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- EMDB-8163: Structure of the F-actin-tropomyosin complex (reprocessed) -

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Basic information

Entry
Database: EMDB / ID: EMD-8163
TitleStructure of the F-actin-tropomyosin complex (reprocessed)
Map dataNone
Sample
  • Complex: F-actin-tropomyosin complex
    • Protein or peptide: ACTIN, ALPHA SKELETAL MUSCLE
    • Protein or peptide: TROPOMYOSIN ALPHA-1
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 speciesRabbit (rabbit) / Mus musculus (house mouse)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.6 Å
Authorsvon der Ecken J / Raunser S
Funding support Germany, 2 items
OrganizationGrant numberCountry
Max Planck Society Germany
Behrens-Weise foundation Germany
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
DepositionApr 27, 2016-
Header (metadata) releaseMay 25, 2016-
Map releaseJun 15, 2016-
UpdateFeb 12, 2020-
Current statusFeb 12, 2020Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.05
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.05
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-5jlf
  • Surface level: 0.05
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-5jlf
  • Surface level: 0.05
  • Imaged by UCSF Chimera
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  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-5jlf
  • Imaged by Jmol
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_8163.png

Downloads & links

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Map

FileDownload / File: emd_8163.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationNone
Voxel sizeX=Y=Z: 1.1 Å
Density
Contour LevelBy AUTHOR: 0.05 / Movie #1: 0.05
Minimum - Maximum-0.11789155 - 0.26992235
Average (Standard dev.)-0.000066911 (±0.010931748)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-129-128-123
Dimensions256256256
Spacing256256256
CellA=B=C: 281.6 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.11.11.1
M x/y/z256256256
origin x/y/z0.0000.0000.000
length x/y/z281.600281.600281.600
α/β/γ90.00090.00090.000
start NX/NY/NZ-38-19-20
NX/NY/NZ858082
MAP C/R/S123
start NC/NR/NS-128-129-123
NC/NR/NS256256256
D min/max/mean-0.1180.270-0.000

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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
  • Complex: F-actin-tropomyosin complex
    • Protein or peptide: ACTIN, ALPHA SKELETAL MUSCLE
    • Protein or peptide: TROPOMYOSIN ALPHA-1

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Supramolecule #1: F-actin-tropomyosin complex

SupramoleculeName: F-actin-tropomyosin complex / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all / Details: Filament

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Macromolecule #1: ACTIN, ALPHA SKELETAL MUSCLE

MacromoleculeName: ACTIN, ALPHA SKELETAL MUSCLE / type: protein_or_peptide / ID: 1 / Enantiomer: LEVO
Source (natural)Organism: Rabbit (rabbit)
SequenceString: DEDETTALVC DNGSGLVKAG FAGDDAPRAV FPSIVGRPRH QGVMVGMGQK DSYVGDEAQS KRGILTLKYP IE(HIC)GII TNW DDMEKIWHHT FYNELRVAPE EHPTLLTEAP LNPKANREKM TQIMFETFNV PAMYVAIQAV LSLYASGRTT GIVLDSG DG VTHNVPIYEG ...String:
DEDETTALVC DNGSGLVKAG FAGDDAPRAV FPSIVGRPRH QGVMVGMGQK DSYVGDEAQS KRGILTLKYP IE(HIC)GII TNW DDMEKIWHHT FYNELRVAPE EHPTLLTEAP LNPKANREKM TQIMFETFNV PAMYVAIQAV LSLYASGRTT GIVLDSG DG VTHNVPIYEG YALPHAIMRL DLAGRDLTDY LMKILTERGY SFVTTAEREI VRDIKEKLCY VALDFENEMA TAASSSSL E KSYELPDGQV ITIGNERFRC PETLFQPSFI GMESAGIHET TYNSIMKCDI DIRKDLYANN VMSGGTTMYP GIADRMQKE ITALAPSTMK IKIIAPPERK YSVWIGGSIL ASLSTFQQMW ITKQEYDEAG PSIVHRKCF

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Macromolecule #2: TROPOMYOSIN ALPHA-1

MacromoleculeName: TROPOMYOSIN ALPHA-1 / type: protein_or_peptide / ID: 2 / Enantiomer: LEVO
Source (natural)Organism: Mus musculus (house mouse)
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: (UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK) (UNK)(UNK)(UNK)(UNK)(UNK)(UNK) (UNK)(UNK)(UNK) (UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK) (UNK)(UNK)(UNK) (UNK)(UNK)(UNK)(UNK)(UNK) ...String:
(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK) (UNK)(UNK)(UNK)(UNK)(UNK)(UNK) (UNK)(UNK)(UNK) (UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK) (UNK)(UNK)(UNK) (UNK)(UNK)(UNK)(UNK)(UNK)(UNK) (UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK) (UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK) (UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK) (UNK)(UNK) (UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK) (UNK)(UNK)(UNK)(UNK) (UNK)(UNK)(UNK)(UNK)(UNK) (UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK) (UNK) (UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK) (UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK) (UNK) (UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK) (UNK)(UNK)(UNK)(UNK)(UNK) (UNK)(UNK)(UNK)(UNK) (UNK)(UNK)(UNK)

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation statefilament

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Sample preparation

BufferpH: 7.5
Component:
ConcentrationFormula
5.0 mMTris-HClTris
1.0 mMDTT
100.0 mMKCl
2.0 mMMgCl2
Sugar embeddingMaterial: I
GridModel: C-flat-2/1 / Material: COPPER / Mesh: 300 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE
VitrificationCryogen name: ETHANE / Chamber humidity: 90 % / Instrument: GATAN CRYOPLUNGE 3
Details: Sample was applied to a glow-discharged holey carbon grid, incubated for 10 s and manually blotted for 3 s 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 opticsCalibrated defocus max: 2.6 µm / Calibrated defocus min: 0.8 µm / Illumination mode: OTHER / Imaging mode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 59000
DetailsCs corrected microscope
Image recordingFilm or detector model: FEI FALCON II (4k x 4k) / Detector mode: COUNTING / Digitization - Frames/image: 2-8 / Average exposure time: 0.475 sec. / Average electron dose: 16.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 119000
CTF correctionSoftware: (Name: CTFFIND (ver. 4), RELION (ver. 1.3))
Startup modelType of model: EMDB MAP
EMDB ID:

6124

Initial angle assignmentType: NOT APPLICABLE / Software - Name: RELION (ver. 1.3)
Final angle assignmentType: NOT APPLICABLE / Software - Name: RELION (ver. 1.3)
Final reconstructionApplied symmetry - Helical parameters - Δz: 27.5 Å
Applied symmetry - Helical parameters - Δ&Phi: 166.9 °
Applied symmetry - Helical parameters - Axial symmetry: C1 (asymmetric)
Resolution.type: BY AUTHOR / Resolution: 3.6 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 1.3)
Details: THE TROPOMYOSIN MAP FILTERED TO 6.5 ANGSTROM WAS MERGED WITH THE FINAL F-ACTIN MAP (3.6 ANGSTROM) TO OBTAIN A MAP OF THE ENTIRE F-ACTIN-TROPOMYOSIN COMPLEX.
Number images used: 91000

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Atomic model buiding 1

Initial modelPDB ID:

3j8a


Chain - Chain ID -:

db-a


Chain - Chain ID - 1: B / Chain - Chain ID - 2: C / Chain - Chain ID - 3: D / Chain - Chain ID - 4: E
RefinementSpace: RECIPROCAL / Protocol: BACKBONE TRACE / Overall B value: 98
Output model

PDB-5jlf:
Structure of the F-actin-tropomyosin complex (Reprocessed)

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Atomic model buiding 2

Initial modelPDB ID:

3j8a


Chain - Chain ID -:

db-f


Chain - Chain ID - 1: G
Detailstropomyosin fitting
RefinementProtocol: RIGID BODY FIT
Output model

PDB-5jlf:
Structure of the F-actin-tropomyosin complex (Reprocessed)

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