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- EMDB-5752: Cryogenic electron microscopy of cardiac actin:tropomyosin -

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Basic information

Entry
Database: EMDB / ID: EMD-5752
TitleCryogenic electron microscopy of cardiac actin:tropomyosin
Map dataReconstruction of rabbit skeletal F-actin and bovine cardiac (striated) tropomyosin. The map is filtered at 15 Angstrom resolution.
Sample
  • Sample: rabbit skeletal F-actin bound to bovine cardiac tropomyosin
  • Protein or peptide: Actin, alpha skeletal muscle
  • Protein or peptide: Tropomyosin alpha-1
Keywordscoiled-coil / actin-binding protein / cytoskeleton
Function / homology
Function and homology information


Striated Muscle Contraction / Smooth Muscle Contraction / cardiac myofibril / cytoskeletal motor activator activity / myosin heavy chain binding / tropomyosin binding / actin filament bundle / troponin I binding / filamentous actin / myofibril ...Striated Muscle Contraction / Smooth Muscle Contraction / cardiac myofibril / cytoskeletal motor activator activity / myosin heavy chain binding / tropomyosin binding / actin filament bundle / troponin I binding / filamentous actin / myofibril / mesenchyme migration / actin filament bundle assembly / skeletal muscle myofibril / striated muscle thin filament / skeletal muscle thin filament assembly / actin monomer binding / skeletal muscle fiber development / cardiac muscle contraction / stress fiber / titin binding / actin filament polymerization / actin filament organization / sarcomere / filopodium / actin filament / Hydrolases; Acting on acid anhydrides; Acting on acid anhydrides to facilitate cellular and subcellular movement / disordered domain specific binding / calcium-dependent protein binding / actin filament binding / lamellipodium / actin cytoskeleton / actin binding / cell body / hydrolase activity / protein heterodimerization activity / protein domain specific binding / calcium ion binding / positive regulation of gene expression / magnesium ion binding / protein homodimerization activity / ATP binding / identical protein binding / cytoplasm
Similarity search - Function
Tropomyosins signature. / Tropomyosin / Tropomyosin / Tropomyosin / Actins signature 1. / Actin, conserved site / Actins signature 2. / Actin/actin-like conserved site / Actins and actin-related proteins signature. / Actin ...Tropomyosins signature. / Tropomyosin / 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 family / Actin / ATPase, nucleotide binding domain
Similarity search - Domain/homology
Actin, alpha skeletal muscle / Tropomyosin alpha-1 chain
Similarity search - Component
Biological speciesOryctolagus cuniculus (rabbit) / Bos taurus (domestic cattle)
Methodsingle particle reconstruction / cryo EM / Resolution: 15.0 Å
AuthorsSousa DR / Stagg SM / Stroupe ME
CitationJournal: J Mol Biol / Year: 2013
Title: Cryo-EM structures of the actin:tropomyosin filament reveal the mechanism for the transition from C- to M-state.
Authors: Duncan R Sousa / Scott M Stagg / M Elizabeth Stroupe /
Abstract: Tropomyosin (Tm) is a key factor in the molecular mechanisms that regulate the binding of myosin motors to actin filaments (F-Actins) in most eukaryotic cells. This regulation is achieved by the ...Tropomyosin (Tm) is a key factor in the molecular mechanisms that regulate the binding of myosin motors to actin filaments (F-Actins) in most eukaryotic cells. This regulation is achieved by the azimuthal repositioning of Tm along the actin (Ac):Tm:troponin (Tn) thin filament to block or expose myosin binding sites on Ac. In striated muscle, including involuntary cardiac muscle, Tm regulates muscle contraction by coupling Ca(2+) binding to Tn with myosin binding to the thin filament. In smooth muscle, the switch is the posttranslational modification of the myosin. Depending on the activation state of Tn and the binding state of myosin, Tm can occupy the blocked, closed, or open position on Ac. Using native cryogenic 3DEM (three-dimensional electron microscopy), we have directly resolved and visualized cardiac and gizzard muscle Tm on filamentous Ac in the position that corresponds to the closed state. From the 8-Å-resolution structure of the reconstituted Ac:Tm filament formed with gizzard-derived Tm, we discuss two possible mechanisms for the transition from closed to open state and describe the role Tm plays in blocking myosin tight binding in the closed-state position.
History
DepositionAug 26, 2013-
Header (metadata) releaseSep 18, 2013-
Map releaseSep 18, 2013-
UpdateMar 2, 2016-
Current statusMar 2, 2016Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.453
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 0.453
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_5752_1.jpg

Downloads & links

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Map

FileDownload / File: emd_5752.map.gz / Format: CCP4 / Size: 7.8 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationReconstruction of rabbit skeletal F-actin and bovine cardiac (striated) tropomyosin. The map is filtered at 15 Angstrom resolution.
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.48 Å/pix.
x 128 pix.
= 189.056 Å		1.48 Å/pix.
x 128 pix.
= 189.056 Å		1.48 Å/pix.
x 128 pix.
= 189.056 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.477 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.453 / Movie #1: 0.453
Minimum - Maximum0.0 - 9.16484642
Average (Standard dev.)0.08226459 (±0.63447458)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin646464
Dimensions128128128
Spacing128128128
CellA=B=C: 189.056 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.4771.4771.477
M x/y/z128128128
origin x/y/z0.0000.0000.000
length x/y/z189.056189.056189.056
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS646464
NC/NR/NS128128128
D min/max/mean0.0009.1650.082

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Supplemental data

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Sample components

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Entire : rabbit skeletal F-actin bound to bovine cardiac tropomyosin

EntireName: rabbit skeletal F-actin bound to bovine cardiac tropomyosin
Components
  • Sample: rabbit skeletal F-actin bound to bovine cardiac tropomyosin
  • Protein or peptide: Actin, alpha skeletal muscle
  • Protein or peptide: Tropomyosin alpha-1

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Supramolecule #1000: rabbit skeletal F-actin bound to bovine cardiac tropomyosin

SupramoleculeName: rabbit skeletal F-actin bound to bovine cardiac tropomyosin
type: sample / ID: 1000
Oligomeric state: fourteen actin monomers bind to two tropomyosin coiled-coils
Number unique components: 2

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Macromolecule #1: Actin, alpha skeletal muscle

MacromoleculeName: Actin, alpha skeletal muscle / type: protein_or_peptide / ID: 1 / Name.synonym: Alpha-actin-1
Details: F-actin filaments were formed and tropomyosin was bound to the filaments.
Oligomeric state: filament / Recombinant expression: No / Database: NCBI
Source (natural)Organism: Oryctolagus cuniculus (rabbit) / synonym: Rabbit / Tissue: skeletal muscle
Molecular weightExperimental: 728 KDa
SequenceUniProtKB: Actin, alpha skeletal muscle / GO: skeletal muscle thin filament assembly / InterPro: Actin family

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Macromolecule #2: Tropomyosin alpha-1

MacromoleculeName: Tropomyosin alpha-1 / type: protein_or_peptide / ID: 2 / Name.synonym: Alpha-tropomyosin, Tropomyosin-1
Details: F-actin filaments were formed and tropomyosin was bound to the filaments.
Oligomeric state: filament / Recombinant expression: No / Database: NCBI
Source (natural)Organism: Bos taurus (domestic cattle) / synonym: Bovine / Tissue: cardiac
Molecular weightExperimental: 728 KDa
SequenceUniProtKB: Tropomyosin alpha-1 chain / GO: actin binding / InterPro: Tropomyosin

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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

Concentration0.5 mg/mL
BufferpH: 7.1
Details: 70 mM NaCl, 3 mM MgCl2, 0.2 mM EGTA, 5 mM NaH2PO4, 5 mM PIPES buffer
GridDetails: Quantifoil 1.2/1.3 sized holes
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 120 K / Instrument: FEI VITROBOT MARK IV
Method: Blot with calcium-free filter paper for 2.5 seconds before plunging.

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Electron microscopy

MicroscopeFEI TITAN KRIOS
DateMar 25, 2010
Image recordingCategory: CCD / Film or detector model: GATAN ULTRASCAN 4000 (4k x 4k) / Number real images: 500 / Average electron dose: 30 e/Å2
Details: Images are recorded on a Gatan Ultrascan 4000 CCD and are available upon request to MES (mestroupe@bio.fsu.edu)
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 101555 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 4.0 µm / Nominal defocus min: 1.5 µm / Nominal magnification: 59000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

DetailsFilaments were selected by hand via points along each filament and then boxed at even intervals.
CTF correctionDetails: Micrograph
Final reconstructionApplied symmetry - Helical parameters - Δz: 27.5 Å
Applied symmetry - Helical parameters - Δ&Phi: 166.6 °
Applied symmetry - Helical parameters - Axial symmetry: C1 (asymmetric)
Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 15.0 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: Matlab, SPIDER, EMAN / Number images used: 49318

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Atomic model buiding 1

Initial modelPDB ID:

4a7h


Chain - #0 - Chain ID: B / Chain - #1 - Chain ID: H
SoftwareName: Chimera Fit-in-Map
DetailsThe F-actin and tropomyosin were fitted separately.
RefinementSpace: REAL / Protocol: RIGID BODY FIT / Target criteria: Cross correlation

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