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

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Basic information

Entry
Database: PDB / ID: 8efi
TitleHelical reconstruction of the human cardiac actin-tropomyosin-myosin complex in the rigor form
Components
  • Actin, alpha cardiac muscle 1
  • Myosin-7
  • Tropomyosin alpha-1 chain
KeywordsMOTOR PROTEIN / actin / tropomyosin / myosin / cardiac
Function / homology
Function and homology information


regulation of slow-twitch skeletal muscle fiber contraction / regulation of the force of skeletal muscle contraction / positive regulation of heart rate by epinephrine / muscle thin filament tropomyosin / bleb / actin-myosin filament sliding / muscle myosin complex / regulation of muscle contraction / regulation of the force of heart contraction / transition between fast and slow fiber ...regulation of slow-twitch skeletal muscle fiber contraction / regulation of the force of skeletal muscle contraction / positive regulation of heart rate by epinephrine / muscle thin filament tropomyosin / bleb / actin-myosin filament sliding / muscle myosin complex / regulation of muscle contraction / regulation of the force of heart contraction / transition between fast and slow fiber / myosin filament / ruffle organization / adult heart development / Striated Muscle Contraction / cardiac muscle hypertrophy in response to stress / muscle filament sliding / myosin complex / myosin II complex / structural constituent of muscle / sarcomere organization / ventricular cardiac muscle tissue morphogenesis / microfilament motor activity / heart contraction / myosin binding / regulation of heart contraction / negative regulation of vascular associated smooth muscle cell migration / myofibril / mesenchyme migration / negative regulation of vascular associated smooth muscle cell proliferation / Smooth Muscle Contraction / skeletal muscle contraction / striated muscle contraction / ATP metabolic process / cytoskeletal protein binding / cardiac muscle contraction / stress fiber / positive regulation of stress fiber assembly / cytoskeleton organization / positive regulation of cell adhesion / muscle contraction / regulation of heart rate / actin filament organization / negative regulation of cell migration / sarcomere / cellular response to reactive oxygen species / filopodium / actin filament / wound healing / structural constituent of cytoskeleton / ruffle membrane / Z disc / actin filament binding / regulation of cell shape / lamellipodium / actin cytoskeleton / actin binding / cell body / cytoskeleton / calmodulin binding / protein heterodimerization activity / positive regulation of gene expression / protein homodimerization activity / ATP binding / identical protein binding / cytoplasm / cytosol
Similarity search - Function
Methane Monooxygenase Hydroxylase; Chain G, domain 1 - #60 / 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 ...Methane Monooxygenase Hydroxylase; Chain G, domain 1 - #60 / 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. / ATPase, substrate binding domain, subdomain 4 / Short calmodulin-binding motif containing conserved Ile and Gln residues. / IQ motif, EF-hand binding site / Myosin head, motor domain / Myosin head (motor domain) / Myosin motor domain profile. / Myosin. Large ATPases. / Actin; Chain A, domain 4 / IQ motif profile. / Kinesin motor domain superfamily / Actins signature 1. / Actin, conserved site / Actins signature 2. / Actin/actin-like conserved site / Actins and actin-related proteins signature. / Actin / Actin family / Actin / Methane Monooxygenase Hydroxylase; Chain G, domain 1 / ATPase, nucleotide binding domain / Alpha-Beta Complex / Up-down Bundle / P-loop containing nucleoside triphosphate hydrolase / Mainly Alpha / Alpha Beta
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: 3.4 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: Myosin loop-4 is critical for optimal tropomyosin repositioning on actin during muscle activation and relaxation.
Authors: Matthew H Doran / Michael J Rynkiewicz / Elumalai Pavadai / Skylar M L Bodt / David Rasicci / Jeffrey R Moore / Christopher M Yengo / Esther Bullitt / William Lehman /
Abstract: During force-generating steps of the muscle crossbridge cycle, the tip of the myosin motor, specifically loop-4, contacts the tropomyosin cable of actin filaments. In the current study, we determined ...During force-generating steps of the muscle crossbridge cycle, the tip of the myosin motor, specifically loop-4, contacts the tropomyosin cable of actin filaments. In the current study, we determined the corresponding effect of myosin loop-4 on the regulatory positioning of tropomyosin on actin. To accomplish this, we compared high-resolution cryo-EM structures of myosin S1-decorated thin filaments containing either wild-type or a loop-4 mutant construct, where the seven-residue portion of myosin loop-4 that contacts tropomyosin was replaced by glycine residues, thus removing polar side chains from residues 366-372. Cryo-EM analysis of fully decorated actin-tropomyosin filaments with wild-type and mutant S1, yielded 3.4-3.6 Å resolution reconstructions, with even higher definition at the actin-myosin interface. Loop-4 densities both in wild-type and mutant S1 were clearly identified, and side chains were resolved in the wild-type structure. Aside from loop-4, actin and myosin structural domains were indistinguishable from each other when filaments were decorated with either mutant or wild-type S1. In marked contrast, the position of tropomyosin on actin in the two reconstructions differed by 3 to 4 Å. In maps of filaments containing the mutant, tropomyosin was located closer to the myosin-head and thus moved in the direction of the C-state conformation adopted by myosin-free thin filaments. Complementary interaction energy measurements showed that tropomyosin in the mutant thin filaments sits on actin in a local energy minimum, whereas tropomyosin is positioned by wild-type S1 in an energetically unfavorable location. We propose that the high potential energy associated with tropomyosin positioning in wild-type filaments favors an effective transition to B- and C-states following release of myosin from the thin filaments during relaxation.
History
DepositionSep 8, 2022Deposition site: RCSB / Processing site: RCSB
Revision 1.0Nov 23, 2022Provider: repository / Type: Initial release
Revision 1.1Dec 14, 2022Group: Database references / Refinement description
Category: citation / citation_author / pdbx_initial_refinement_model
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
Revision 1.2Jun 19, 2024Group: Data collection / Refinement description
Category: chem_comp_atom / chem_comp_bond / em_3d_fitting_list
Item: _em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id ..._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
Revision 1.3Nov 13, 2024Group: Data collection / Structure summary
Category: em_admin / em_entity_assembly_molwt / pdbx_entry_details
Item: _em_admin.last_update / _em_entity_assembly_molwt.entity_assembly_id ..._em_admin.last_update / _em_entity_assembly_molwt.entity_assembly_id / _em_entity_assembly_molwt.id / _pdbx_entry_details.has_protein_modification

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
M: Myosin-7
B: Actin, alpha cardiac muscle 1
C: Actin, alpha cardiac muscle 1
D: Actin, alpha cardiac muscle 1
E: Actin, alpha cardiac muscle 1
F: Actin, alpha cardiac muscle 1
O: Tropomyosin alpha-1 chain
P: Tropomyosin alpha-1 chain
hetero molecules


Theoretical massNumber of molelcules
Total (without water)501,55518
Polymers499,2988
Non-polymers2,25810
Water00
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 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: 223445.984 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: MYH7, MYHCB / 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-ADP / ADENOSINE-5'-DIPHOSPHATE


Mass: 427.201 Da / Num. of mol.: 5 / 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.: 5 / Source method: obtained synthetically / Formula: Mg
Has ligand of interestN
Has protein modificationN

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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 in the rigor form.COMPLEXCardiac actomyosin-tropomyosin complex.#1-#30MULTIPLE SOURCES
2Cardiac F-actin complexCOMPLEXF-actin forms the backbone of the complex#21NATURAL
3Human 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
22NO
33
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 supportGrid material: GOLD / Grid mesh size: 200 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: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 80000 X / Nominal defocus max: 2000 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
Image scansWidth: 539 / Height: 539

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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
13RELION3.1.13D reconstruction
CTF correctionType: PHASE FLIPPING ONLY
Helical symmertyAngular rotation/subunit: -166.4 ° / Axial rise/subunit: 27.9 Å / Axial symmetry: C1
Particle selectionNum. of particles selected: 1045903
3D reconstructionResolution: 3.4 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 176178 / Symmetry type: HELICAL
Atomic model buildingProtocol: OTHER / Space: REAL
Atomic model buildingPDB-ID: 6X5Z

6x5z


Accession code: 6X5Z / Source name: PDB / Type: experimental model
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00323737
ELECTRON MICROSCOPYf_angle_d0.54832092
ELECTRON MICROSCOPYf_dihedral_angle_d4.2853291
ELECTRON MICROSCOPYf_chiral_restr0.0423549
ELECTRON MICROSCOPYf_plane_restr0.0044135

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