+Open data
-Basic information
Entry | Database: EMDB / ID: EMD-6446 | |||||||||
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Title | Cryo-EM reconstruction of the vinculin-actin interface | |||||||||
Map data | Reconstruction of vinculin tail domain bound to F-actin | |||||||||
Sample |
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Keywords | actin / 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 / muscle alpha-actinin binding / dystroglycan binding / MAP2K and MAPK activation / alpha-catenin binding / vinculin 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 / tropomyosin binding / myosin heavy chain binding / mesenchyme migration / troponin I binding / alpha-actinin binding / filamentous actin / actin filament bundle / brush border / skeletal muscle thin filament assembly / actin filament bundle assembly / striated muscle thin filament / skeletal muscle myofibril / actin monomer binding / skeletal muscle fiber development / stress fiber / titin binding / regulation of cell migration / actin filament polymerization / filopodium / cell projection / Neutrophil degranulation / actin filament / morphogenesis of an epithelium / adherens junction / Hydrolases; Acting on acid anhydrides; Acting on acid anhydrides to facilitate cellular and subcellular movement / neuromuscular junction / sarcolemma / Z disc / beta-catenin binding / calcium-dependent protein binding / cell-cell junction / actin filament binding / actin cytoskeleton / lamellipodium / cell body / scaffold protein binding / mitochondrial inner membrane / cytoskeleton / cell adhesion / hydrolase activity / cadherin binding / protein domain specific binding / focal adhesion / ubiquitin protein ligase binding / calcium ion 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 | |||||||||
Biological species | Oryctolagus cuniculus (rabbit) / Gallus gallus (chicken) | |||||||||
Method | helical reconstruction / cryo EM / Resolution: 8.5 Å | |||||||||
Authors | Kim LY / Thompson PM / Lee HT / Pershad M / Campbell SL / Alushin GM | |||||||||
Citation | Journal: 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 |
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-Structure visualization
Movie |
Movie viewer |
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Structure viewer | EM map: SurfViewMolmilJmol/JSmol |
Supplemental images |
-Downloads & links
-EMDB archive
Map data | emd_6446.map.gz | 25.4 MB | EMDB map data format | |
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Header (meta data) | emd-6446-v30.xml emd-6446.xml | 13.1 KB 13.1 KB | Display Display | EMDB header |
Images | emd_6446.png | 204 KB | ||
Archive directory | http://ftp.pdbj.org/pub/emdb/structures/EMD-6446 ftp://ftp.pdbj.org/pub/emdb/structures/EMD-6446 | HTTPS FTP |
-Validation report
Summary document | emd_6446_validation.pdf.gz | 315.1 KB | Display | EMDB validaton report |
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Full document | emd_6446_full_validation.pdf.gz | 314.6 KB | Display | |
Data in XML | emd_6446_validation.xml.gz | 5.9 KB | Display | |
Arichive directory | https://ftp.pdbj.org/pub/emdb/validation_reports/EMD-6446 ftp://ftp.pdbj.org/pub/emdb/validation_reports/EMD-6446 | HTTPS FTP |
-Related structure data
Related structure data | 3jbiMC 6447C 6448C 6449C 6450C 6451C 3jbjC 3jbkC M: atomic model generated by this map C: citing same article (ref.) |
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Similar structure data |
-Links
EMDB pages | EMDB (EBI/PDBe) / EMDataResource |
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Related items in Molecule of the Month |
-Map
File | Download / File: emd_6446.map.gz / Format: CCP4 / Size: 29.8 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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Annotation | Reconstruction of vinculin tail domain bound to F-actin | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Projections & slices | Image control
Images are generated by Spider. | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Voxel size | X=Y=Z: 2.18 Å | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Details | EMDB XML:
CCP4 map header:
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-Supplemental data
-Sample components
-Entire : Vinculin tail domain bound to F-actin
Entire | Name: Vinculin tail domain bound to F-actin |
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Components |
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-Supramolecule #1000: Vinculin tail domain bound to F-actin
Supramolecule | Name: Vinculin tail domain 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
Macromolecule | Name: skeletal muscle actin / type: protein_or_peptide / ID: 1 / Name.synonym: actin / Oligomeric state: helical filament / Recombinant expression: No / Database: NCBI |
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Source (natural) | Organism: Oryctolagus cuniculus (rabbit) / synonym: Rabbit / Tissue: Muscle / Location in cell: Cytoplasm, cytoskeleton |
Molecular weight | Theoretical: 41.8 KDa |
Sequence | UniProtKB: Actin, alpha skeletal muscle |
-Macromolecule #2: Vinculin tail domain, residues 879-1061
Macromolecule | Name: Vinculin tail domain, residues 879-1061 / type: protein_or_peptide / ID: 2 / Name.synonym: Vt / Oligomeric state: helical / Recombinant expression: Yes |
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Source (natural) | Organism: Gallus gallus (chicken) / synonym: Chicken / Organelle: Focal adhesion / Location in cell: Cytoplasmic |
Molecular weight | Theoretical: 28.1 KDa |
Recombinant expression | Organism: Escherichia coli BL21(DE3) (bacteria) / Recombinant strain: BL21(DE3) |
Sequence | UniProtKB: Vinculin |
-Experimental details
-Structure determination
Method | cryo EM |
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Processing | helical reconstruction |
Aggregation state | filament |
-Sample preparation
Concentration | 0.0125 mg/mL |
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Buffer | pH: 7 / Details: 50 mM KCl, 1 mM MgCl2, 1 mM EGTA, 10 mM imidazole |
Grid | Details: 200 mesh 1.2 / 1.3 C-flat |
Vitrification | Cryogen 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 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 Vt was then applied and incubated for 60 seconds. 3 microliters of solution was removed, then an additional 3 microliters of Vt applied. After 60 seconds, 3 microliters of solution was removed, then the grid was blotted for 2 seconds before plunging. |
-Electron microscopy
Microscope | FEI TECNAI 20 |
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Alignment procedure | Legacy - Astigmatism: Objective lens astigmatism was corrected at 100,000 times magnification. |
Date | May 22, 2014 |
Image recording | Category: CCD / Film or detector model: GATAN ULTRASCAN 4000 (4k x 4k) / Number real images: 1384 / Average electron dose: 25 e/Å2 |
Electron beam | Acceleration voltage: 120 kV / Electron source: FIELD EMISSION GUN |
Electron optics | Calibrated 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 stage | Specimen holder model: GATAN LIQUID NITROGEN |
-Image processing
Details | Multi-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). |
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Final reconstruction | Applied symmetry - Helical parameters - Δz: 27.80 Å Applied symmetry - Helical parameters - Δ&Phi: 166.82 ° Applied symmetry - Helical parameters - Axial symmetry: C1 (asymmetric) Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 8.5 Å / Resolution method: OTHER / Software - Name: CTFFIND3, EMAN2/SPARX, FREALIGN |
CTF correction | Details: FREALIGN (per segment) |
-Atomic model buiding 1
Initial model | PDB ID: Chain - Chain ID: A |
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Software | Name: Chimera, MDFF |
Details | Components were initially rigid body fit using Chimera, followed by flexible fitting with MDFF. |
Refinement | Space: REAL / Protocol: FLEXIBLE FIT |
Output model | PDB-3jbi: |