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- EMDB-6447: Cryo-EM reconstruction of the metavinculin-actin interface -

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Basic information

Entry
Database: EMDB / ID: EMD-6447
TitleCryo-EM reconstruction of the metavinculin-actin interface
Map dataReconstruction of metavinculin tail domain bound to F-actin
Sample
  • Sample: Metavinculin tail domain bound to F-actin
  • Protein or peptide: skeletal muscle actin
  • Protein or peptide: Metavinculin tail domain, residues 858-1129
Keywordsactin / metavinculin / vinculin / cell migration / adhesion / mechanosensation / cytoskeleton
Function / homology
Function and homology information


regulation of protein localization to adherens junction / outer dense plaque of desmosome / inner dense plaque of desmosome / podosome ring / terminal web / cell-substrate junction / epithelial cell-cell adhesion / zonula adherens / dystroglycan binding / alpha-catenin binding ...regulation of protein localization to adherens junction / outer dense plaque of desmosome / inner dense plaque of desmosome / podosome ring / terminal web / cell-substrate junction / epithelial cell-cell adhesion / zonula adherens / dystroglycan binding / alpha-catenin binding / fascia adherens / cell-cell contact zone / apical junction assembly / costamere / regulation of establishment of endothelial barrier / adherens junction assembly / axon extension / protein localization to cell surface / lamellipodium assembly / regulation of focal adhesion assembly / cytoskeletal motor activator activity / maintenance of blood-brain barrier / tropomyosin binding / myosin heavy chain binding / troponin I binding / mesenchyme migration / filamentous actin / actin filament bundle / Turbulent (oscillatory, disturbed) flow shear stress activates signaling by PIEZO1 and integrins in endothelial cells / brush border / striated muscle thin filament / actin filament bundle assembly / skeletal muscle thin filament assembly / skeletal muscle myofibril / actin monomer binding / Smooth Muscle Contraction / stress fiber / skeletal muscle fiber development / titin binding / actin filament polymerization / negative regulation of cell migration / cell-matrix adhesion / filopodium / cell projection / morphogenesis of an epithelium / actin filament / adherens junction / Signaling by high-kinase activity BRAF mutants / MAP2K and MAPK activation / Hydrolases; Acting on acid anhydrides; Acting on acid anhydrides to facilitate cellular and subcellular movement / sarcolemma / beta-catenin binding / platelet aggregation / specific granule lumen / calcium-dependent protein binding / Signaling by RAF1 mutants / Signaling by moderate kinase activity BRAF mutants / Paradoxical activation of RAF signaling by kinase inactive BRAF / Signaling downstream of RAS mutants / Signaling by BRAF and RAF1 fusions / Signaling by ALK fusions and activated point mutants / cell-cell junction / extracellular vesicle / Platelet degranulation / lamellipodium / High laminar flow shear stress activates signaling by PIEZO1 and PECAM1:CDH5:KDR in endothelial cells / actin binding / cell body / secretory granule lumen / molecular adaptor activity / ficolin-1-rich granule lumen / cytoskeleton / cell adhesion / hydrolase activity / cadherin binding / membrane raft / protein domain specific binding / focal adhesion / ubiquitin protein ligase binding / calcium ion binding / Neutrophil degranulation / positive regulation of gene expression / structural molecule activity / magnesium ion binding / protein-containing complex / extracellular exosome / extracellular region / ATP binding / identical protein binding / plasma membrane / cytosol / cytoplasm
Similarity search - Function
Vinculin repeated domain signature. / Vinculin / Vinculin, conserved site / Vinculin family talin-binding region signature. / Vinculin/alpha-catenin / Vinculin family / Alpha-catenin/vinculin-like superfamily / Actins signature 1. / Actin, conserved site / Actins signature 2. ...Vinculin repeated domain signature. / Vinculin / Vinculin, conserved site / Vinculin family talin-binding region signature. / Vinculin/alpha-catenin / Vinculin family / Alpha-catenin/vinculin-like 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 / ATPase, nucleotide binding domain
Similarity search - Domain/homology
Vinculin / Actin, alpha skeletal muscle
Similarity search - Component
Biological speciesOryctolagus cuniculus (rabbit) / Homo sapiens (human)
Methodhelical reconstruction / cryo EM / Resolution: 8.2 Å
AuthorsKim LY / Thompson PM / Lee HT / Pershad M / Campbell SL / Alushin GM
CitationJournal: J Mol Biol / Year: 2016
Title: The Structural Basis of Actin Organization by Vinculin and Metavinculin.
Authors: Laura Y Kim / Peter M Thompson / Hyunna T Lee / Mihir Pershad / Sharon L Campbell / Gregory M Alushin /
Abstract: Vinculin is an essential adhesion protein that links membrane-bound integrin and cadherin receptors through their intracellular binding partners to filamentous actin, facilitating mechanotransduction. ...Vinculin is an essential adhesion protein that links membrane-bound integrin and cadherin receptors through their intracellular binding partners to filamentous actin, facilitating mechanotransduction. Here we present an 8.5-Å-resolution cryo-electron microscopy reconstruction and pseudo-atomic model of the vinculin tail (Vt) domain bound to F-actin. Upon actin engagement, the N-terminal "strap" and helix 1 are displaced from the Vt helical bundle to mediate actin bundling. We find that an analogous conformational change also occurs in the H1' helix of the tail domain of metavinculin (MVt) upon actin binding, a muscle-specific splice isoform that suppresses actin bundling by Vt. These data support a model in which metavinculin tunes the actin bundling activity of vinculin in a tissue-specific manner, providing a mechanistic framework for understanding metavinculin mutations associated with hereditary cardiomyopathies.
History
DepositionSep 3, 2015-
Header (metadata) releaseSep 23, 2015-
Map releaseNov 4, 2015-
UpdateFeb 17, 2016-
Current statusFeb 17, 2016Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 6.5
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 6.5
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-3jbk
  • Surface level: 6.5
  • Imaged by UCSF Chimera
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  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-3jbk
  • Imaged by Jmol
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_6447.png

Downloads & links

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Map

FileDownload / File: emd_6447.map.gz / Format: CCP4 / Size: 29.8 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationReconstruction of metavinculin tail domain bound to F-actin
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
2.18 Å/pix.
x 200 pix.
= 436. Å		2.18 Å/pix.
x 200 pix.
= 436. Å		2.18 Å/pix.
x 200 pix.
= 436. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 2.18 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 6.5 / Movie #1: 6.5
Minimum - Maximum-7.91757345 - 19.67712212
Average (Standard dev.)0.0 (±1.0)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-69-69-42
Dimensions200200200
Spacing200200200
CellA=B=C: 436.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z2.182.182.18
M x/y/z200200200
origin x/y/z0.0000.0000.000
length x/y/z436.000436.000436.000
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS-69-69-42
NC/NR/NS200200200
D min/max/mean-7.91819.6770.000

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Supplemental data

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Sample components

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Entire : Metavinculin tail domain bound to F-actin

EntireName: Metavinculin tail domain bound to F-actin
Components
  • Sample: Metavinculin tail domain bound to F-actin
  • Protein or peptide: skeletal muscle actin
  • Protein or peptide: Metavinculin tail domain, residues 858-1129

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Supramolecule #1000: Metavinculin tail domain bound to F-actin

SupramoleculeName: Metavinculin tail domain bound to F-actin / type: sample / ID: 1000
Details: Helical filament with one metavinculin tail domain bound per actin protomer
Oligomeric state: One metavinculin tail domain per actin protomer
Number unique components: 2

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Macromolecule #1: skeletal muscle actin

MacromoleculeName: skeletal muscle actin / type: protein_or_peptide / ID: 1 / Name.synonym: actin / Oligomeric state: helical filament / Recombinant expression: No / Database: NCBI
Source (natural)Organism: Oryctolagus cuniculus (rabbit) / synonym: Rabbit / Tissue: Muscle / Location in cell: Cytoplasm, cytoskeleton
Molecular weightTheoretical: 41.8 KDa
SequenceUniProtKB: Actin, alpha skeletal muscle

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Macromolecule #2: Metavinculin tail domain, residues 858-1129

MacromoleculeName: Metavinculin tail domain, residues 858-1129 / type: protein_or_peptide / ID: 2 / Name.synonym: MVt / Oligomeric state: helical / Recombinant expression: Yes
Source (natural)Organism: Homo sapiens (human) / synonym: Human / Organelle: Focal adhesion / Location in cell: Cytoplasmic
Molecular weightTheoretical: 29.6 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria) / Recombinant strain: BL21(DE3) / Recombinant plasmid: 2HR-T (Addgene #29718)
SequenceUniProtKB: Vinculin

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Experimental details

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Structure determination

Methodcryo EM
Processinghelical reconstruction
Aggregation statefilament

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Sample preparation

Concentration0.0125 mg/mL
BufferpH: 7 / Details: 50 mM KCl, 1 mM MgCl2, 1 mM EGTA, 10 mM imidazole
GridDetails: 200 mesh 1.2 / 1.3 C-flat
VitrificationCryogen name: ETHANE / Chamber humidity: 90 % / Instrument: LEICA EM GP
Method: 3 microliters of 0.3 micromolar actin was applied to the grid and incubated for 60 seconds at 25 degrees C. 3 microliters of 10 micromolar MVt was then applied and incubated for 60 seconds. 3 ...Method: 3 microliters of 0.3 micromolar actin was applied to the grid and incubated for 60 seconds at 25 degrees C. 3 microliters of 10 micromolar MVt was then applied and incubated for 60 seconds. 3 microliters of solution was removed, then an additional 3 microliters of MVt applied. After 60 seconds, 3 microliters of solution was removed, then the grid was blotted for 2 seconds before plunging.

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Electron microscopy

MicroscopeFEI TECNAI 20
Alignment procedureLegacy - Astigmatism: Objective lens astigmatism was corrected at 100,000 times magnification.
DateSep 10, 2014
Image recordingCategory: CCD / Film or detector model: GATAN ULTRASCAN 4000 (4k x 4k) / Number real images: 671 / Average electron dose: 25 e/Å2
Electron beamAcceleration voltage: 120 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 137615 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.0 mm / Nominal defocus max: 3.0 µm / Nominal defocus min: 1.5 µm / Nominal magnification: 100000
Sample stageSpecimen holder model: GATAN LIQUID NITROGEN

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Image processing

DetailsMulti-model IHRSR was performed using EMAN2 / SPARX to select for bound segments, followed by single-model IHRSR with EMAN2 / SPARX and final reconstruction with FREALIGN (fixed helical parameters).
Final reconstructionApplied symmetry - Helical parameters - Δz: 27.80 Å
Applied symmetry - Helical parameters - Δ&Phi: 166.75 °
Applied symmetry - Helical parameters - Axial symmetry: C1 (asymmetric)
Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 8.2 Å / Resolution method: OTHER / Software - Name: CTFFIND3, EMAN2/SPARX, FREALIGN
CTF correctionDetails: FREALIGN (per segment)

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Atomic model buiding 1

Initial modelPDB ID:

1qkr


Chain - Chain ID: A
SoftwareName: Chimera, MDFF
DetailsComponents were initially rigid body fit using Chimera, followed by flexible fitting with MDFF.
RefinementSpace: REAL / Protocol: FLEXIBLE FIT
Output model

PDB-3jbk:
Cryo-EM reconstruction of the metavinculin-actin interface

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Atomic model buiding 2

Initial modelPDB ID:

3j8a


Chain - Chain ID: A
SoftwareName: MDFF
DetailsComponents were initially rigid body fit using Chimera, followed by flexible fitting with MDFF.
RefinementSpace: REAL / Protocol: FLEXIBLE FIT
Output model

PDB-3jbk:
Cryo-EM reconstruction of the metavinculin-actin interface

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