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- EMDB-6450: Unsharpened cryo-EM reconstruction of the Vt-GFP-actin interface ... -

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Basic information

Entry
Database: EMDB / ID: EMD-6450
TitleUnsharpened cryo-EM reconstruction of the Vt-GFP-actin interface for difference map calculation
Map dataUnsharpened reconstruction of C-terminal GFP fusion to the vinculin tail domain (Vt-GFP) bound to actin for difference map calculation
Sample
  • Sample: Vt-GFP bound to F-actin
  • Protein or peptide: skeletal muscle actin
  • Protein or peptide: Vinculin tail domain, residues 879-1061
Keywordsactin / vinculin / cell migration / adhesion / mechanosensation / cytoskeleton
Function / homology
Function and homology information


muscle tendon junction / Platelet degranulation / Smooth Muscle Contraction / regulation of protein localization to adherens junction / outer dense plaque of desmosome / inner dense plaque of desmosome / podosome ring / terminal web / epithelial cell-cell adhesion / zonula adherens ...muscle tendon junction / Platelet degranulation / Smooth Muscle Contraction / regulation of protein localization to adherens junction / outer dense plaque of desmosome / inner dense plaque of desmosome / podosome ring / terminal web / epithelial cell-cell adhesion / zonula adherens / fascia adherens / MAP2K and MAPK activation / dystroglycan binding / muscle alpha-actinin binding / alpha-catenin binding / vinculin binding / cell-cell contact zone / apical junction assembly / costamere / regulation of establishment of endothelial barrier / axon extension / adherens junction assembly / protein localization to cell surface / lamellipodium assembly / regulation of focal adhesion assembly / cytoskeletal motor activator activity / myosin heavy chain binding / tropomyosin binding / actin filament bundle / troponin I binding / filamentous actin / mesenchyme migration / alpha-actinin binding / brush border / actin filament bundle assembly / skeletal muscle myofibril / striated muscle thin filament / skeletal muscle thin filament assembly / actin monomer binding / skeletal muscle fiber development / stress fiber / titin binding / actin filament polymerization / regulation of cell migration / Neutrophil degranulation / morphogenesis of an epithelium / cell projection / actin filament / adherens junction / filopodium / neuromuscular junction / sarcolemma / beta-catenin binding / Hydrolases; Acting on acid anhydrides; Acting on acid anhydrides to facilitate cellular and subcellular movement / Z disc / calcium-dependent protein binding / actin filament binding / cell-cell junction / lamellipodium / actin cytoskeleton / cell body / scaffold protein binding / cytoskeleton / cell adhesion / mitochondrial inner membrane / hydrolase activity / cadherin binding / protein domain specific binding / focal adhesion / calcium ion binding / ubiquitin protein ligase binding / positive regulation of gene expression / structural molecule activity / magnesium ion binding / protein homodimerization activity / protein-containing complex / ATP binding / identical protein binding / plasma membrane / 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) / Gallus gallus (chicken)
Methodhelical reconstruction / cryo EM / Resolution: 19.2 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: 3.5
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 3.5
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_6450.png

Downloads & links

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Map

FileDownload / File: emd_6450.map.gz / Format: CCP4 / Size: 62.5 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationUnsharpened reconstruction of C-terminal GFP fusion to the vinculin tail domain (Vt-GFP) bound to actin for difference map calculation
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
2.18 Å/pix.
x 256 pix.
= 558.08 Å		2.18 Å/pix.
x 256 pix.
= 558.08 Å		2.18 Å/pix.
x 256 pix.
= 558.08 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: 3.5 / Movie #1: 3.5
Minimum - Maximum-3.68592596 - 14.095081329999999
Average (Standard dev.)0.00000001 (±1.0)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-128-128-128
Dimensions256256256
Spacing256256256
CellA=B=C: 558.08 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z2.182.182.18
M x/y/z256256256
origin x/y/z0.0000.0000.000
length x/y/z558.080558.080558.080
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS-128-128-128
NC/NR/NS256256256
D min/max/mean-3.68614.0950.000

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Supplemental data

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Sample components

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Entire : Vt-GFP bound to F-actin

EntireName: Vt-GFP bound to F-actin
Components
  • Sample: Vt-GFP bound to F-actin
  • Protein or peptide: skeletal muscle actin
  • Protein or peptide: Vinculin tail domain, residues 879-1061

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Supramolecule #1000: Vt-GFP bound to F-actin

SupramoleculeName: Vt-GFP bound to F-actin / type: sample / ID: 1000
Details: Helical filament with one vinculin tail domain bound per actin protomer
Oligomeric state: One vinculin 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: Vinculin tail domain, residues 879-1061

MacromoleculeName: Vinculin tail domain, residues 879-1061 / type: protein_or_peptide / ID: 2 / Name.synonym: Vt-GFP / Details: C-terminal GFP fusion / Oligomeric state: helical / Recombinant expression: Yes
Source (natural)Organism: Gallus gallus (chicken) / synonym: Chicken / Organelle: Focal adhesion / Location in cell: Cytoplasmic
Molecular weightTheoretical: 55.1 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria) / Recombinant strain: BL21(DE3) / Recombinant plasmid: H6-msfGFP (Addgene #29725)
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 Vt-GFP was then applied and incubated for 60 seconds. ...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 Vt-GFP was then applied and incubated for 60 seconds. 3 microliters of solution was removed, then an additional 3 microliters of Vt-GFP 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.
DateMay 12, 2014
Image recordingCategory: CCD / Film or detector model: GATAN ULTRASCAN 4000 (4k x 4k) / Number real images: 260 / 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: 27.87 Å
Applied symmetry - Helical parameters - Δ&Phi: 166.60 °
Applied symmetry - Helical parameters - Axial symmetry: C1 (asymmetric)
Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 19.2 Å / Resolution method: OTHER / Software - Name: CTFFIND3, EMAN2/SPARX, FREALIGN
CTF correctionDetails: FREALIGN (per segment)

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