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- EMDB-1465: Three-dimensional structure of vertebrate cardiac muscle myosin f... -

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Basic information

Entry
Database: EMDB / ID: EMD-1465
TitleThree-dimensional structure of vertebrate cardiac muscle myosin filaments.
Map dataReconstruction of cardiac myosin filaments (C-zone) under relaxing conditions. The reconstruction (filtered to 4.0 nm resolution) shows two 42.9 nm repeats. The main globular features are myosin heads, which are arranged in three crowns within each 42.9 nm repeat, following a perturbed helical path. Each crown has 3-fold rotational symmetry. Smaller features with a periodicity of about 4 nm can also be observed. Those features represent the accessory proteins titin and myosin-binding protein-C.
Sample
  • Sample: Cardiac myosin filaments from mouse ventricle muscle
  • Protein or peptide: Myosin
Biological speciesMus musculus (house mouse)
Methodhelical reconstruction / negative staining / Resolution: 32.0 Å
AuthorsZoghbi ME / Woodhead JL / Moss RL / Craig R
CitationJournal: Proc Natl Acad Sci U S A / Year: 2008
Title: Three-dimensional structure of vertebrate cardiac muscle myosin filaments.
Authors: Maria E Zoghbi / John L Woodhead / Richard L Moss / Roger Craig /
Abstract: Contraction of the heart results from interaction of the myosin and actin filaments. Cardiac myosin filaments consist of the molecular motor myosin II, the sarcomeric template protein, titin, and the ...Contraction of the heart results from interaction of the myosin and actin filaments. Cardiac myosin filaments consist of the molecular motor myosin II, the sarcomeric template protein, titin, and the cardiac modulatory protein, myosin binding protein C (MyBP-C). Inherited hypertrophic cardiomyopathy (HCM) is a disease caused mainly by mutations in these proteins. The structure of cardiac myosin filaments and the alterations caused by HCM mutations are unknown. We have used electron microscopy and image analysis to determine the three-dimensional structure of myosin filaments from wild-type mouse cardiac muscle and from a MyBP-C knockout model for HCM. Three-dimensional reconstruction of the wild-type filament reveals the conformation of the myosin heads and the organization of titin and MyBP-C at 4 nm resolution. Myosin heads appear to interact with each other intramolecularly, as in off-state smooth muscle myosin [Wendt T, Taylor D, Trybus KM, Taylor K (2001) Proc Natl Acad Sci USA 98:4361-4366], suggesting that all relaxed muscle myosin IIs may adopt this conformation. Titin domains run in an elongated strand along the filament surface, where they appear to interact with part of MyBP-C and with the myosin backbone. In the knockout filament, some of the myosin head interactions are disrupted, suggesting that MyBP-C is important for normal relaxation of the filament. These observations provide key insights into the role of the myosin filament in cardiac contraction, assembly, and disease. The techniques we have developed should be useful in studying the structural basis of other myosin-related HCM diseases.
History
DepositionDec 18, 2007-
Header (metadata) releaseJan 18, 2008-
Map releaseApr 7, 2008-
UpdateMay 26, 2011-
Current statusMay 26, 2011Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.04
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.04
  • Imaged by UCSF Chimera
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Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

1465.gif

emd_1465.tif

emd_1465_1.tif

Downloads & links

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Map

FileDownload / File: emd_1465.map.gz / Format: CCP4 / Size: 2.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationReconstruction of cardiac myosin filaments (C-zone) under relaxing conditions. The reconstruction (filtered to 4.0 nm resolution) shows two 42.9 nm repeats. The main globular features are myosin heads, which are arranged in three crowns within each 42.9 nm repeat, following a perturbed helical path. Each crown has 3-fold rotational symmetry. Smaller features with a periodicity of about 4 nm can also be observed. Those features represent the accessory proteins titin and myosin-binding protein-C.
Voxel sizeX=Y=Z: 5.7 Å
Density
Contour Level1: 0.625 / Movie #1: 0.04
Minimum - Maximum-1.04307 - 1.44742
Average (Standard dev.)-0.129344 (±0.384131)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions1536670
Spacing1536670
CellA: 872.1 Å / B: 376.2 Å / C: 399 Å
α=β=γ: 90 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z5.75.75.7
M x/y/z1536670
origin x/y/z0.0000.0000.000
length x/y/z872.100376.200399.000
α/β/γ90.00090.00090.000
start NX/NY/NZ29-50166
NX/NY/NZ106122134
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS6615370
D min/max/mean-1.0431.447-0.129

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Supplemental data

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Sample components

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Entire : Cardiac myosin filaments from mouse ventricle muscle

EntireName: Cardiac myosin filaments from mouse ventricle muscle
Components
  • Sample: Cardiac myosin filaments from mouse ventricle muscle
  • Protein or peptide: Myosin

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Supramolecule #1000: Cardiac myosin filaments from mouse ventricle muscle

SupramoleculeName: Cardiac myosin filaments from mouse ventricle muscle / type: sample / ID: 1000 / Number unique components: 1

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Macromolecule #1: Myosin

MacromoleculeName: Myosin / type: protein_or_peptide / ID: 1 / Name.synonym: Myosin / Details: Filament is a polymer of myosin / Oligomeric state: polymer / Recombinant expression: No / Database: NCBI
Source (natural)Organism: Mus musculus (house mouse) / Strain: 129SVE / synonym: mouse / Tissue: cardiac ventricle / Location in cell: cytoplasm
Molecular weightExperimental: 520 KDa / Theoretical: 520 KDa

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Experimental details

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Structure determination

Methodnegative staining
Processinghelical reconstruction
Aggregation statefilament

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Sample preparation

BufferpH: 7.2
Details: 100 mM NaCl, 2 mM EGTA, 5 mM MgCl2, 1 mM DTT, 10 mM Imidazole, 5 mM MgATP, 0.1 mM blebbistatin,pH 7.2
StainingType: NEGATIVE
Details: A drop of filament suspension was placed on an electron microscope grid coated with a thin layer of carbon supported by a thicker holey carbon film. The grid was rinsed sequentially with 6 ...Details: A drop of filament suspension was placed on an electron microscope grid coated with a thin layer of carbon supported by a thicker holey carbon film. The grid was rinsed sequentially with 6 drops of relaxing rinse (in mM: 140 NaAc, 1 MgAc2, 1 EGTA, 5 Imidazole, 1 sodium azide, 1 MgATP, pH 7.0 ) and 5 drops of 2% uranyl acetate. Staining was carried out at room temperature with solutions pre-warmed to 37o C.
GridDetails: carbon holey grids (400 mesh copper)
VitrificationCryogen name: NONE

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Electron microscopy

MicroscopeFEI/PHILIPS CM120T
Alignment procedureLegacy - Astigmatism: corrected at 240,000 x
DetailsGrids were observed in a Philips CM120 electron microscope (FEI, Hillsboro, OR) under low dose conditions. Only filaments on thin carbon over holes were photographed .
DateMay 30, 2006
Image recordingCategory: CCD / Film or detector model: TVIPS TEMCAM-F224 (2k x 2k) / Number real images: 300
Details: Images were acquired on a CDD camera at 5.7 A/pixel
Bits/pixel: 8
Electron beamAcceleration voltage: 80 kV / Electron source: LAB6
Electron opticsCalibrated magnification: 42000 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.0 mm / Nominal defocus max: 1.1 µm / Nominal defocus min: 0.9 µm / Nominal magnification: 42000
Sample stageSpecimen holder: Eucentric / Specimen holder model: OTHER

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Image processing

DetailsThe filament shows perturbations from a perfect helical structure.
Final reconstructionApplied symmetry - Helical parameters - Axial symmetry: C3 (3 fold cyclic)
Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 32.0 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: SPIDER
Details: Since these filaments are not perfectly helical, we used single particle analysis for their reconstruction. Filaments were oriented vertically and the region between the 3rd and 10th 42.9 nm ...Details: Since these filaments are not perfectly helical, we used single particle analysis for their reconstruction. Filaments were oriented vertically and the region between the 3rd and 10th 42.9 nm repeats from the bare zone (where MyBP-C is present) was computationally cut. Those selected filament regions were converted to SPIDER format (EM2EM; Image Science and Imperial College, London), and cut into segments 3x42.9 nm long in SPIDER (v11.2, Wadsworth Center, Albany, NY). Relative rotations of different filament segments were determined before back-projection by matching filament images against 2D projections of 3D models rotated around their long axis at known angles. C3 symmetry was imposed during reconstruction. A total of 2564 segments (2600 particles) were used for the reconstruction.

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Atomic model buiding 1

Initial modelPDB ID:

1i84

DetailsAtomic structure of the myosin heads (pdb 1i84; with the modifications introduced by Woodhead et al., 2005. Nature. 436:1195)was fitted manually to the globular features of the reconstruction using UCSF Chimera.
RefinementProtocol: RIGID BODY FIT

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