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- EMDB-6448: Cryo-EM reconstruction of F-actin -

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Basic information

Entry
Database: EMDB / ID: EMD-6448
TitleCryo-EM reconstruction of F-actin
Map dataControl reconstruction of F-actin alone
Sample
  • Sample: F-actinActin
  • Protein or peptide: skeletal muscle actin
Keywordsactin / cell migration / adhesion / mechanosensation / cytoskeleton
Function / homology
Function and homology information


cytoskeletal motor activator activity / tropomyosin binding / myosin heavy chain binding / mesenchyme migration / troponin I binding / actin filament bundle / filamentous actin / actin filament bundle assembly / skeletal muscle thin filament assembly / striated muscle thin filament ...cytoskeletal motor activator activity / tropomyosin binding / myosin heavy chain binding / mesenchyme migration / troponin I binding / actin filament bundle / filamentous actin / actin filament bundle assembly / skeletal muscle thin filament assembly / striated muscle thin filament / skeletal muscle myofibril / actin monomer binding / skeletal muscle fiber development / stress fiber / titin binding / actin filament polymerization / filopodium / actin filament / Hydrolases; Acting on acid anhydrides; Acting on acid anhydrides to facilitate cellular and subcellular movement / calcium-dependent protein binding / lamellipodium / cell body / hydrolase activity / protein domain specific binding / calcium ion binding / positive regulation of gene expression / magnesium ion binding / ATP binding / identical protein binding / cytoplasm
Similarity search - Function
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
Actin, alpha skeletal muscle
Similarity search - Component
Biological speciesOryctolagus cuniculus (rabbit)
Methodhelical reconstruction / cryo EM / Resolution: 7.6 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: 7.5
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 7.5
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-3jbj
  • Surface level: 7.5
  • Imaged by UCSF Chimera
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  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-3jbj
  • Imaged by Jmol
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_6448.png

Downloads & links

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Map

FileDownload / File: emd_6448.map.gz / Format: CCP4 / Size: 29.8 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationControl reconstruction of F-actin alone
Voxel sizeX=Y=Z: 2.18 Å
Density
Contour LevelBy AUTHOR: 7.5 / Movie #1: 7.5
Minimum - Maximum-5.74246216 - 23.804840089999999
Average (Standard dev.)0.0 (±1.00000012)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-79-79-42
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-79-79-42
NC/NR/NS200200200
D min/max/mean-5.74223.805-0.000

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Supplemental data

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Sample components

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Entire : F-actin

EntireName: F-actinActin
Components
  • Sample: F-actinActin
  • Protein or peptide: skeletal muscle actin

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Supramolecule #1000: F-actin

SupramoleculeName: F-actin / type: sample / ID: 1000 / Details: Helical filament of F-actin / Oligomeric state: Helical / Number unique components: 1

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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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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. The grid was blotted for 2 seconds before plunging.

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Electron microscopy

MicroscopeFEI TECNAI 20
Electron beamAcceleration voltage: 120 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 137615 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / 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
Alignment procedureLegacy - Astigmatism: Objective lens astigmatism was corrected at 100,000 times magnification.
DateMay 13, 2014
Image recordingCategory: CCD / Film or detector model: GATAN ULTRASCAN 4000 (4k x 4k) / Number real images: 1257 / Average electron dose: 25 e/Å2

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Image processing

CTF correctionDetails: FREALIGN (per segment)
Final reconstructionApplied symmetry - Helical parameters - Δz: 27.80 Å
Applied symmetry - Helical parameters - Δ&Phi: 166.67 °
Applied symmetry - Helical parameters - Axial symmetry: C1 (asymmetric)
Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 7.6 Å / Resolution method: OTHER / Software - Name: CTFFIND3, EMAN2/SPARX, FREALIGN
DetailsSingle-model IHRSR was performed with EMAN2 / SPARX and final reconstruction with FREALIGN (fixed helical parameters).

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Atomic model buiding 1

Initial modelPDB ID:

3j8a


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-3jbj:
Cryo-EM reconstruction of F-actin

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