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- PDB-7pmg: Cryo-EM structure of the actomyosin-V complex in the post-rigor t... -

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Basic information

Entry
Database: PDB / ID: 7pmg
TitleCryo-EM structure of the actomyosin-V complex in the post-rigor transition state (AppNHp, central 1er, class 3)
Components
  • Actin, alpha skeletal muscle
  • Myosin light chain 6B
  • Phalloidin
  • Unconventional myosin-Va
KeywordsMOTOR PROTEIN / myosin / cytoskeleton / F-actin / phalloidin
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 II complex / vesicle transport along actin filament / myosin complex / cytoskeletal motor activator activity / microfilament motor activity ...minus-end directed microfilament motor activity / unconventional myosin complex / insulin-responsive compartment / muscle myosin complex / muscle filament sliding / myosin II complex / vesicle transport along actin filament / myosin complex / cytoskeletal motor activator activity / microfilament motor activity / structural constituent of muscle / mesenchyme migration / tropomyosin binding / myosin heavy chain binding / troponin I binding / actin filament bundle / skeletal muscle thin filament assembly / striated muscle thin filament / filamentous actin / Smooth Muscle Contraction / actin filament bundle assembly / actin monomer binding / cytoskeletal motor activity / skeletal muscle myofibril / skeletal muscle fiber development / skeletal muscle tissue development / stress fiber / titin binding / actin filament polymerization / actin filament organization / filopodium / muscle contraction / actin filament / vesicle-mediated transport / protein localization to plasma membrane / cell body / cellular response to insulin stimulus / calcium-dependent protein binding / actin cytoskeleton / actin filament binding / lamellipodium / calmodulin binding / vesicle / Golgi membrane / protein domain specific binding / calcium ion binding / positive regulation of gene expression / magnesium ion binding / extracellular exosome / ATP binding / identical protein binding / cytosol / cytoplasm
Similarity search - Function
Myosin 5a, cargo-binding domain / Class V myosin, motor domain / Dilute domain / Dilute domain profile. / DIL domain / DIL / IQ calmodulin-binding motif / Myosin N-terminal SH3-like domain profile. / Myosin, N-terminal, SH3-like / Short calmodulin-binding motif containing conserved Ile and Gln residues. ...Myosin 5a, cargo-binding domain / Class V myosin, motor domain / Dilute domain / Dilute domain profile. / DIL domain / DIL / IQ calmodulin-binding motif / Myosin N-terminal SH3-like domain profile. / Myosin, N-terminal, SH3-like / Short calmodulin-binding motif containing conserved Ile and Gln residues. / Myosin head (motor domain) / Myosin. Large ATPases. / Myosin motor domain profile. / Myosin head, motor domain / IQ motif profile. / IQ motif, EF-hand binding site / Actins signature 1. / Actin, conserved site / Actins signature 2. / Actin/actin-like conserved site / Actins and actin-related proteins signature. / Actin / Actin family / Actin / Kinesin motor domain superfamily / ATPase, nucleotide binding domain / EF-hand, calcium binding motif / EF-hand calcium-binding domain profile. / EF-hand domain / EF-hand domain pair / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
Myosin light chain 6B / Actin, alpha skeletal muscle / : / ADENOSINE-5'-DIPHOSPHATE / PHOSPHOAMINOPHOSPHONIC ACID-ADENYLATE ESTER / Unconventional myosin-Va
Similarity search - Component
Biological speciesGallus gallus (chicken)
Homo sapiens (human)
Oryctolagus cuniculus (rabbit)
Amanita phalloides (death cap)
MethodELECTRON MICROSCOPY / helical reconstruction / cryo EM / Resolution: 3.3 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 2, 2021Deposition site: PDBE / Processing site: PDBE
Revision 1.0Dec 22, 2021Provider: repository / Type: Initial release

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Structure visualization

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  • Deposited structure unit
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  • Superimposition on EM map
  • EMDB-13532
  • Imaged by UCSF Chimera
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Structure viewerMolecule:
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Assembly

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


Theoretical massNumber of molelcules
Total (without water)152,3799
Polymers151,3734
Non-polymers1,0065
Water0
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area7240 Å2
ΔGint-70 kcal/mol
Surface area56720 Å2
MethodPISA

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Components

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Protein , 3 types, 3 molecules ACB

#1: 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
#2: 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
#4: 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

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Protein/peptide , 1 types, 1 molecules H

#3: Protein/peptide Phalloidin / /


Type: Peptide-like / Class: Toxin / Mass: 808.899 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Amanita phalloides (death cap) / References: BIRD: PRD_002366

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Non-polymers , 3 types, 5 molecules

#5: Chemical ChemComp-ANP / PHOSPHOAMINOPHOSPHONIC ACID-ADENYLATE ESTER


Mass: 506.196 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C10H17N6O12P3 / Comment: AMP-PNP, energy-carrying molecule analogue*YM
#6: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: Mg
#7: Chemical ChemComp-ADP / ADENOSINE-5'-DIPHOSPHATE / Adenosine diphosphate


Mass: 427.201 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C10H15N5O10P2 / Comment: ADP, energy-carrying molecule*YM

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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: Actomyosin-V complex in the post-rigor transition stateMyofibril
Type: COMPLEX
Details: Aged ADP-bound F-actin stabilized with phalloidin and decorated with myosin-Va-LC in a post-rigor transition state (Mg2+-AppNHp)
Entity ID: #1-#4 / Source: MULTIPLE SOURCES
Molecular weightExperimental value: NO
Buffer solutionpH: 7.5
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES / Details: Rise 27.8 A, Twist -167.3 degrees
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 % / Details: On grid decoration, two data sets combined

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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 FIELDBright-field microscopy / Cs: 0 mm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 15 sec. / Electron dose: 81 e/Å2 / Detector mode: SUPER-RESOLUTION / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of grids imaged: 2 / Num. of real images: 12475
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
Helical symmertyAngular rotation/subunit: -167.3 ° / Axial rise/subunit: 27.8 Å / Axial symmetry: C1
3D reconstructionResolution: 3.3 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 365722 / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingSpace: REAL
Details: Starting from average model and ChimeraX-Isolde session
Atomic model buildingPDB-ID: 7PMD
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.01410406
ELECTRON MICROSCOPYf_angle_d1.1214063
ELECTRON MICROSCOPYf_dihedral_angle_d19.4743872
ELECTRON MICROSCOPYf_chiral_restr0.0691536
ELECTRON MICROSCOPYf_plane_restr0.0081794

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