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- EMDB-13522: Cryo-EM structure of the actomyosin-V complex in the strong-ADP s... -

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Basic information

Entry
Database: EMDB / ID: EMD-13522
TitleCryo-EM structure of the actomyosin-V complex in the strong-ADP state (central 3er/2er)
Map dataSharpened map of the central three F-actin and two myosin-V-LC molecules filtered to local resolution
Sample
  • Complex: Actomyosin-V complex in the strong-ADP state
    • Protein or peptide: Myosin light chain 6B
    • Protein or peptide: Unconventional myosin-Va
    • Protein or peptide: Actin, alpha skeletal muscle
    • Protein or peptide: Phalloidin
  • Ligand: MAGNESIUM ION
  • Ligand: ADENOSINE-5'-DIPHOSPHATE
Function / homology
Function and homology information


minus-end directed microfilament motor activity / unconventional myosin complex / insulin-responsive compartment / vesicle transport along actin filament / muscle myosin complex / muscle filament sliding / myosin II complex / myosin complex / structural constituent of muscle / cytoskeletal motor activator activity ...minus-end directed microfilament motor activity / unconventional myosin complex / insulin-responsive compartment / vesicle transport along actin filament / muscle myosin complex / muscle filament sliding / myosin II complex / myosin complex / structural constituent of muscle / cytoskeletal motor activator activity / microfilament motor activity / tropomyosin binding / myosin heavy chain binding / mesenchyme migration / troponin I binding / filamentous actin / actin filament bundle / cytoskeletal motor activity / skeletal muscle thin filament assembly / actin filament bundle assembly / striated muscle thin filament / skeletal muscle myofibril / actin monomer binding / Smooth Muscle Contraction / stress fiber / skeletal muscle fiber development / vesicle-mediated transport / titin binding / skeletal muscle tissue development / muscle contraction / actin filament polymerization / filopodium / actin filament organization / protein localization to plasma membrane / 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 / vesicle / calmodulin binding / hydrolase activity / protein domain specific binding / Golgi membrane / calcium ion binding / positive regulation of gene expression / magnesium ion binding / 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. ...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
Myosin light chain 6B / Actin, alpha skeletal muscle / Unconventional myosin-Va
Similarity search - Component
Biological speciesHomo sapiens (human) / Gallus gallus (chicken) / Rabbit (rabbit) / Amanita phalloides (death cap)
Methodhelical reconstruction / cryo EM / Resolution: 3.0 Å
AuthorsPospich S / Sweeney HL / Houdusse A / Raunser S
Funding support Germany, European Union, France, United States, 5 items
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, 2021-
Header (metadata) releaseDec 22, 2021-
Map releaseDec 22, 2021-
UpdateDec 22, 2021-
Current statusDec 22, 2021Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.01
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.01
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  • Surface view with fitted model
  • Atomic models: PDB-7pm6
  • Surface level: 0.01
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_13522.png

Downloads & links

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Map

FileDownload / File: emd_13522.map.gz / Format: CCP4 / Size: 125 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationSharpened map of the central three F-actin and two myosin-V-LC molecules filtered to local resolution
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.06 Å/pix.
x 320 pix.
= 339.2 Å		1.06 Å/pix.
x 320 pix.
= 339.2 Å		1.06 Å/pix.
x 320 pix.
= 339.2 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.06 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.01 / Movie #1: 0.01
Minimum - Maximum-0.01514043 - 0.056154225
Average (Standard dev.)0.00012904614 (±0.0013860627)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions320320320
Spacing320320320
CellA=B=C: 339.19998 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.061.061.06
M x/y/z320320320
origin x/y/z0.0000.0000.000
length x/y/z339.200339.200339.200
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS320320320
D min/max/mean-0.0150.0560.000

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Supplemental data

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Mask #1

Fileemd_13522_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: Sharpened map of the central three F-actin and...

Fileemd_13522_additional_1.map
AnnotationSharpened map of the central three F-actin and two myosin-V-LC molecules filtered to nominal resolution
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: Denoised map of the central three F-actin and...

Fileemd_13522_additional_2.map
AnnotationDenoised map of the central three F-actin and two myosin-V-LC molecules (LAFTER)
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map of full filament

Fileemd_13522_half_map_1.map
AnnotationHalf map of full filament
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map of full filament

Fileemd_13522_half_map_2.map
AnnotationHalf map of full filament
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Actomyosin-V complex in the strong-ADP state

EntireName: Actomyosin-V complex in the strong-ADP state
Components
  • Complex: Actomyosin-V complex in the strong-ADP state
    • Protein or peptide: Myosin light chain 6B
    • Protein or peptide: Unconventional myosin-Va
    • Protein or peptide: Actin, alpha skeletal muscle
    • Protein or peptide: Phalloidin
  • Ligand: MAGNESIUM ION
  • Ligand: ADENOSINE-5'-DIPHOSPHATE

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Supramolecule #1: Actomyosin-V complex in the strong-ADP state

SupramoleculeName: Actomyosin-V complex in the strong-ADP state / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#4
Details: Aged ADP-bound F-actin stabilized with phalloidin and decorated with myosin-Va-LC in the strong-ADP state (Mg2+-ADP)

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Macromolecule #1: Myosin light chain 6B

MacromoleculeName: Myosin light chain 6B / type: protein_or_peptide / ID: 1 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 17.090277 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString:
MIEFNKDQLE EFKEAFELFD RVGDGKILYS QCGDVMRALG QNPTNAEVLK VLGNPKSDEL KSRRVDFETF LPMLQAVAKN RGQGTYEDY LEGFRVFDKE GNGKVMGAEL RHVLTTLGEK MTEEEVETVL AGHEDSNGCI NYEAFLKHIL SV

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Macromolecule #2: Unconventional myosin-Va

MacromoleculeName: Unconventional myosin-Va / type: protein_or_peptide / ID: 2 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Gallus gallus (chicken)
Molecular weightTheoretical: 91.363953 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: MAASELYTKY ARVWIPDPEE VWKSAELLKD YKPGDKVLQL RLEEGKDLEY CLDPKTKELP PLRNPDILVG ENDLTALSYL HEPAVLHNL KVRFIDSKLI YTYCGIVLVA INPYEQLPIY GEDIINAYSG QNMGDMDPHI FAVAEEAYKQ MARDERNQSI I VSGESGAG ...String:
MAASELYTKY ARVWIPDPEE VWKSAELLKD YKPGDKVLQL RLEEGKDLEY CLDPKTKELP PLRNPDILVG ENDLTALSYL HEPAVLHNL KVRFIDSKLI YTYCGIVLVA INPYEQLPIY GEDIINAYSG QNMGDMDPHI FAVAEEAYKQ MARDERNQSI I VSGESGAG KTVSAKYAMR YFATVSGSAS EANVEEKVLA SNPIMESIGN AKTTRNDNSS RFGKYIEIGF DKRYRIIGAN MR TYLLEKS RVVFQAEEER NYHIFYQLCA SAALPEFKTL RLGNANYFHY TKQGGSPVID GIDDAKEMVN TRQACTLLGI SDS YQMGIF RILAGILHLG NVEFASRDSD SCAIPPKHDP LTIFCDLMGV DYEEMAHWLC HRKLATATET YIKPISKLHA INAR DALAK HIYANLFNWI VDHVNKALHS TVKQHSFIGV LDIYGFETFE INSFEQFCIN YANEKLQQQF NMHVFKLEQE EYMKE QIPW TLIDFYDNQP CINLIEAKMG VLDLLDEECK MPKGSDDTWA QKLYNTHLNK CALFEKPRLS NKAFIIKHFA DKVEYQ CEG FLEKNKDTVY EEQIKVLKSS KKFKLLPELF QDEEKAISPT SATPSGRVPL SRTPVKPAKA RPGQTSKEHK KTVGHQF RN SLHLLMETLN ATTPHYVRCI KPNDFKFPFT FDEKRAVQQL RACGVLETIR ISAAGFPSRW TYQEFFSRYR VLMKQKDV L SDRKQTCKNV LEKLILDKDK YQFGKTKIFF RAGQVAYLEK IRADKLRAAC IRIQKTIRGW LMRKKYMRMR R

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Macromolecule #3: Actin, alpha skeletal muscle

MacromoleculeName: Actin, alpha skeletal muscle / type: protein_or_peptide / ID: 3 / Number of copies: 3 / Enantiomer: LEVO
Source (natural)Organism: Rabbit (rabbit)
Molecular weightTheoretical: 42.109973 KDa
SequenceString: MCDEDETTAL VCDNGSGLVK AGFAGDDAPR AVFPSIVGRP RHQGVMVGMG QKDSYVGDEA QSKRGILTLK YPIE(HIC)G IIT NWDDMEKIWH HTFYNELRVA PEEHPTLLTE APLNPKANRE KMTQIMFETF NVPAMYVAIQ AVLSLYASGR TTGIVLD SG DGVTHNVPIY ...String:
MCDEDETTAL VCDNGSGLVK AGFAGDDAPR AVFPSIVGRP RHQGVMVGMG QKDSYVGDEA QSKRGILTLK YPIE(HIC)G IIT NWDDMEKIWH HTFYNELRVA PEEHPTLLTE APLNPKANRE KMTQIMFETF NVPAMYVAIQ AVLSLYASGR TTGIVLD SG DGVTHNVPIY EGYALPHAIM RLDLAGRDLT DYLMKILTER GYSFVTTAER EIVRDIKEKL CYVALDFENE MATAASSS S LEKSYELPDG QVITIGNERF RCPETLFQPS FIGMESAGIH ETTYNSIMKC DIDIRKDLYA NNVMSGGTTM YPGIADRMQ KEITALAPST MKIKIIAPPE RKYSVWIGGS ILASLSTFQQ MWITKQEYDE AGPSIVHRKC F

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Macromolecule #4: Phalloidin

MacromoleculeName: Phalloidin / type: protein_or_peptide / ID: 4 / Number of copies: 3 / Enantiomer: LEVO
Source (natural)Organism: Amanita phalloides (death cap)
Molecular weightTheoretical: 808.899 Da
SequenceString:
W(EEP)A(DTH)C(HYP)A

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Macromolecule #5: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 5 / Number of copies: 5 / Formula: MG
Molecular weightTheoretical: 24.305 Da

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Macromolecule #6: ADENOSINE-5'-DIPHOSPHATE

MacromoleculeName: ADENOSINE-5'-DIPHOSPHATE / type: ligand / ID: 6 / Number of copies: 5 / 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
Processinghelical reconstruction
Aggregation statefilament

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Sample preparation

BufferpH: 7.5
GridModel: Quantifoil R2/1 / Material: COPPER / Mesh: 300 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 286 K / Instrument: FEI VITROBOT MARK III / Details: On grid decoration.
DetailsRise 27.8 A, Twist -167.3 degrees

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Specialist opticsEnergy filter - Name: GIF Bioquantum / Energy filter - Slit width: 20 eV
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: SUPER-RESOLUTION / Number grids imaged: 1 / Number real images: 5908 / Average exposure time: 15.0 sec. / Average electron dose: 82.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: SPOT SCAN / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.0 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: SPHIRE / Number images used: 871844
CTF correctionSoftware - Name: Gctf
Startup modelType of model: PDB ENTRY
PDB model - PDB ID:

5jlh


Details: Actomyosin only
Final angle assignmentType: NOT APPLICABLE / Software - Name: SPHIRE
Details: No helical symmetry was applied. See original publication for details.
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

7pm5

DetailsAn initial model of phalloidin was generated using elBow within Phenix inputting the SMILES string.
RefinementSpace: REAL
Output model

PDB-7pm6:
Cryo-EM structure of the actomyosin-V complex in the strong-ADP state (central 3er/2er)

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