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- PDB-8efh: Helical reconstruction of the human cardiac actin-tropomyosin-myo... -

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Basic information

Entry
Database: PDB / ID: 8efh
TitleHelical reconstruction of the human cardiac actin-tropomyosin-myosin complex in complex with ADP-Mg2+
Components
  • Actin, alpha cardiac muscle 1
  • Tropomyosin alpha-1 chain
  • beta-cardiac myosin II
KeywordsMOTOR PROTEIN / actin / tropomyosin / myosin / cardiac
Function / homology
Function and homology information


positive regulation of heart rate by epinephrine / muscle thin filament tropomyosin / regulation of slow-twitch skeletal muscle fiber contraction / RHOB GTPase cycle / Striated Muscle Contraction / regulation of the force of skeletal muscle contraction / RHOA GTPase cycle / actin-myosin filament sliding / muscle myosin complex / bleb ...positive regulation of heart rate by epinephrine / muscle thin filament tropomyosin / regulation of slow-twitch skeletal muscle fiber contraction / RHOB GTPase cycle / Striated Muscle Contraction / regulation of the force of skeletal muscle contraction / RHOA GTPase cycle / actin-myosin filament sliding / muscle myosin complex / bleb / negative regulation of vascular associated smooth muscle cell migration / regulation of muscle contraction / muscle filament sliding / regulation of the force of heart contraction / transition between fast and slow fiber / myosin filament / ruffle organization / myosin II complex / adult heart development / cardiac muscle hypertrophy in response to stress / Striated Muscle Contraction / positive regulation of ATP-dependent activity / myosin complex / regulation of heart contraction / structural constituent of muscle / sarcomere organization / microfilament motor activity / ventricular cardiac muscle tissue morphogenesis / myosin binding / myofibril / mesenchyme migration / heart contraction / negative regulation of vascular associated smooth muscle cell proliferation / skeletal muscle contraction / positive regulation of cell adhesion / striated muscle contraction / Smooth Muscle Contraction / stress fiber / ATP metabolic process / cytoskeleton organization / cardiac muscle contraction / positive regulation of stress fiber assembly / cytoskeletal protein binding / regulation of heart rate / sarcomere / negative regulation of cell migration / filopodium / muscle contraction / actin filament organization / actin filament / wound healing / structural constituent of cytoskeleton / ruffle membrane / Z disc / cellular response to reactive oxygen species / actin filament binding / actin cytoskeleton / lamellipodium / cell body / actin binding / regulation of cell shape / cytoskeleton / calmodulin binding / protein heterodimerization activity / positive regulation of gene expression / 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. / 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 / 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 / Actin, alpha cardiac muscle 1 / Tropomyosin alpha-1 chain / Myosin-7
Similarity search - Component
Biological speciesHomo sapiens (human)
Sus scrofa (pig)
MethodELECTRON MICROSCOPY / helical reconstruction / cryo EM / Resolution: 3.3 Å
AuthorsDoran, M.H. / Lehman, W. / Rynkiewicz, M.J.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Heart, Lung, and Blood Institute (NIH/NHLBI)R01HL036153 United States
CitationJournal: J Gen Physiol / Year: 2023
Title: Conformational changes linked to ADP release from human cardiac myosin bound to actin-tropomyosin.
Authors: Matthew H Doran / Michael J Rynkiewicz / David Rasicci / Skylar M L Bodt / Meaghan E Barry / Esther Bullitt / Christopher M Yengo / Jeffrey R Moore / William Lehman /
Abstract: Following binding to the thin filament, β-cardiac myosin couples ATP-hydrolysis to conformational rearrangements in the myosin motor that drive myofilament sliding and cardiac ventricular ...Following binding to the thin filament, β-cardiac myosin couples ATP-hydrolysis to conformational rearrangements in the myosin motor that drive myofilament sliding and cardiac ventricular contraction. However, key features of the cardiac-specific actin-myosin interaction remain uncertain, including the structural effect of ADP release from myosin, which is rate-limiting during force generation. In fact, ADP release slows under experimental load or in the intact heart due to the afterload, thereby adjusting cardiac muscle power output to meet physiological demands. To further elucidate the structural basis of this fundamental process, we used a combination of cryo-EM reconstruction methodologies to determine structures of the human cardiac actin-myosin-tropomyosin filament complex at better than 3.4 Å-resolution in the presence and in the absence of Mg2+·ADP. Focused refinements of the myosin motor head and its essential light chains in these reconstructions reveal that small changes in the nucleotide-binding site are coupled to significant rigid body movements of the myosin converter domain and a 16-degree lever arm swing. Our structures provide a mechanistic framework to understand the effect of ADP binding and release on human cardiac β-myosin, and offer insights into the force-sensing mechanism displayed by the cardiac myosin motor.
History
DepositionSep 8, 2022Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jan 11, 2023Provider: repository / Type: Initial release
Revision 1.1Jan 25, 2023Group: Database references / Category: citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.journal_volume / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: beta-cardiac myosin II
F: Actin, alpha cardiac muscle 1
B: Actin, alpha cardiac muscle 1
C: Actin, alpha cardiac muscle 1
D: Actin, alpha cardiac muscle 1
E: Actin, alpha cardiac muscle 1
O: Tropomyosin alpha-1 chain
P: Tropomyosin alpha-1 chain
hetero molecules


Theoretical massNumber of molelcules
Total (without water)377,71820
Polymers375,0098
Non-polymers2,70912
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: microscopy, The assembly forms filaments under cryoelectron microscopy images.
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein beta-cardiac myosin II / 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: 99157.695 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: MYH7, MYHCB / Cell line (production host): C2C12 / Production host: Mus musculus (house mouse) / References: UniProt: P12883
#2: Protein
Actin, alpha cardiac muscle 1 / / Cardiac muscle alpha actin 1


Mass: 42064.891 Da / Num. of mol.: 5 / Source method: isolated from a natural source / Source: (natural) Sus scrofa (pig) / References: UniProt: B6VNT8
#3: 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
Source: (gene. exp.) Homo sapiens (human) / Gene: TPM1, C15orf13, TMSA / Production host: Escherichia coli (E. coli) / References: UniProt: P09493
#4: Chemical
ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 6 / Source method: obtained synthetically / Formula: Mg / Feature type: SUBJECT OF INVESTIGATION
#5: Chemical
ChemComp-ADP / ADENOSINE-5'-DIPHOSPHATE / Adenosine diphosphate


Mass: 427.201 Da / Num. of mol.: 6 / Source method: obtained synthetically / Formula: C10H15N5O10P2 / Feature type: SUBJECT OF INVESTIGATION / Comment: ADP, energy-carrying molecule*YM
Has ligand of interestY

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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

Component
IDNameTypeDetailsEntity IDParent-IDSource
1Human cardiac actin-tropomyosin-beta-myosin II complex bound to ADPMg2+.COMPLEXCardiac actomyosin-tropomyosin complex with ADPMg2+ bound to the myosin motor head.#1-#30MULTIPLE SOURCES
2Cardiac F-actin complexCOMPLEXF-actin forms the backbone of the complex#21NATURAL
3Human beta-cardiac myosin IICOMPLEXThe motor domain of the myosin saturates the actin filament.#11RECOMBINANT
4Human cardiac tropomyosinCOMPLEXTropomyosin wraps around the F-actin core.#31RECOMBINANT
Molecular weight
IDEntity assembly-IDExperimental value
11NO
21NO
31NO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
32Sus scrofa (pig)9823
43Homo sapiens (human)9606
54Homo sapiens (human)9606
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-IDCell
43Mus musculus (house mouse)10090C2C12
54Escherichia coli (E. coli)562
Buffer solutionpH: 7
SpecimenConc.: 0.13 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: 15 mA was used in the Pelco Easiglow glow discharge machine
Grid material: GOLD / Grid mesh size: 400 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK III / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 283 K

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: TFS KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: SPOT SCAN
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 80000 X / Nominal defocus max: 3000 nm / Nominal defocus min: 700 nm / Cs: 2.7 mm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 3.12 sec. / Electron dose: 53.7 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) / Num. of grids imaged: 4 / Num. of real images: 3961

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Processing

SoftwareName: PHENIX / Version: 1.19.1_4122: / Classification: refinement
EM software
IDNameVersionCategory
1RELION3.1.1particle selection
2Leginonimage acquisition
4CTFFINDCTF correction
7PHENIX1.18model fitting
9PHENIX1.18model refinement
10RELION3.1.1initial Euler assignment
11RELION3.1.1final Euler assignment
13RELION3D reconstruction
CTF correctionType: PHASE FLIPPING ONLY
Helical symmertyAngular rotation/subunit: -166.8 ° / Axial rise/subunit: 27.9 Å / Axial symmetry: C1
Particle selectionNum. of particles selected: 628787
3D reconstructionResolution: 3.3 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 131194 / Algorithm: BACK PROJECTION / Num. of class averages: 1 / Symmetry type: HELICAL
Atomic model buildingProtocol: OTHER / Space: REAL
Atomic model buildingPDB-ID: 6X5Z

6x5z

Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00223710
ELECTRON MICROSCOPYf_angle_d0.52932064
ELECTRON MICROSCOPYf_dihedral_angle_d4.4133249
ELECTRON MICROSCOPYf_chiral_restr0.0413546
ELECTRON MICROSCOPYf_plane_restr0.0044128

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