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- EMDB-7117: CryoEM structure of Myosin VI-Actin complex in the ADP state -

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Basic information

Entry
Database: EMDB / ID: EMD-7117
TitleCryoEM structure of Myosin VI-Actin complex in the ADP state
Map dataCryoEM density map of myosin VI-actin complex in the ADP state, B factor sharpened to -200.
Sample
  • Complex: myosin VI-actin in the ADP state
    • Complex: myosin VI
      • Protein or peptide: Unconventional myosin-VI
    • Complex: actin
      • Protein or peptide: Actin, alpha skeletal muscle
  • Ligand: MAGNESIUM ION
  • Ligand: ADENOSINE-5'-DIPHOSPHATE
KeywordsCytoskeleton / Filament / Complex / CONTRACTILE PROTEIN
Function / homology
Function and homology information


regulation of secretion / actin filament-based movement / inner ear auditory receptor cell differentiation / vesicle transport along actin filament / myosin complex / clathrin-coated vesicle / cytoskeletal motor activator activity / microfilament motor activity / inner ear morphogenesis / tropomyosin binding ...regulation of secretion / actin filament-based movement / inner ear auditory receptor cell differentiation / vesicle transport along actin filament / myosin complex / clathrin-coated vesicle / cytoskeletal motor activator activity / microfilament motor activity / inner ear morphogenesis / tropomyosin binding / myosin heavy chain binding / mesenchyme migration / troponin I binding / actin filament bundle / filamentous actin / actin filament bundle assembly / skeletal muscle thin filament assembly / microvillus / cytoskeletal motor activity / striated muscle thin filament / skeletal muscle myofibril / actin monomer binding / DNA damage response, signal transduction by p53 class mediator / skeletal muscle fiber development / stress fiber / clathrin-coated pit / titin binding / ruffle / actin filament polymerization / filopodium / actin filament organization / actin filament / ADP binding / sensory perception of sound / Hydrolases; Acting on acid anhydrides; Acting on acid anhydrides to facilitate cellular and subcellular movement / intracellular protein transport / ruffle membrane / endocytosis / calcium-dependent protein binding / actin filament binding / actin cytoskeleton / lamellipodium / cell body / cell cortex / cytoplasmic vesicle / nuclear membrane / vesicle / calmodulin binding / hydrolase activity / protein domain specific binding / calcium ion binding / positive regulation of gene expression / perinuclear region of cytoplasm / Golgi apparatus / magnesium ion binding / nucleoplasm / ATP binding / identical protein binding / nucleus / cytosol / cytoplasm
Similarity search - Function
: / Myosin VI, lever arm / Myosin VI, cargo binding domain / Class VI myosin, motor domain / Myosin VI cargo binding domain / Myosin S1 fragment, N-terminal / Myosin, N-terminal, SH3-like / Myosin N-terminal SH3-like domain profile. / Myosin head, motor domain / Myosin head (motor domain) ...: / Myosin VI, lever arm / Myosin VI, cargo binding domain / Class VI myosin, motor domain / Myosin VI cargo binding domain / Myosin S1 fragment, N-terminal / Myosin, N-terminal, SH3-like / Myosin N-terminal SH3-like domain profile. / Myosin head, motor domain / Myosin head (motor domain) / Myosin motor domain profile. / Myosin. Large ATPases. / Actins signature 1. / Actin, conserved site / Actins signature 2. / Kinesin motor domain superfamily / 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
Unconventional myosin-VI / Actin, alpha skeletal muscle / Unconventional myosin-VI
Similarity search - Component
Biological speciesSus scrofa (pig) / Oryctolagus cuniculus (rabbit)
Methodhelical reconstruction / cryo EM / Resolution: 5.5 Å
AuthorsGurel PG / Alushin GM
Funding support United States, 1 items
OrganizationGrant numberCountry
National Institutes of Health/Office of the Director5DP5OD017885 United States
CitationJournal: Nat Nanotechnol / Year: 2018
Title: Controllable molecular motors engineered from myosin and RNA.
Authors: Tosan Omabegho / Pinar S Gurel / Clarence Y Cheng / Laura Y Kim / Paul V Ruijgrok / Rhiju Das / Gregory M Alushin / Zev Bryant /
Abstract: Engineering biomolecular motors can provide direct tests of structure-function relationships and customized components for controlling molecular transport in artificial systems or in living cells . ...Engineering biomolecular motors can provide direct tests of structure-function relationships and customized components for controlling molecular transport in artificial systems or in living cells . Previously, synthetic nucleic acid motors and modified natural protein motors have been developed in separate complementary strategies to achieve tunable and controllable motor function. Integrating protein and nucleic-acid components to form engineered nucleoprotein motors may enable additional sophisticated functionalities. However, this potential has only begun to be explored in pioneering work harnessing DNA scaffolds to dictate the spacing, number and composition of tethered protein motors . Here, we describe myosin motors that incorporate RNA lever arms, forming hybrid assemblies in which conformational changes in the protein motor domain are amplified and redirected by nucleic acid structures. The RNA lever arm geometry determines the speed and direction of motor transport and can be dynamically controlled using programmed transitions in the lever arm structure . We have characterized the hybrid motors using in vitro motility assays, single-molecule tracking, cryo-electron microscopy and structural probing . Our designs include nucleoprotein motors that reversibly change direction in response to oligonucleotides that drive strand-displacement reactions. In multimeric assemblies, the controllable motors walk processively along actin filaments at speeds of 10-20 nm s. Finally, to illustrate the potential for multiplexed addressable control, we demonstrate sequence-specific responses of RNA variants to oligonucleotide signals.
History
DepositionNov 17, 2017-
Header (metadata) releaseJan 10, 2018-
Map releaseJan 10, 2018-
UpdateMar 13, 2024-
Current statusMar 13, 2024Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 5.8
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 5.8
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6bnq
  • Surface level: 5.8
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6bnw
  • Surface level: 5.8
  • Imaged by UCSF Chimera
  • Download
  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-6bnq
  • Imaged by Jmol
  • Download
  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-6bnw
  • Imaged by Jmol
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_7117_1.png

emd_7117_2.png

Downloads & links

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Map

FileDownload / File: emd_7117.map.gz / Format: CCP4 / Size: 512 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationCryoEM density map of myosin VI-actin complex in the ADP state, B factor sharpened to -200.
Voxel sizeX=Y=Z: 1.27 Å
Density
Contour LevelBy AUTHOR: 5.8 / Movie #1: 5.8
Minimum - Maximum-7.0485396 - 18.042619999999999
Average (Standard dev.)-0.000000001499693 (±0.99999994)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-256-256-256
Dimensions512512512
Spacing512512512
CellA=B=C: 650.24 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.271.271.27
M x/y/z512512512
origin x/y/z0.0000.0000.000
length x/y/z650.240650.240650.240
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS-256-256-256
NC/NR/NS512512512
D min/max/mean-7.04918.043-0.000

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Supplemental data

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Mask #1

Fileemd_7117_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Mask #2

Fileemd_7117_msk_2.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: Un-sharpened density map of myosin VI-actin complex

Fileemd_7117_additional.map
AnnotationUn-sharpened density map of myosin VI-actin complex
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map of the full reconstruction of myosin...

Fileemd_7117_half_map_1.map
AnnotationHalf map of the full reconstruction of myosin VI-actin complex in the ADP state
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map of the full reconstruction of myosin...

Fileemd_7117_half_map_2.map
AnnotationHalf map of the full reconstruction of myosin VI-actin complex in the ADP state
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : myosin VI-actin in the ADP state

EntireName: myosin VI-actin in the ADP state
Components
  • Complex: myosin VI-actin in the ADP state
    • Complex: myosin VI
      • Protein or peptide: Unconventional myosin-VI
    • Complex: actin
      • Protein or peptide: Actin, alpha skeletal muscle
  • Ligand: MAGNESIUM ION
  • Ligand: ADENOSINE-5'-DIPHOSPHATE

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Supramolecule #1: myosin VI-actin in the ADP state

SupramoleculeName: myosin VI-actin in the ADP state / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#2
Molecular weightTheoretical: 122 KDa

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Supramolecule #2: myosin VI

SupramoleculeName: myosin VI / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1
Source (natural)Organism: Sus scrofa (pig)

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Supramolecule #3: actin

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

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Macromolecule #1: Unconventional myosin-VI

MacromoleculeName: Unconventional myosin-VI / type: protein_or_peptide / ID: 1 / Number of copies: 6 / Enantiomer: LEVO
Source (natural)Organism: Sus scrofa (pig)
Molecular weightTheoretical: 79.837367 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: EDGKPVWAPH PTDGFQVGNI VDIGPDSLTI EPLNQKGKTF LALINQVFPA EEDSKKDVED NCSLMYLNEA TLLHNIKVRY SKDRIYTYV ANILIAVNPY FDIPKIYSSE TIKSYQGKSL GTMPPHVFAI ADKAFRDMKV LKLSQSIIVS GESGAGKTEN T KFVLRYLT ...String:
EDGKPVWAPH PTDGFQVGNI VDIGPDSLTI EPLNQKGKTF LALINQVFPA EEDSKKDVED NCSLMYLNEA TLLHNIKVRY SKDRIYTYV ANILIAVNPY FDIPKIYSSE TIKSYQGKSL GTMPPHVFAI ADKAFRDMKV LKLSQSIIVS GESGAGKTEN T KFVLRYLT ESYGTGQDID DRIVEANPLL EAFGNAKTVR NNNSSRFGKF VEIHFNEKSS VVGGFVSHYL LEKSRICVQG KE ERNYHIF YRLCAGASED IRERLHLSSP DNFRYLNRGC TRYFANKETD KQILQNRKSP EYLKAGSLKD PLLDDHGDFI RMC TAMKKI GLDDEEKLDL FRVVAGVLHL GNIDFEEAGS TSGGCNLKNK STQALEYCAE LLGLDQDDLR VSLTTRVMLT TAGG AKGTV IKVPLKVEQA NNARDALAKT VYSHLFDHVV NRVNQCFPFE TSSYFIGVLD IAGFEYFEHN SFEQFCINYC NEKLQ QFFN ERILKEEQEL YQKEGLGVNE VHYVDNQDCI DLIEARLVGI LDILDEENRL PQPSDQHFTS AVHQKHKDHF RLSIPR KSK LAIHRNIRDD EGFIIRHFAG AVCYETTQFV EKNNDALHMS LESLICESRD KFIRELFESS TNNNKDTKQK AGKLSFI SV GNKFKTQLNL LLDKLRSTGA SFIRCIKPNL KMTSHHFEGA QILSQLQCSG MVSVLDLMQG GF

UniProtKB: Unconventional myosin-VI

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Macromolecule #2: Actin, alpha skeletal muscle

MacromoleculeName: Actin, alpha skeletal muscle / type: protein_or_peptide / ID: 2 / Number of copies: 8 / Enantiomer: LEVO
Source (natural)Organism: Oryctolagus cuniculus (rabbit) / Tissue: Skeletal Muscle
Molecular weightTheoretical: 41.560266 KDa
SequenceString: MCDEDETTAL VCDNGSGLVK AGFAGDDAPR AVFPSIVGRP RHQGVMVGMG QKDSYVGDEA QSKRGILTLK YPIEHGIITN WDDMEKIWH HTFYNELRVA PEEHPTLLTE APLNPKANRE KMTQIMFETF NVPAMYVAIQ AVLSLYASGR TTGIVLDSGD G VTHNVPIY ...String:
MCDEDETTAL VCDNGSGLVK AGFAGDDAPR AVFPSIVGRP RHQGVMVGMG QKDSYVGDEA QSKRGILTLK YPIEHGIITN WDDMEKIWH HTFYNELRVA PEEHPTLLTE APLNPKANRE KMTQIMFETF NVPAMYVAIQ AVLSLYASGR TTGIVLDSGD G VTHNVPIY EGYALPHAIM RLDLAGRDLT DYLMKILTER GYSFVTTAER EIVRDIKEKL CYVALDFENE MATAASSSSL EK SYELPDG QVITIGNERF RCPETLFQPS FIGMESAGIH ETTYNSIMKC DIDIRKDLYA NNVMSGGTTM YPGIADRMQK EIT ALAPST MKIKIIAPPE RKYSVWIGGS ILASLSTFQQ MWITKQEYDE AGPSIVH

UniProtKB: Actin, alpha skeletal muscle

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Macromolecule #3: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 3 / Number of copies: 8 / Formula: MG
Molecular weightTheoretical: 24.305 Da

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Macromolecule #4: ADENOSINE-5'-DIPHOSPHATE

MacromoleculeName: ADENOSINE-5'-DIPHOSPHATE / type: ligand / ID: 4 / Number of copies: 8 / Formula: ADP
Molecular weightTheoretical: 427.201 Da
Chemical component information

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

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Experimental details

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Structure determination

Methodcryo EM
Processinghelical reconstruction
Aggregation statefilament

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Sample preparation

Concentration0.45 mg/mL
BufferpH: 7.5
Component:
ConcentrationFormulaName
50.0 mMKClpotassium chloride
1.0 mMMgCl2magnesium chloride
1.0 mMC14H24N2O10EGTA
10.0 mMC3H4N2imidazole
2.0 mMC4H11NO3Tris hydrochloride
5.0 mMC4H10O2S2dithiothreitol
200.0 mMC10H16N5O13P3adenosine triphosphate
0.01 %NaN3sodium azide
5.0 mMMg-ADPmagnesium-adenosine diphosphate

Details: Buffer was filtered through 0.44 um filter and degassed.
GridModel: C-flat-1.2/1.3 / Material: COPPER / Mesh: 200 / Pretreatment - Type: PLASMA CLEANING / Pretreatment - Time: 6 sec. / Pretreatment - Atmosphere: OTHER
VitrificationCryogen name: ETHANE / Chamber humidity: 95 % / Chamber temperature: 298 K / Instrument: LEICA EM GP
Details: Sample was applied to a glow-discharged holey carbon grid. 3 uL actin was incubated for 60 seconds. 3 uL of myosin VI was added and incubated for 60 seconds. 3 uL solution was removed. An ...Details: Sample was applied to a glow-discharged holey carbon grid. 3 uL actin was incubated for 60 seconds. 3 uL of myosin VI was added and incubated for 60 seconds. 3 uL solution was removed. An additional 3 uL of myosin VI was applied. After 60 seconds, 3 uL solution was removed, and the grid was blotted for 3 seconds from the backside with filter paper..
Details0.45 mg/mL myosin VI was added to 0.025 mg/mL actin

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Electron microscopy

MicroscopeFEI TECNAI 20
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 100.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.0 mm / Nominal defocus max: 3.0 µm / Nominal defocus min: 1.5 µm / Nominal magnification: 29000
Sample stageSpecimen holder model: GATAN 626 SINGLE TILT LIQUID NITROGEN CRYO TRANSFER HOLDER
Cooling holder cryogen: NITROGEN
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Digitization - Dimensions - Width: 3838 pixel / Digitization - Dimensions - Height: 3710 pixel / Digitization - Frames/image: 1-24 / Number grids imaged: 2 / Number real images: 377 / Average exposure time: 0.25 sec. / Average electron dose: 1.5 e/Å2

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Image processing

Startup modelType of model: EMDB MAP
EMDB ID:

1990

Final angle assignmentType: NOT APPLICABLE / Software - Name: FREALIGN (ver. 9.11)
Final reconstructionNumber classes used: 1
Applied symmetry - Helical parameters - Δz: 28.06 Å
Applied symmetry - Helical parameters - Δ&Phi: -166.69 °
Applied symmetry - Helical parameters - Axial symmetry: C1 (asymmetric)
Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 5.5 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: FREALIGN (ver. 9.11) / Number images used: 36114
FSC plot (resolution estimation)

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Atomic model buiding 1

DetailsInitial models were assembled from 8 actins (3J8A) and 6 myosins (4PFO) through rigid body docking in Chimera, followed by flexible fitting with DireX. Resulting models were subjected to MDFF.
RefinementSpace: REAL / Protocol: FLEXIBLE FIT / Overall B value: 200
Output model

PDB-6bnq:
CryoEM structure of Myosin VI-Actin complex in the ADP state

PDB-6bnw:
CryoEM structure of Myosin VI-Actin complex in the ADP state, backbone-averaged with side chains truncated to alanine

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