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

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Entry
Database: EMDB / ID: 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.
Samplemyosin VI-actin in the ADP state
Function/homologyminus-end directed microfilament motor activity / Trafficking of AMPA receptors / unconventional myosin complex / Gap junction degradation / Gap junction degradation / Myosin VI, cargo binding domain / Class VI myosin, motor domain / regulation of secretion / Myosin VI cargo binding domain / DNA-directed RNA polymerase II, holoenzyme ...minus-end directed microfilament motor activity / Trafficking of AMPA receptors / unconventional myosin complex / Gap junction degradation / Gap junction degradation / Myosin VI, cargo binding domain / Class VI myosin, motor domain / regulation of secretion / Myosin VI cargo binding domain / DNA-directed RNA polymerase II, holoenzyme / actin filament-based movement / clathrin-coated endocytic vesicle / Trafficking of AMPA receptors / positive regulation of actin-dependent ATPase activity / mesenchyme migration / Myosin N-terminal SH3-like domain profile. / myosin complex / tropomyosin binding / myosin heavy chain binding / Myosin S1 fragment, N-terminal / troponin I binding / skeletal muscle thin filament assembly / striated muscle thin filament / actin filament bundle / Actins signature 1. / Actin, conserved site / Actins signature 2. / filamentous actin / Myosin motor domain profile. / Myosin head, motor domain / Actin/actin-like conserved site / Actins and actin-related proteins signature. / Actin family / actin monomer binding / skeletal muscle fiber development / DNA damage response, signal transduction by p53 class mediator / actin filament bundle assembly / clathrin-coated pit / motor activity / skeletal muscle myofibril / Myosin head (motor domain) / stress fiber / ADP binding / titin binding / clathrin-coated vesicle membrane / actin filament polymerization / Kinesin motor domain superfamily / actin filament / Actin / endocytic vesicle / filopodium / ruffle / calcium-dependent protein binding / intracellular protein transport / sensory perception of sound / cell body / microvillus / ruffle membrane / cell cortex / lysosomal membrane / endocytosis / actin filament binding / actin binding / lamellipodium / apical part of cell / cytoplasmic vesicle / nuclear membrane / calmodulin binding / cadherin binding / response to drug / protein domain specific binding / positive regulation of gene expression / magnesium ion binding / calcium ion binding / perinuclear region of cytoplasm / Golgi apparatus / positive regulation of transcription by RNA polymerase II / P-loop containing nucleoside triphosphate hydrolase / membrane / extracellular exosome / nucleoplasm / ATP binding / identical protein binding / plasma membrane / nucleus / cytosol / cytoplasm / Unconventional myosin-VI / Actin, alpha skeletal muscle / Unconventional myosin-VI / Unconventional myosin-VI
Function and homology information
SourceSus scrofa / mammal / image: Sus scrofa domestica
Oryctolagus cuniculus / mammal / European rabbit /
Methodhelical reconstruction, at 5.5 Å resolution
AuthorsGurel PG / Alushin GM
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
Abstract: Despite extensive scrutiny of the myosin superfamily, the lack of high-resolution structures of actin-bound states has prevented a complete description of its mechanochemical cycle and limited ...Despite extensive scrutiny of the myosin superfamily, the lack of high-resolution structures of actin-bound states has prevented a complete description of its mechanochemical cycle and limited insight into how sequence and structural diversification of the motor domain gives rise to specialized functional properties. Here we present cryo-EM structures of the unique minus-end directed myosin VI motor domain in rigor (4.6 Å) and Mg-ADP (5.5 Å) states bound to F-actin. Comparison to the myosin IIC-F-actin rigor complex reveals an almost complete lack of conservation of residues at the actin-myosin interface despite preservation of the primary sequence regions composing it, suggesting an evolutionary path for motor specialization. Additionally, analysis of the transition from ADP to rigor provides a structural rationale for force sensitivity in this step of the mechanochemical cycle. Finally, we observe reciprocal rearrangements in actin and myosin accompanying the transition between these states, supporting a role for actin structural plasticity during force generation by myosin VI.
#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
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.
Validation ReportPDB-ID: 6bnq

SummaryFull report
PDB-ID: 6bnw

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: 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
  • Download
  • Surface view with fitted model
  • Atomic models: : PDB-6bnw
  • Surface level: 5.8
  • Imaged by UCSF CHIMERA
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  • Simplified surface model + fitted atomic model
  • Atomic models: PDB-6bnq
  • Imaged by Jmol
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  • Simplified surface model + fitted atomic model
  • Atomic models: PDB-6bnw
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3D viewer
Supplemental images

Downloads & links

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Map

Fileemd_7117.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 package.

Voxel sizeX=Y=Z: 1.27 Å
Density
Contour Level:5.8 (by author), 5.8 (movie #1):
Minimum - Maximum-7.0485396 - 18.04262
Average (Standard dev.)-1.499693E-9 (0.99999994)
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-7.04918.043-0.000

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Supplemental data

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Mask #1

Fileemd_7117_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 of the myosin VI-actin complex in the ADP state, masked at 90A to exclude lower density portions of the density map including the lever arm
Space group number1

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Mask #2

Fileemd_7117_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 of the myosin VI-actin complex in the ADP state, masked at 90A to exclude lower density portions of the density map including the lever arm
Space group number1

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Mask #2~

Fileemd_7117_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 ADP state

EntireName: myosin VI-actin in the ADP state / Number of components: 7
MassTheoretical: 122 kDa

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Component #1: protein, myosin VI-actin in the ADP state

ProteinName: myosin VI-actin in the ADP state / Recombinant expression: No
MassTheoretical: 122 kDa

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Component #2: protein, myosin VI

ProteinName: myosin VI / Recombinant expression: No
SourceSpecies: Sus scrofa / mammal / image: Sus scrofa domestica
Source (engineered)Expression System: Spodoptera frugiperda / arthropod / Fall armyworm

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Component #3: protein, actin

ProteinName: actin / Recombinant expression: No
SourceSpecies: Oryctolagus cuniculus / mammal / European 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 / mammal / image: Sus scrofa domestica
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 / mammal / European 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

Specimen statefilament
Helical parametersAxial symmetry: C1 (asymmetric) / Delta z: 28.06 Å / Delta phi: -166.69 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: 377 / Sampling size: 5 microns

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Image processing

ProcessingMethod: helical reconstruction
3D reconstructionAlgorithm: FOURIER SPACE / Software: FREALIGN / Resolution: 5.5 Å / Resolution method: FSC 0.143 CUT-OFF
FSC plot (resolution assessment)

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Atomic model buiding

Output model

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