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- EMDB-19013: CryoEM structure of the primed actomyosin-5a complex -

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Entry
Database: EMDB / ID: EMD-19013
TitleCryoEM structure of the primed actomyosin-5a complex
Map data
Sample
  • Complex: Primed actomyosin-5a complex
    • Complex: Unconventional myosin-5a
      • Protein or peptide: Unconventional myosin-Va
    • Complex: F-actin
      • Protein or peptide: Actin, alpha skeletal muscle
  • Ligand: PHOSPHATE ION
  • Ligand: MAGNESIUM ION
  • Ligand: ADENOSINE-5'-DIPHOSPHATE
KeywordsMyosin / Actin / Actomyosin / Primed actomyosin / MOTOR PROTEIN
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
Actin, alpha skeletal muscle / Unconventional myosin-Va
Similarity search - Component
Biological speciesMus musculus (house mouse) / Oryctolagus cuniculus (rabbit)
Methodsingle particle reconstruction / cryo EM / Resolution: 4.4 Å
AuthorsKlebl DP / McMillan SN / Risi C / Forgacs E / Virok B / Atherton JL / Stofella M / Winkelmann DA / Sobott F / Galkin VE ...Klebl DP / McMillan SN / Risi C / Forgacs E / Virok B / Atherton JL / Stofella M / Winkelmann DA / Sobott F / Galkin VE / Knight PJ / Muench SP / Scarff CA / White HD
Funding support United Kingdom, United States, 5 items
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, 2023-
Header (metadata) releaseDec 11, 2024-
Map releaseDec 11, 2024-
UpdateApr 23, 2025-
Current statusApr 23, 2025Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_19013.png

Downloads & links

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Map

FileDownload / File: emd_19013.map.gz / Format: CCP4 / Size: 178 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

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AxesZ (Sec.)Y (Row.)X (Col.)
1.07 Å/pix.
x 360 pix.
= 385.2 Å		1.07 Å/pix.
x 360 pix.
= 385.2 Å		1.07 Å/pix.
x 360 pix.
= 385.2 Å

Surface

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Images are generated by Spider.

Voxel sizeX=Y=Z: 1.07 Å
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Contour LevelBy AUTHOR: 0.085
Minimum - Maximum-0.006143573 - 2.0572615
Average (Standard dev.)0.0010570161 (±0.024475748)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions360360360
Spacing360360360
CellA=B=C: 385.2 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_19013_msk_1.map
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Additional map: #2

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Additional map: #1

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Half map: #2

Fileemd_19013_half_map_1.map
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Sample components

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Entire : Primed actomyosin-5a complex

EntireName: Primed actomyosin-5a complex
Components
  • Complex: Primed actomyosin-5a complex
    • Complex: Unconventional myosin-5a
      • Protein or peptide: Unconventional myosin-Va
    • Complex: F-actin
      • Protein or peptide: Actin, alpha skeletal muscle
  • Ligand: PHOSPHATE ION
  • Ligand: MAGNESIUM ION
  • Ligand: ADENOSINE-5'-DIPHOSPHATE

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Supramolecule #1: Primed actomyosin-5a complex

SupramoleculeName: Primed actomyosin-5a complex / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#2
Details: F-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 ...Details: F-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
Source (natural)Organism: Mus musculus (house mouse)

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Supramolecule #2: Unconventional myosin-5a

SupramoleculeName: Unconventional myosin-5a / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1
Source (natural)Organism: Mus musculus (house mouse)

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Supramolecule #3: F-actin

SupramoleculeName: F-actin / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #2
Source (natural)Organism: Oryctolagus cuniculus (rabbit)

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Macromolecule #1: Unconventional myosin-Va

MacromoleculeName: Unconventional myosin-Va / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Mus musculus (house mouse)
Molecular weightTheoretical: 92.247883 KDa
Recombinant expressionOrganism: Santiria sp. sf9 (plant)
SequenceString: MAASELYTKF ARVWIPDPEE VWKSAELLKD YKPGDKVLLL HLEEGKDLEY RLDPKTGELP HLRNPDILVG ENDLTALSYL HEPAVLHNL RVRFIDSKLI YTYCGIVLVA INPYEQLPIY GEDIINAYSG QNMGDMDPHI FAVAEEAYKQ MARDERNQSI I VSGESGAG ...String:
MAASELYTKF ARVWIPDPEE VWKSAELLKD YKPGDKVLLL HLEEGKDLEY RLDPKTGELP HLRNPDILVG ENDLTALSYL HEPAVLHNL RVRFIDSKLI YTYCGIVLVA INPYEQLPIY GEDIINAYSG QNMGDMDPHI FAVAEEAYKQ MARDERNQSI I VSGESGAG KTVSAKYAMR YFATVSGSAS EANVEEKVLA SNPIMESIGN AKTTRNDNAS RFGKYIEIGF DKRYRIIGAN MR TYLLEKS RVVFQAEEER NYHIFYQLCA SAKLPEFKML RLGNADSFHY TKQGGSPMIE GVDDAKEMAH TRQACTLLGI SES YQMGIF RILAGILHLG NVGFASRDSD SCTIPPKHEP LTIFCDLMGV DYEEMCHWLC HRKLATATET YIKPISKLQA TNAR DALAK HIYAKLFNWI VDHVNQALHS AVKQHSFIGV LDIYGFETFE INSFEQFCIN YANEKLQQQF NMHVFKLEQE EYMKE QIPW TLIDFYDNQP CINLIESKLG ILDLLDEECK MPKGTDDTWA QKLYNTHLNK CALFEKPRMS NKAFIIKHFA DKVEYQ CEG FLEKNKDTVF EEQIKVLKSS KFKMLPELFQ AISPTSATSS GRTPLTRVPV KPTKGRPGQT AKEHKKTVGH QFRNSLH LL METLNATTPH YVRCIKPNDF KFPFTFDEKR AVQQLRACGV LETIRISAAG FPSRWTYQEF FSRYRVLMKQ KDVLGDRK Q TCKNVLEKLI LDKDKYQFGK TKIFFRAGQV AYLEKLRADK LRAACIRIQK TIRGWLLRKR YLCMQRAAIT VQDYKDDDD K

UniProtKB: Unconventional myosin-Va

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Macromolecule #2: Actin, alpha skeletal muscle

MacromoleculeName: Actin, alpha skeletal muscle / type: protein_or_peptide / ID: 2 / Number of copies: 3 / Enantiomer: LEVO
EC number: Hydrolases; Acting on acid anhydrides; Acting on acid anhydrides to facilitate cellular and subcellular movement
Source (natural)Organism: Oryctolagus cuniculus (rabbit)
Molecular weightTheoretical: 41.888652 KDa
SequenceString: (ACE)DEDETTALV CDNGSGLVKA GFAGDDAPRA VFPSIVGRPR HQGVMVGMGQ KDSYVGDEAQ SKRGILTLKY PIEHGI ITN WDDMEKIWHH TFYNELRVAP EEHPTLLTEA PLNPKANREK MTQIMFETFN VPAMYVAIQA VLSLYASGRT TGIVLDS GD GVTHNVPIYE ...String:
(ACE)DEDETTALV CDNGSGLVKA GFAGDDAPRA VFPSIVGRPR HQGVMVGMGQ KDSYVGDEAQ SKRGILTLKY PIEHGI ITN WDDMEKIWHH TFYNELRVAP EEHPTLLTEA PLNPKANREK MTQIMFETFN VPAMYVAIQA VLSLYASGRT TGIVLDS GD GVTHNVPIYE GYALPHAIMR LDLAGRDLTD YLMKILTERG YSFVTTAERE IVRDIKEKLC YVALDFENEM ATAASSSS L EKSYELPDGQ VITIGNERFR CPETLFQPSF IGMESAGIHE TTYNSIMKCD IDIRKDLYAN NVMSGGTTMY PGIADRMQK EITALAPSTM KIKIIAPPER KYSVWIGGSI LASLSTFQQM WITKQEYDEA GPSIVHRKCF

UniProtKB: Actin, alpha skeletal muscle

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Macromolecule #3: PHOSPHATE ION

MacromoleculeName: PHOSPHATE ION / type: ligand / ID: 3 / Number of copies: 1 / Formula: PO4
Molecular weightTheoretical: 94.971 Da
Chemical component information

ChemComp-PO4:
PHOSPHATE ION

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Macromolecule #4: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 4 / Number of copies: 4 / Formula: MG
Molecular weightTheoretical: 24.305 Da

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Macromolecule #5: ADENOSINE-5'-DIPHOSPHATE

MacromoleculeName: ADENOSINE-5'-DIPHOSPHATE / type: ligand / ID: 5 / Number of copies: 4 / Formula: ADP
Molecular weightTheoretical: 427.201 Da
Chemical component information

ChemComp-ADP:
ADENOSINE-5'-DIPHOSPHATE / ADP, energy-carrying molecule*YM

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation statefilament

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Sample preparation

BufferpH: 7
Component:
ConcentrationFormulaName
0.25 mMATPAdenosine triphosphate
10.0 mMMOPS3-(N-morpholino)propanesulfonic acid
38.0 mMKAcPotassium acetate
25.0 mMKClPotassium chloride
2.0 mMMgCl2Magnesium Chloride
0.1 mMEGTAEGTA
GridModel: Quantifoil / Material: COPPER / Mesh: 300 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 60 sec. / Pretreatment - Atmosphere: AIR / Pretreatment - Pressure: 0.01 kPa
Details: 300 mesh copper grids with copper nano-wires (SPT Labtech)
VitrificationCryogen name: ETHANE / Chamber humidity: 60 % / Chamber temperature: 293 K / Instrument: HOMEMADE PLUNGER
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.
DetailsActin-myosin mixture comprising 13 uM myosin (pre-mixed with ATP) and 13 uM actin

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K2 QUANTUM (4k x 4k) / Detector mode: COUNTING / Number grids imaged: 3 / Number real images: 9692 / Average exposure time: 7.0 sec. / Average electron dose: 61.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 4.1 µm / Nominal defocus min: 2.0 µm / Nominal magnification: 130000
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 715731
Startup modelType of model: NONE
Final reconstructionResolution.type: BY AUTHOR / Resolution: 4.4 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 93374
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

4zg4


Chain - Source name: PDB / Chain - Initial model type: experimental model
RefinementSpace: REAL / Protocol: FLEXIBLE FIT
Output model

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

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