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- PDB-8r9v: CryoEM structure of the primed actomyosin-5a complex -

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Basic information

Entry
Database: PDB / ID: 8r9v
TitleCryoEM structure of the primed actomyosin-5a complex
Components
  • Actin, alpha skeletal muscle
  • Unconventional myosin-Va
KeywordsMOTOR PROTEIN / Myosin / Actin / Actomyosin / Primed actomyosin
Function / homology
Function and homology information


establishment of endoplasmic reticulum localization to postsynapse / regulation of postsynaptic cytosolic calcium ion concentration / melanosome localization / endoplasmic reticulum localization / locomotion involved in locomotory behavior / melanin metabolic process / vesicle transport along actin filament / unconventional myosin complex / insulin-responsive compartment / developmental pigmentation ...establishment of endoplasmic reticulum localization to postsynapse / regulation of postsynaptic cytosolic calcium ion concentration / melanosome localization / endoplasmic reticulum localization / locomotion involved in locomotory behavior / melanin metabolic process / vesicle transport along actin filament / unconventional myosin complex / insulin-responsive compartment / developmental pigmentation / melanosome transport / secretory granule localization / Regulation of MITF-M-dependent genes involved in pigmentation / Regulation of actin dynamics for phagocytic cup formation / actin filament-based movement / melanin biosynthetic process / hair follicle maturation / melanocyte differentiation / actomyosin / long-chain fatty acid biosynthetic process / myosin complex / insulin secretion / odontogenesis / intermediate filament / cytoskeletal motor activator activity / pigmentation / microfilament motor activity / myosin heavy chain binding / tropomyosin binding / troponin I binding / filamentous actin / exocytosis / mesenchyme migration / actin filament bundle / cytoskeletal motor activity / actin filament bundle assembly / skeletal muscle myofibril / striated muscle thin filament / skeletal muscle thin filament assembly / actin monomer binding / photoreceptor outer segment / smooth endoplasmic reticulum / stress fiber / skeletal muscle fiber development / vesicle-mediated transport / titin binding / actin filament polymerization / myelination / visual perception / secretory granule / protein localization to plasma membrane / filopodium / actin filament / synapse organization / small GTPase binding / Hydrolases; Acting on acid anhydrides; Acting on acid anhydrides to facilitate cellular and subcellular movement / cellular response to insulin stimulus / calcium-dependent protein binding / disordered domain specific binding / melanosome / lamellipodium / actin binding / cell body / chemical synaptic transmission / hydrolase activity / postsynapse / calmodulin binding / protein domain specific binding / neuronal cell body / calcium ion binding / positive regulation of gene expression / glutamatergic synapse / magnesium ion binding / Golgi apparatus / ATP hydrolysis activity / ATP binding / identical protein binding / cytosol / cytoplasm
Similarity search - Function
Myosin 5a, cargo-binding domain / Class V myosin, motor domain / Dilute domain / DIL domain / Dilute domain profile. / DIL / IQ calmodulin-binding motif / Myosin, N-terminal, SH3-like / Myosin N-terminal SH3-like domain profile. / Short calmodulin-binding motif containing conserved Ile and Gln residues. ...Myosin 5a, cargo-binding domain / Class V myosin, motor domain / Dilute domain / DIL domain / Dilute domain profile. / DIL / IQ calmodulin-binding motif / Myosin, N-terminal, SH3-like / Myosin N-terminal SH3-like domain profile. / 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. / 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 / ATPase, nucleotide binding domain / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
ADENOSINE-5'-DIPHOSPHATE / PHOSPHATE ION / Actin, alpha skeletal muscle / Unconventional myosin-Va
Similarity search - Component
Biological speciesMus musculus (house mouse)
Oryctolagus cuniculus (rabbit)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4.4 Å
AuthorsKlebl, D.P. / McMillan, S.N. / Risi, C. / Forgacs, E. / Virok, B. / Atherton, J.L. / Stofella, M. / Winkelmann, D.A. / Sobott, F. / Galkin, V.E. ...Klebl, D.P. / McMillan, S.N. / Risi, C. / Forgacs, E. / Virok, B. / Atherton, J.L. / Stofella, M. / Winkelmann, D.A. / Sobott, F. / Galkin, V.E. / Knight, P.J. / Muench, S.P. / Scarff, C.A. / White, H.D.
Funding support United Kingdom, United States, 5items
OrganizationGrant numberCountry
Wellcome Trust United Kingdom
Biotechnology and Biological Sciences Research Council (BBSRC) United Kingdom
American Heart Association United States
Medical Research Council (MRC, United Kingdom) United Kingdom
British Heart Foundation United Kingdom
CitationJournal: Nature / Year: 2025
Title: Swinging lever mechanism of myosin directly shown by time-resolved cryo-EM.
Authors: David P Klebl / Sean N McMillan / Cristina Risi / Eva Forgacs / Betty Virok / Jennifer L Atherton / Sarah A Harris / Michele Stofella / Donald A Winkelmann / Frank Sobott / Vitold E Galkin / ...Authors: David P Klebl / Sean N McMillan / Cristina Risi / Eva Forgacs / Betty Virok / Jennifer L Atherton / Sarah A Harris / Michele Stofella / Donald A Winkelmann / Frank Sobott / Vitold E Galkin / Peter J Knight / Stephen P Muench / Charlotte A Scarff / Howard D White /
Abstract: Myosins produce force and movement in cells through interactions with F-actin. Generation of movement is thought to arise through actin-catalysed conversion of myosin from an ATP-generated primed ...Myosins produce force and movement in cells through interactions with F-actin. Generation of movement is thought to arise through actin-catalysed conversion of myosin from an ATP-generated primed (pre-powerstroke) state to a post-powerstroke state, accompanied by myosin lever swing. However, the initial, primed actomyosin state has never been observed, and the mechanism by which actin catalyses myosin ATPase activity is unclear. Here, to address these issues, we performed time-resolved cryogenic electron microscopy (cryo-EM) of a myosin-5 mutant having slow hydrolysis product release. Primed actomyosin was predominantly captured 10 ms after mixing primed myosin with F-actin, whereas post-powerstroke actomyosin predominated at 120 ms, with no abundant intermediate states detected. For detailed interpretation, cryo-EM maps were fitted with pseudo-atomic models. Small but critical changes accompany the primed motor binding to actin through its lower 50-kDa subdomain, with the actin-binding cleft open and phosphate release prohibited. Amino-terminal actin interactions with myosin promote rotation of the upper 50-kDa subdomain, closing the actin-binding cleft, and enabling phosphate release. The formation of interactions between the upper 50-kDa subdomain and actin creates the strong-binding interface needed for effective force production. The myosin-5 lever swings through 93°, predominantly along the actin axis, with little twisting. The magnitude of lever swing matches the typical step length of myosin-5 along actin. These time-resolved structures demonstrate the swinging lever mechanism, elucidate structural transitions of the power stroke, and resolve decades of conjecture on how myosins generate movement.
History
DepositionNov 30, 2023Deposition site: PDBE / Processing site: PDBE
Revision 1.0Dec 11, 2024Provider: repository / Type: Initial release
Revision 1.1Apr 23, 2025Group: Data collection / Database references / Category: citation / citation_author / em_admin
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_ASTM / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year / _em_admin.last_update

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Unconventional myosin-Va
B: Actin, alpha skeletal muscle
C: Actin, alpha skeletal muscle
D: Actin, alpha skeletal muscle
hetero molecules


Theoretical massNumber of molelcules
Total (without water)219,81513
Polymers217,9144
Non-polymers1,9019
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

#1: Protein Unconventional myosin-Va / Dilute myosin heavy chain / non-muscle


Mass: 92247.883 Da / Num. of mol.: 1 / Mutation: S217A, Deletion DDEK 594-597
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mus musculus (house mouse) / Gene: Myo5a, Dilute / Production host: Santiria sp. sf9 (plant) / References: UniProt: Q99104
#2: Protein Actin, alpha skeletal muscle / Alpha-actin-1


Mass: 41888.652 Da / Num. of mol.: 3 / Source method: isolated from a natural source / Source: (natural) Oryctolagus cuniculus (rabbit)
References: UniProt: P68135, Hydrolases; Acting on acid anhydrides; Acting on acid anhydrides to facilitate cellular and subcellular movement
#3: Chemical ChemComp-PO4 / PHOSPHATE ION


Mass: 94.971 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: PO4 / Feature type: SUBJECT OF INVESTIGATION
#4: Chemical
ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: Mg / Feature type: SUBJECT OF INVESTIGATION
#5: Chemical
ChemComp-ADP / ADENOSINE-5'-DIPHOSPHATE


Mass: 427.201 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: C10H15N5O10P2 / Feature type: SUBJECT OF INVESTIGATION / Comment: ADP, energy-carrying molecule*YM
Has ligand of interestY
Has protein modificationY

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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

Component
IDNameTypeDetailsEntity IDParent-IDSource
1Primed actomyosin-5a complexCOMPLEXF-actin decorated with primed actomyosin-5a - F-actin was mixed with myosin-5a (S1 1 IQ motif, residues 1-797, S217A, DDEK 594-597 deletion) that had been pre-incubated with ATP, and vitrified at 10 ms post-mixing#1-#20MULTIPLE SOURCES
2Unconventional myosin-5aCOMPLEX#11RECOMBINANT
3F-actinCOMPLEX#21NATURAL
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
21Mus musculus (house mouse)10090
32Mus musculus (house mouse)10090
43Oryctolagus cuniculus (rabbit)9986
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-ID
21Santiria sp. sf9 (plant)1737608
32Santiria sp. sf9 (plant)1737608
43Santiria sp. sf9 (plant)1737608
Buffer solutionpH: 7
Buffer component
IDConc.NameFormulaBuffer-ID
10.25 mMAdenosine triphosphateATP1
210 mM3-(N-morpholino)propanesulfonic acidMOPS1
338 mMPotassium acetateKAc1
425 mMPotassium chlorideKCl1
52 mMMagnesium ChlorideMgCl21
60.1 mMEGTAEGTA1
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Details: Actin-myosin mixture comprising 13 uM myosin (pre-mixed with ATP) and 13 uM actin
Specimen supportDetails: 300 mesh copper grids with copper nano-wires (SPT Labtech)
Grid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil
VitrificationInstrument: HOMEMADE PLUNGER / Cryogen name: ETHANE / Humidity: 60 % / Chamber temperature: 293 K
Details: F-actin was mixed with myosin-5a (S1 1 IQ motif, residues 1-797, S217A, DDEK 594-597 deletion) that had been pre-incubated with ATP, and vitrified at 10 ms post-mixing

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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: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 130000 X / Nominal defocus max: 4100 nm / Nominal defocus min: 2000 nm
Image recordingAverage exposure time: 7 sec. / Electron dose: 61 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 QUANTUM (4k x 4k) / Num. of grids imaged: 3 / Num. of real images: 9692

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Processing

CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 715731
3D reconstructionResolution: 4.4 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 93374 / Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT / Space: REAL
Atomic model buildingPDB-ID: 4ZG4

4zg4


Accession code: 4ZG4 / Source name: PDB / Type: experimental model

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