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- PDB-7pm2: Cryo-EM structure of the actomyosin-V complex in the rigor state ... -

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Basic information

Entry
Database: PDB / ID: 7pm2
TitleCryo-EM structure of the actomyosin-V complex in the rigor state (central 1er, young JASP-stabilized F-actin, class 4)
Components
  • Actin, alpha skeletal muscle
  • Myosin light chain 6B
  • Unconventional myosin-Va
KeywordsMOTOR PROTEIN / myosin / cytoskeleton / F-actin / jasplakinolide
Function / homology
Function and homology information


minus-end directed microfilament motor activity / unconventional myosin complex / insulin-responsive compartment / muscle myosin complex / muscle filament sliding / myosin complex / myosin II complex / structural constituent of muscle / cytoskeletal motor activator activity / myosin heavy chain binding ...minus-end directed microfilament motor activity / unconventional myosin complex / insulin-responsive compartment / muscle myosin complex / muscle filament sliding / myosin complex / myosin II complex / structural constituent of muscle / cytoskeletal motor activator activity / myosin heavy chain binding / microfilament motor activity / tropomyosin binding / actin filament bundle / troponin I binding / filamentous actin / mesenchyme migration / cytoskeletal motor activity / actin filament bundle assembly / skeletal muscle myofibril / striated muscle thin filament / skeletal muscle thin filament assembly / actin monomer binding / Smooth Muscle Contraction / skeletal muscle tissue development / skeletal muscle fiber development / vesicle-mediated transport / stress fiber / titin binding / actin filament polymerization / muscle contraction / actin filament organization / protein localization to plasma membrane / filopodium / actin filament / Hydrolases; Acting on acid anhydrides; Acting on acid anhydrides to facilitate cellular and subcellular movement / cellular response to insulin stimulus / calcium-dependent protein binding / actin filament binding / actin cytoskeleton / lamellipodium / cell body / calmodulin binding / hydrolase activity / Golgi membrane / protein domain specific binding / calcium ion binding / positive regulation of gene expression / magnesium ion binding / ATP hydrolysis activity / extracellular exosome / ATP binding / identical protein binding / membrane / 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 / EF-hand, calcium binding motif / ATPase, nucleotide binding domain / EF-hand calcium-binding domain profile. / EF-hand domain / EF-hand domain pair / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
Jasplakinolide / ADENOSINE-5'-DIPHOSPHATE / PHOSPHATE ION / Myosin light chain 6B / Actin, alpha skeletal muscle / Unconventional myosin-Va
Similarity search - Component
Biological speciesHomo sapiens (human)
Gallus gallus (chicken)
Oryctolagus cuniculus (rabbit)
MethodELECTRON MICROSCOPY / helical reconstruction / cryo EM / Resolution: 3.6 Å
AuthorsPospich, S. / Sweeney, H.L. / Houdusse, A. / Raunser, S.
Funding support Germany, European Union, France, United States, 5items
OrganizationGrant numberCountry
Max Planck Society Germany
European CommissionERC-2019-SyG 856118European Union
Centre National de la Recherche Scientifique (CNRS) France
Agence Nationale de la Recherche (ANR)ANR-17-CE11-0029-01 France
National Institutes of Health/National Institute on Deafness and Other Communication Disorders (NIH/NIDCD)R01-DC009100 United States
CitationJournal: Elife / Year: 2021
Title: High-resolution structures of the actomyosin-V complex in three nucleotide states provide insights into the force generation mechanism.
Authors: Sabrina Pospich / H Lee Sweeney / Anne Houdusse / Stefan Raunser /
Abstract: The molecular motor myosin undergoes a series of major structural transitions during its force-producing motor cycle. The underlying mechanism and its coupling to ATP hydrolysis and actin binding are ...The molecular motor myosin undergoes a series of major structural transitions during its force-producing motor cycle. The underlying mechanism and its coupling to ATP hydrolysis and actin binding are only partially understood, mostly due to sparse structural data on actin-bound states of myosin. Here, we report 26 high-resolution cryo-EM structures of the actomyosin-V complex in the strong-ADP, rigor, and a previously unseen post-rigor transition state that binds the ATP analog AppNHp. The structures reveal a high flexibility of myosin in each state and provide valuable insights into the structural transitions of myosin-V upon ADP release and binding of AppNHp, as well as the actomyosin interface. In addition, they show how myosin is able to specifically alter the structure of F-actin.
History
DepositionSep 1, 2021Deposition site: PDBE / Processing site: PDBE
Revision 1.0Dec 22, 2021Provider: repository / Type: Initial release

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Structure visualization

Movie
  • Deposited structure unit
  • Imaged by Jmol
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  • Superimposition on EM map
  • EMDB-13510
  • Imaged by UCSF Chimera
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Structure viewerMolecule:
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Assembly

Deposited unit
B: Myosin light chain 6B
A: Unconventional myosin-Va
C: Actin, alpha skeletal muscle
hetero molecules


Theoretical massNumber of molelcules
Total (without water)151,8207
Polymers150,5643
Non-polymers1,2564
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area6150 Å2
ΔGint-60 kcal/mol
Surface area57920 Å2
MethodPISA

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Components

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Protein , 3 types, 3 molecules BAC

#1: Protein Myosin light chain 6B / Myosin light chain 1 slow-twitch muscle A isoform / MLC1sa / Smooth muscle and nonmuscle myosin ...Myosin light chain 1 slow-twitch muscle A isoform / MLC1sa / Smooth muscle and nonmuscle myosin light chain alkali 6B


Mass: 17090.277 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: MYL6B, MLC1SA / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: P14649
#2: Protein Unconventional myosin-Va / Dilute myosin heavy chain / non-muscle / Myosin heavy chain p190 / Myosin-V


Mass: 91363.953 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Gallus gallus (chicken) / Gene: MYO5A / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: Q02440
#3: Protein Actin, alpha skeletal muscle / Alpha-actin-1


Mass: 42109.973 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Oryctolagus cuniculus (rabbit) / References: UniProt: P68135

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Non-polymers , 4 types, 4 molecules

#4: Chemical ChemComp-ADP / ADENOSINE-5'-DIPHOSPHATE


Mass: 427.201 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C10H15N5O10P2 / Comment: ADP, energy-carrying molecule*YM
#5: Chemical ChemComp-PO4 / PHOSPHATE ION


Mass: 94.971 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: PO4
#6: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Mg
#7: Chemical ChemComp-9UE / Jasplakinolide


Mass: 709.670 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C36H45BrN4O6

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Details

Has ligand of interestN

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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

ComponentName: Young actomyosin-V complex in the rigor state / Type: COMPLEX
Details: Young ADP-Pi-bound F-actin stabilized with jasplakinolide and decorated with myosin-Va-LC in the rigor state (nucleotide-free)
Entity ID: #1-#3 / Source: MULTIPLE SOURCES
Molecular weightExperimental value: NO
Buffer solutionpH: 7.5
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES / Details: Signal subtracted
Specimen supportGrid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R2/1
VitrificationInstrument: FEI VITROBOT MARK III / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 286 K / Details: On grid decoration

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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 FIELD / Cs: 0 mm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 15 sec. / Electron dose: 80 e/Å2 / Detector mode: SUPER-RESOLUTION / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 3336
EM imaging opticsEnergyfilter name: GIF Bioquantum / Energyfilter slit width: 20 eV / Spherical aberration corrector: Cs-corrected microscope
Image scansMovie frames/image: 40

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Processing

SoftwareName: PHENIX / Version: 1.17.1_3660: / Classification: refinement
EM software
IDNameVersionCategory
1crYOLOparticle selection
2EPUimage acquisition
4GctfCTF correction
7UCSF Chimera1.15model fitting
9SPHIREinitial Euler assignment
10SPHIREfinal Euler assignment
11RELIONclassification
12SPHIRE3D reconstruction
13PHENIX1.17model refinement
14ISOLDE0.91model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3.6 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 107174 / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingProtocol: OTHER / Space: REAL
Details: Starting from average model and ChimeraX-Isolde session.
Atomic model buildingPDB-ID: 7PLY

7ply

Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00410372
ELECTRON MICROSCOPYf_angle_d0.83314008
ELECTRON MICROSCOPYf_dihedral_angle_d19.6473877
ELECTRON MICROSCOPYf_chiral_restr0.0521527
ELECTRON MICROSCOPYf_plane_restr0.0071793

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