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- EMDB-6124: Structure of the F-actin-tropomyosin complex -

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Basic information

Entry
Database: EMDB / ID: EMD-6124
TitleStructure of the F-actin-tropomyosin complex
Map dataReconstruction of F-actin tropomyosin complex
Sample
  • Sample: F-actin-tropomyosin complex
  • Protein or peptide: F-actin (alpha)
  • Protein or peptide: tropomyosin (alpha)
KeywordsF-actin / tropomyosin / filament / protein polymers / muscle / thin filament / cytoskeleton
Function / homology
Function and homology information


positive regulation of heart rate by epinephrine / muscle thin filament tropomyosin / Striated Muscle Contraction / Smooth Muscle Contraction / bleb / negative regulation of vascular associated smooth muscle cell migration / muscle filament sliding / actin filament capping / ruffle organization / sarcomere organization ...positive regulation of heart rate by epinephrine / muscle thin filament tropomyosin / Striated Muscle Contraction / Smooth Muscle Contraction / bleb / negative regulation of vascular associated smooth muscle cell migration / muscle filament sliding / actin filament capping / ruffle organization / sarcomere organization / cytoskeletal motor activator activity / ventricular cardiac muscle tissue morphogenesis / tropomyosin binding / myofibril / myosin heavy chain binding / mesenchyme migration / troponin I binding / actin filament bundle / filamentous actin / negative regulation of vascular associated smooth muscle cell proliferation / actin filament bundle assembly / skeletal muscle thin filament assembly / striated muscle thin filament / skeletal muscle myofibril / actin monomer binding / positive regulation of cell adhesion / skeletal muscle fiber development / stress fiber / titin binding / cardiac muscle contraction / positive regulation of stress fiber assembly / cytoskeletal protein binding / actin filament polymerization / negative regulation of cell migration / filopodium / actin filament organization / actin filament / Hydrolases; Acting on acid anhydrides; Acting on acid anhydrides to facilitate cellular and subcellular movement / wound healing / structural constituent of cytoskeleton / ruffle membrane / calcium-dependent protein binding / disordered domain specific binding / actin filament binding / actin cytoskeleton / lamellipodium / cell body / actin binding / regulation of cell shape / in utero embryonic development / hydrolase activity / protein heterodimerization activity / protein domain specific binding / calcium ion binding / positive regulation of gene expression / magnesium ion binding / protein homodimerization activity / protein-containing complex / ATP binding / identical protein binding / cytosol / cytoplasm
Similarity search - Function
Tropomyosins signature. / Tropomyosin / Tropomyosin / Actins signature 1. / Actin, conserved site / Actins signature 2. / Actin/actin-like conserved site / Actins and actin-related proteins signature. / Actin / Actin family ...Tropomyosins signature. / Tropomyosin / Tropomyosin / 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
Tropomyosin alpha-1 chain / Actin, alpha skeletal muscle
Similarity search - Component
Biological speciesOryctolagus cuniculus (rabbit) / Mus musculus (house mouse)
Methodhelical reconstruction / cryo EM / Resolution: 3.7 Å
Authorsvon der Ecken J / Mueller M / Lehman W / Manstein DJ / Penczek PA / Raunser S
CitationJournal: Nature / Year: 2015
Title: Structure of the F-actin-tropomyosin complex.
Authors: Julian von der Ecken / Mirco Müller / William Lehman / Dietmar J Manstein / Pawel A Penczek / Stefan Raunser /
Abstract: Filamentous actin (F-actin) is the major protein of muscle thin filaments, and actin microfilaments are the main component of the eukaryotic cytoskeleton. Mutations in different actin isoforms lead ...Filamentous actin (F-actin) is the major protein of muscle thin filaments, and actin microfilaments are the main component of the eukaryotic cytoskeleton. Mutations in different actin isoforms lead to early-onset autosomal dominant non-syndromic hearing loss, familial thoracic aortic aneurysms and dissections, and multiple variations of myopathies. In striated muscle fibres, the binding of myosin motors to actin filaments is mainly regulated by tropomyosin and troponin. Tropomyosin also binds to F-actin in smooth muscle and in non-muscle cells and stabilizes and regulates the filaments there in the absence of troponin. Although crystal structures for monomeric actin (G-actin) are available, a high-resolution structure of F-actin is still missing, hampering our understanding of how disease-causing mutations affect the function of thin muscle filaments and microfilaments. Here we report the three-dimensional structure of F-actin at a resolution of 3.7 Å in complex with tropomyosin at a resolution of 6.5 Å, determined by electron cryomicroscopy. The structure reveals that the D-loop is ordered and acts as a central region for hydrophobic and electrostatic interactions that stabilize the F-actin filament. We clearly identify map density corresponding to ADP and Mg(2+) and explain the possible effect of prominent disease-causing mutants. A comparison of F-actin with G-actin reveals the conformational changes during filament formation and identifies the D-loop as their key mediator. We also confirm that negatively charged tropomyosin interacts with a positively charged groove on F-actin. Comparison of the position of tropomyosin in F-actin-tropomyosin with its position in our previously determined F-actin-tropomyosin-myosin structure reveals a myosin-induced transition of tropomyosin. Our results allow us to understand the role of individual mutations in the genesis of actin- and tropomyosin-related diseases and will serve as a strong foundation for the targeted development of drugs.
History
DepositionOct 8, 2014-
Header (metadata) releaseOct 29, 2014-
Map releaseDec 10, 2014-
UpdateMar 11, 2015-
Current statusMar 11, 2015Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 3.7
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 3.7
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-3j8a
  • Surface level: 3.7
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_6124.jpg

Downloads & links

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Map

FileDownload / File: emd_6124.map.gz / Format: CCP4 / Size: 6.4 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationReconstruction of F-actin tropomyosin complex
Voxel sizeX=Y=Z: 1.12 Å
Density
Contour LevelBy AUTHOR: 3.7 / Movie #1: 3.7
Minimum - Maximum-8.4796648 - 16.16378212
Average (Standard dev.)0.03826505 (±1.49326706)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-44-51-89
Dimensions92103182
Spacing92103182
CellA: 115.36 Å / B: 103.04 Å / C: 203.84 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.121.121.12
M x/y/z10392182
origin x/y/z0.0000.0000.000
length x/y/z115.360103.040203.840
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ969680
MAP C/R/S123
start NC/NR/NS-51-44-89
NC/NR/NS10392182
D min/max/mean-8.48016.1640.038

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Supplemental data

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Sample components

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Entire : F-actin-tropomyosin complex

EntireName: F-actin-tropomyosin complex
Components
  • Sample: F-actin-tropomyosin complex
  • Protein or peptide: F-actin (alpha)
  • Protein or peptide: tropomyosin (alpha)

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Supramolecule #1000: F-actin-tropomyosin complex

SupramoleculeName: F-actin-tropomyosin complex / type: sample / ID: 1000 / Oligomeric state: filament / Number unique components: 2

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Macromolecule #1: F-actin (alpha)

MacromoleculeName: F-actin (alpha) / type: protein_or_peptide / ID: 1 / Number of copies: 5 / Oligomeric state: filament / Recombinant expression: No / Database: NCBI
Source (natural)Organism: Oryctolagus cuniculus (rabbit) / synonym: rabbit / Tissue: muscle
SequenceUniProtKB: Actin, alpha skeletal muscle

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Macromolecule #2: tropomyosin (alpha)

MacromoleculeName: tropomyosin (alpha) / type: protein_or_peptide / ID: 2 / Oligomeric state: filament (dimer) / Recombinant expression: Yes
Source (natural)Organism: Mus musculus (house mouse) / synonym: mouse / Tissue: muscle
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceUniProtKB: Tropomyosin alpha-1 chain

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Experimental details

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Structure determination

Methodcryo EM
Processinghelical reconstruction
Aggregation statefilament

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Sample preparation

BufferpH: 7.5 / Details: 5 mM Tris, 1 mM DTT, 100 mM KCL, 2 mM MgCl2
GridDetails: C-flats 2/1 copper 300 mesh, Protochips, glow-discharged
VitrificationCryogen name: ETHANE / Chamber humidity: 90 % / Chamber temperature: 106 K / Instrument: GATAN CRYOPLUNGE 3
Method: Sample was applied to grid, incubated for 10 seconds, and manually blotted for 3 seconds from the backside with filter paper.

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 2.6 µm / Nominal defocus min: 0.8 µm / Nominal magnification: 59000 / Cs: mm
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Cs0
DetailsCs-corrected microscope
DateOct 17, 2013
Image recordingCategory: CCD / Film or detector model: FEI FALCON II (4k x 4k) / Number real images: 1311 / Average electron dose: 14.6 e/Å2
Details: Every image is the average of seven frames recorded by the direct electron detector after beam-induced motion correction. Only 689 images were used for the refinement.
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

CTF correctionDetails: each micrograph
Final reconstructionApplied symmetry - Helical parameters - Δz: 27.5 Å
Applied symmetry - Helical parameters - Δ&Phi: 166.4 °
Applied symmetry - Helical parameters - Axial symmetry: C1 (asymmetric)
Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 3.7 Å / Resolution method: OTHER / Software - Name: SPARX
Details: The tropomyosin map filtered to 6.5 Angstrom was merged with the final F-actin map (3.7 Angstrom) to obtain a map of the entire F-actin tropomyosin complex.

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Atomic model buiding 1

Initial modelPDB ID:

4a7n


Chain - #0 - Chain ID: A / Chain - #1 - Chain ID: B / Chain - #2 - Chain ID: C / Chain - #3 - Chain ID: D / Chain - #4 - Chain ID: E
SoftwareName: SPARX, PHENIX, COOT
RefinementSpace: RECIPROCAL / Protocol: FLEXIBLE FIT / Overall B value: 55.4 / Target criteria: R-factor
Output model

PDB-3j8a:
Structure of the F-actin-tropomyosin complex

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