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- EMDB-7116: CryoEM structure of MyosinVI-actin complex in the rigor (nucleoti... -

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Entry
Database: EMDB / ID: 7116
TitleCryoEM structure of MyosinVI-actin complex in the rigor (nucleotide-free) state
Map dataCryo-EM structure of myosin VI-actin complex, B factor sharpened to -150
Samplemyosin VI-actin in the rigor (nucleotide-free) state:
myosin VI / actin / Unconventional myosin-VI / Actin, alpha skeletal muscle / (ligand) x 2
Function / homologyStimuli-sensing channels / Myosin S1 fragment, N-terminal / Actin / Calmodulin / Kinesin motor domain superfamily / Class VI myosin, motor domain / Myosin VI, cargo binding domain / Phase 0 - rapid depolarisation / VEGFR2 mediated vascular permeability / P-loop containing nucleoside triphosphate hydrolase ...Stimuli-sensing channels / Myosin S1 fragment, N-terminal / Actin / Calmodulin / Kinesin motor domain superfamily / Class VI myosin, motor domain / Myosin VI, cargo binding domain / Phase 0 - rapid depolarisation / VEGFR2 mediated vascular permeability / P-loop containing nucleoside triphosphate hydrolase / Actin/actin-like conserved site / EF-Hand 1, calcium-binding site / EF-hand domain pair / Myosin, N-terminal, SH3-like / EF-hand domain pair / Actin, conserved site / Actin family / Activation of Ca-permeable Kainate Receptor / EF-hand domain / Myosin head, motor domain / Smooth Muscle Contraction / Ca2+ pathway / Reduction of cytosolic Ca++ levels / Sodium/Calcium exchangers / CREB phosphorylation through the activation of CaMKK / FCERI mediated Ca+2 mobilization / Ras activation upon Ca2+ influx through NMDA receptor / Myosin head (motor domain) / Ion homeostasis / Activation of CaMK IV / Protein methylation / Inactivation, recovery and regulation of the phototransduction cascade / eNOS activation / Synthesis of IP3 and IP4 in the cytosol / PKA activation / Tetrahydrobiopterin (BH4) synthesis, recycling, salvage and regulation / Platelet degranulation / Trafficking of AMPA receptors / Cam-PDE 1 activation / Gap junction degradation / Calmodulin induced events / Ion transport by P-type ATPases / Glycogen breakdown (glycogenolysis) / Myosin VI cargo binding domain / RAF/MAP kinase cascade / RHO GTPases activate PAKs / RHO GTPases activate IQGAPs / CaMK IV-mediated phosphorylation of CREB / Myosin N-terminal SH3-like domain profile. / Myosin motor domain profile. / EF-hand calcium-binding domain profile. / Actins and actin-related proteins signature. / Actins signature 2. / Actins signature 1. / CLEC7A (Dectin-1) induces NFAT activation / EF-hand calcium-binding domain. / CREB phosphorylation through the activation of CaMKII / CH domain binding / regulation of secretion / RNA polymerase II, holoenzyme / actin filament-based movement / clathrin-coated endocytic vesicle / positive regulation of actin-dependent ATPase activity / myosin complex / mesenchyme migration / myosin binding / tropomyosin binding / N-terminal myristoylation domain binding / positive regulation by host of symbiont cAMP-mediated signal transduction / myosin heavy chain binding / troponin I binding / skeletal muscle thin filament assembly / striated muscle thin filament / protein phosphatase activator activity / adenylate cyclase activator activity / positive regulation of ryanodine-sensitive calcium-release channel activity / actin filament bundle / positive regulation of cyclic-nucleotide phosphodiesterase activity / filamentous actin / catalytic complex / adenylate cyclase binding / detection of calcium ion / actin monomer binding / skeletal muscle fiber development / DNA damage response, signal transduction by p53 class mediator / negative regulation of ryanodine-sensitive calcium-release channel activity / regulation of cardiac muscle contraction / voltage-gated potassium channel complex / actin filament bundle assembly / calcium channel inhibitor activity / skeletal muscle myofibril / motor activity / regulation of release of sequestered calcium ion into cytosol by sarcoplasmic reticulum / positive regulation of phosphoprotein phosphatase activity / stress fiber / calcium channel complex / ADP binding / sarcomere / titin binding / actin filament polymerization
Function and homology information
SourceSus scrofa (pig) / Oryctolagus cuniculus (rabbit)
Methodhelical reconstruction / cryo EM / 4.6 Å resolution
AuthorsGurel PS / Alushin GA
Citation
Journal: Elife / Year: 2017
Title: Cryo-EM structures reveal specialization at the myosin VI-actin interface and a mechanism of force sensitivity.
Authors: Pinar S Gurel / Laura Y Kim / Paul V Ruijgrok / Tosan Omabegho / Zev Bryant / Gregory M Alushin
#1: Journal: 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
Validation ReportPDB-ID: 6bnp

SummaryFull report
PDB-ID: 6bnv

SummaryFull report
About validation report
DateDeposition: Nov 17, 2017 / Header (metadata) release: Jan 10, 2018 / Map release: Jan 10, 2018 / Last update: Jan 17, 2018

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Structure visualization

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

Downloads & links

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Map

Fileemd_7116.map.gz (map file in CCP4 format, 536871 KB)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
512 pix
1.27 Å/pix.
= 650.24 Å
512 pix
1.27 Å/pix.
= 650.24 Å
512 pix
1.27 Å/pix.
= 650.24 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.27 Å
Density
Contour Level:6 (by author), 6 (movie #1):
Minimum - Maximum-9.431532 - 20.607714
Average (Standard dev.)1.548081E-9 (1)
Details

EMDB XML:

Space Group Number1
Map Geometry
Axis orderXYZ
Dimensions512512512
Origin-256-256-256
Limit255255255
Spacing512512512
CellA=B=C: 650.24 Å
α=β=γ: 90 deg.

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
start NX/NY/NZ
NX/NY/NZ
MAP C/R/S123
start NC/NR/NS-256-256-256
NC/NR/NS512512512
D min/max/mean-9.43220.6080.000

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Supplemental data

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Mask #1

Fileemd_7116_msk_1.map ( map file in CCP4 format, 536871 KB )
Projections & Slices
AxesZYX
Projections
Slices (1/2)
Density Histograms
Data typeImage stored as Reals
Annotation detailsHalf map for the myosin VI-actin reconstruction, masked at 90A to exclude lower resolution regions of the density map including the lever arm
Space group number1

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Mask #2

Fileemd_7116_msk_2.map ( map file in CCP4 format, 536871 KB )
Projections & Slices
AxesZYX
Projections
Slices (1/2)
Density Histograms
Data typeImage stored as Reals
Annotation detailsHalf map for the myosin VI-actin reconstruction, masked at 90A to exclude lower resolution regions of the density map including the lever arm
Space group number1

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Mask #2~

Fileemd_7116_msk_2.map
Projections & Slices
AxesZYX
Projections
Slices (1/2)
Density Histograms

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Sample components

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Entire myosin VI-actin in the rigor (nucleotide-free) state

EntireName: myosin VI-actin in the rigor (nucleotide-free) state / Number of components: 7
MassTheoretical: 42 kDa

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Component #1: protein, myosin VI-actin in the rigor (nucleotide-free) state

ProteinName: myosin VI-actin in the rigor (nucleotide-free) state / Recombinant expression: No
MassTheoretical: 42 kDa

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Component #2: protein, myosin VI

ProteinName: myosin VI / Recombinant expression: No
SourceSpecies: Sus scrofa (pig)
Source (engineered)Expression System: Spodoptera frugiperda (fall armyworm)

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Component #3: protein, actin

ProteinName: actin / Recombinant expression: No
SourceSpecies: Oryctolagus cuniculus (rabbit)

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Component #4: protein, Unconventional myosin-VI

ProteinName: Unconventional myosin-VI / Recombinant expression: No
MassTheoretical: 79.837367 kDa
Source (engineered)Expression System: Sus scrofa (pig) / Vector: pBiex-1

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Component #5: protein, Actin, alpha skeletal muscle

ProteinName: Actin, alpha skeletal muscle / Recombinant expression: No
MassTheoretical: 41.560266 kDa
SourceSpecies: Oryctolagus cuniculus (rabbit)
Source (natural)Organ or tissue: Skeletal Muscle

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Component #6: ligand, MAGNESIUM ION

LigandName: MAGNESIUM ION / Number of Copies: 8 / Recombinant expression: No
MassTheoretical: 2.430505 MDa

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Component #7: ligand, ADENOSINE-5'-DIPHOSPHATE

LigandName: ADENOSINE-5'-DIPHOSPHATE / Number of Copies: 8 / Recombinant expression: No
MassTheoretical: 0.427201 kDa

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Experimental details

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Sample preparation

SpecimenSpecimen state: filament / Method: cryo EM
Helical parametersAxial symmetry: C1 (asymmetric) / Delta z: 28.06 Å / Delta phi: -166.73 deg.
Sample solutionSpecimen conc.: 0.45 mg/ml
Buffer solution: Buffer was filtered through 0.44 um filter and degassed.
pH: 7.5
VitrificationInstrument: LEICA EM GP / Cryogen name: ETHANE / Temperature: 298 K / Humidity: 95 %
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.

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Electron microscopy imaging

ImagingMicroscope: FEI TECNAI 20
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Electron dose: 1.5 e/Å2 / Illumination mode: FLOOD BEAM
LensMagnification: 29000 X (nominal) / Cs: 2 mm / Imaging mode: BRIGHT FIELD / Defocus: 1500 - 3000 nm
Specimen HolderModel: GATAN LIQUID NITROGEN
CameraDetector: GATAN K2 (4k x 4k)

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Image acquisition

Image acquisitionNumber of digital images: 778 / Sampling size: 5 microns

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Image processing

ProcessingMethod: helical reconstruction
3D reconstructionAlgorithm: FOURIER SPACE / Software: FREALIGN / Resolution: 4.6 Å / Resolution method: FSC 0.143 CUT-OFF
FSC plot
(resolution estimation)

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Atomic model buiding

Output model

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