosin-7
  • Tropomyosin alpha-1 chain
KeywordsCONTRACTILE PROTEIN / myosin / tropomyosin / actin / cardiac
Function / homology
Function and homology information


positive regulation of heart rate by epinephrine / muscle thin filament tropomyosin / bleb / Striated Muscle Contraction / negative regulation of vascular associated smooth muscle cell migration / regulation of muscle contraction / muscle filament sliding / myosin filament / ruffle organization / myosin II complex ...positive regulation of heart rate by epinephrine / muscle thin filament tropomyosin / bleb / Striated Muscle Contraction / negative regulation of vascular associated smooth muscle cell migration / regulation of muscle contraction / muscle filament sliding / myosin filament / ruffle organization / myosin II complex / adult heart development / Striated Muscle Contraction / positive regulation of ATP-dependent activity / regulation of heart contraction / structural constituent of muscle / sarcomere organization / microfilament motor activity / ventricular cardiac muscle tissue morphogenesis / myofibril / negative regulation of vascular associated smooth muscle cell proliferation / skeletal muscle thin filament assembly / striated muscle thin filament / positive regulation of cell adhesion / Smooth Muscle Contraction / skeletal muscle fiber development / stress fiber / cytoskeleton organization / cardiac muscle contraction / positive regulation of stress fiber assembly / cytoskeletal protein binding / sarcomere / negative regulation of cell migration / actin filament organization / actin filament / Hydrolases; Acting on acid anhydrides; Acting on acid anhydrides to facilitate cellular and subcellular movement / wound healing / structural constituent of cytoskeleton / ruffle membrane / cellular response to reactive oxygen species / actin filament binding / actin cytoskeleton / actin binding / regulation of cell shape / cytoskeleton / calmodulin binding / hydrolase activity / protein heterodimerization activity / protein homodimerization activity / ATP binding / identical protein binding / cytosol / cytoplasm
Similarity search - Function
Tropomyosins signature. / Tropomyosin / Tropomyosin / 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. ...Tropomyosins signature. / Tropomyosin / Tropomyosin / 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. / Myosin head, motor domain / Myosin head (motor domain) / Myosin motor domain profile. / Myosin. Large ATPases. / IQ motif profile. / IQ motif, EF-hand binding site / Actins signature 1. / Actin, conserved site / Actins signature 2. / Kinesin motor domain superfamily / Actin/actin-like conserved site / Actins and actin-related proteins signature. / Actin / Actin family / Actin / ATPase, nucleotide binding domain / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
ADENOSINE-5'-DIPHOSPHATE / Tropomyosin alpha-1 chain / Actin, alpha skeletal muscle / Myosin-7
Similarity search - Component
Biological speciesHomo sapiens (human)
Gallus gallus (chicken)
Bos taurus (cattle)
MethodELECTRON MICROSCOPY / helical reconstruction / cryo EM / Resolution: 4.24 Å
AuthorsDoran, M.H. / Lehman, W. / Rynkiewicz, M.J. / Bullitt, E.
Funding support United States, European Union, 7items
OrganizationGrant numberCountry
National Institutes of Health/National Heart, Lung, and Blood Institute (NIH/NHLBI)R01HL036153 United States
National Institutes of Health/National Heart, Lung, and Blood Institute (NIH/NHLBI)R01HL123774 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)S10RR25434 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)U24 GM129541 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01GM029090 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)U24GM116787 United States
Horizon 2020 Research and Innovation Programme777204 SILICOFCMEuropean Union
CitationJournal: Biophys J / Year: 2020
Title: Cryo-EM and Molecular Docking Shows Myosin Loop 4 Contacts Actin and Tropomyosin on Thin Filaments.
Authors: Matthew H Doran / Elumalai Pavadai / Michael J Rynkiewicz / Jonathan Walklate / Esther Bullitt / Jeffrey R Moore / Michael Regnier / Michael A Geeves / William Lehman /
Abstract: The motor protein myosin drives muscle and nonmuscle motility by binding to and moving along actin of thin filaments. Myosin binding to actin also modulates interactions of the regulatory protein, ...The motor protein myosin drives muscle and nonmuscle motility by binding to and moving along actin of thin filaments. Myosin binding to actin also modulates interactions of the regulatory protein, tropomyosin, on thin filaments, and conversely tropomyosin affects myosin binding to actin. Insight into this reciprocity will facilitate a molecular level elucidation of tropomyosin regulation of myosin interaction with actin in muscle contraction, and in turn, promote better understanding of nonmuscle cell motility. Indeed, experimental approaches such as fiber diffraction, cryoelectron microscopy, and three-dimensional reconstruction have long been used to define regulatory interaction of tropomyosin and myosin on actin at a structural level. However, their limited resolution has not proven sufficient to determine tropomyosin and myosin contacts at an atomic-level and thus to fully substantiate possible functional contributions. To overcome this deficiency, we have followed a hybrid approach by performing new cryogenic electron microscopy reconstruction of myosin-S1-decorated F-actin-tropomyosin together with atomic scale protein-protein docking of tropomyosin to the EM models. Here, cryo-EM data were derived from filaments reconstituted with α1-actin, cardiac αα-tropomyosin, and masseter muscle β-myosin complexes; masseter myosin, which shares sequence identity with β-cardiac myosin-heavy chain, was used because of its stability in vitro. The data were used to build an atomic model of the tropomyosin cable that fits onto the actin filament between the tip of the myosin head and a cleft on the innermost edge of actin subunits. The docking and atomic scale fitting showed multiple discrete interactions of myosin loop 4 and acidic residues on successive 39-42 residue-long tropomyosin pseudorepeats. The contacts between S1 and tropomyosin on actin appear to compete with and displace ones normally found between actin and tropomyosin on myosin-free thin filaments in relaxed muscle, thus restructuring the filament during myosin-induced activation.
History
DepositionMay 27, 2020Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jul 22, 2020Provider: repository / Type: Initial release
Revision 1.1Aug 12, 2020Group: Database references / Category: citation / Item: _citation.pdbx_database_id_PubMed / _citation.title
Revision 1.2Sep 2, 2020Group: Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first / _citation.page_last
Revision 1.3Mar 6, 2024Group: Data collection / Database references / 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
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

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Structure visualization

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  • Deposited structure unit
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  • Simplified surface model + fitted atomic model
  • EMDB-22067
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  • Superimposition on EM map
  • EMDB-22067
  • Imaged by UCSF Chimera
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Structure viewerMolecule:
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Assembly

Deposited unit
B: Actin, alpha skeletal muscle
O: Tropomyosin alpha-1 chain
G: Myosin-7
P: Tropomyosin alpha-1 chain
A: Actin, alpha skeletal muscle
D: Myosin-7
C: Actin, alpha skeletal muscle
J: Myosin-7
hetero molecules


Theoretical massNumber of molelcules
Total (without water)485,51314
Polymers484,1598
Non-polymers1,3556
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: microscopy
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area25960 Å2
ΔGint-220 kcal/mol
Surface area161510 Å2
SymmetryHelical symmetry: (Circular symmetry: 1 / Dyad axis: no / N subunits divisor: 1 / Num. of operations: 2 / Rise per n subunits: 27.5 Å / Rotation per n subunits: -166.4 °)

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Components

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


Mass: 42096.953 Da / Num. of mol.: 3 / Source method: isolated from a natural source
Details: Residues 1-9 and 377 were disordered and were not modeled.
Source: (natural) Gallus gallus (chicken) / Tissue: Breast Skeletal Muscle / References: UniProt: P68139
#2: Protein Tropomyosin alpha-1 chain / Alpha-tropomyosin / Tropomyosin-1


Mass: 32763.621 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Details: Only the central section (residues 45-210) was modeled.
Source: (gene. exp.) Homo sapiens (human) / Gene: TPM1, C15orf13, TMSA / Production host: Escherichia coli (E. coli) / References: UniProt: P09493
#3: Protein Myosin-7 / / Myosin heavy chain 7 / Myosin heavy chain slow isoform / MyHC-slow / Myosin heavy chain / cardiac ...Myosin heavy chain 7 / Myosin heavy chain slow isoform / MyHC-slow / Myosin heavy chain / cardiac muscle beta isoform / MyHC-beta


Mass: 97446.898 Da / Num. of mol.: 3 / Fragment: S1 fragment (UNP residues 1-850) / Source method: isolated from a natural source
Details: Residues 1-35, 200-216, 624-641, and 777-850 were disordered and not modeled.
Source: (natural) Bos taurus (cattle) / Tissue: Masseter Muscle / References: UniProt: Q9BE39
#4: Chemical ChemComp-ADP / ADENOSINE-5'-DIPHOSPHATE / Adenosine diphosphate


Mass: 427.201 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: C10H15N5O10P2 / Comment: ADP, energy-carrying molecule*YM
#5: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: Mg
Has ligand of interestN

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: HELICAL ARRAY / 3D reconstruction method: helical reconstruction

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Sample preparation

Component
IDNameTypeDetailsEntity IDParent-IDSource
1Helical complex of bovine S1 cardiac myosin and human cardiac tropomyosin-decorated chicken skeletal actin filamentsCOMPLEXBovine myosin was isolated from the masseter muscle and the S1 fragment was generated through proteolytic cleavage.#1-#30MULTIPLE SOURCES
2skeletal actin filamentsORGANELLE OR CELLULAR COMPONENT#11NATURAL
3S1 cardiac myosinCOMPLEX#21NATURAL
4cardiac tropomyosinCOMPLEX#31RECOMBINANT
Molecular weightValue: 0.40322 MDa / Experimental value: NO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-IDTissue
12Gallus gallus (chicken)9031Breast Skeletal Muscle
23Bos taurus (cattle)9913Masseter Muscle
34Homo sapiens (human)9606
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 7
Buffer component
IDConc.NameFormulaBuffer-ID
150 mMSodium AcetateCH3COONa1
23 mMMagnesium ChlorideMgCl21
31 mMDithiothreitol1
410 nMHEPES1
SpecimenConc.: 0.525 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: Glow discharged using a PELCO easiGlow station / Grid material: COPPER / Grid mesh size: 200 divisions/in. / Grid type: Quantifoil R2/1
VitrificationInstrument: FEI VITROBOT MARK III / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 283 K
Details: Thin filaments were reconstituted by first mixing F-actin and tropomyosin to final concentrations of 10 uM actin and 7 uM tropomyosin. Just before applying 1.5 uL actin-tropomyosin to a ...Details: Thin filaments were reconstituted by first mixing F-actin and tropomyosin to final concentrations of 10 uM actin and 7 uM tropomyosin. Just before applying 1.5 uL actin-tropomyosin to a freshly glow discharged holey-carbon grid, the surfactant octyl B-D-glucopyranoside was added to the protein solution to a concentration of 12 nM. The grid sample was manually blotted for 1 second and a 1.5 uL drop of 7.5 uM myosin-S1 sub-fragment was then applied to the blotted grid sample. The sample was immediately blotted for 4 seconds and plunge-frozen in liquid ethane.

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: SPOT SCAN
Electron lensMode: BRIGHT FIELDBright-field microscopy / Calibrated magnification: 130000 X / Nominal defocus max: 2500 nm / Nominal defocus min: 1500 nm
Specimen holderSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 7 sec. / Electron dose: 53 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 2496
EM imaging opticsEnergyfilter name: In-column Omega Filter / Energyfilter slit width: 20 eV
Image scansWidth: 3838 / Height: 3710 / Movie frames/image: 35 / Used frames/image: 1-35

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Processing

EM software
IDNameVersionCategory
1RELION3.0.7particle selection
2EPUimage acquisition
4CTFFIND4.1CTF correction
9RELION3.0.7initial Euler assignment
10RELION3.0.7final Euler assignment
12RELION3D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Helical symmertyAngular rotation/subunit: -166.4 ° / Axial rise/subunit: 27.5 Å / Axial symmetry: C1
Particle selectionNum. of particles selected: 45040
3D reconstructionResolution: 4.24 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 32158 / Details: Performed in the Relion post-processing step / Symmetry type: HELICAL
Atomic model building
IDMethodProtocolSpaceDetailsB value
1flexible fittingFLEXIBLE FITREALversion 1.9.3; initial flexible fitting to align secondary structure using MDFF
2real space refinementOTHERREALversion 1.18-386170.88
3manual refinementOTHERREALmanual refinement of residues to match backbone and sidechain density and to limit clashes, sidechain outliers, and Ramachandran outliers
Atomic model building

3D fitting-ID: 1 / Source name: PDB / Type: experimental model

IDPDB-IDAccession codeDetailsInitial refinement model-ID
16KN8

6kn8

6KN8The F-actin structure was taken from the Yamada et al. map (PDB ID: 6KN8), fitted within its corresponding density of the masseter S1-decorated actin-tropomyosin reconstruction. A homology model of the beta-myosin S1 was then built based on the rigor-state crystal structure of squid muscle myosin S1 (PDB ID: 3I5G). These models were fit using molecular dynamics flexible fitting in order to align secondary structure elements using VMD 1.9.3. Next, we used Phenix’s Cryo-EM Real Space Refinement in order to refine the MDFF result. Finally, Coot was used to manually refine side chain orientations to reduce clashes, Ramachandran outliers, and sidechain outliers. The tropomyosin backbone was fit into its respective density using UCSF Chimera’s Fit in Map tool. The side chains of tropomyosin included in this structure are based on our extensive protein-protein docking studies found in the associated publication.1
23I5G

3i5g

3I5G2

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