EMD-1465
Three-dimensional structure of vertebrate cardiac muscle myosin filaments.
EMD-1465
Helical reconstruction32.0 Å
Deposition: 18/12/2007Map released: 07/04/2008
Last modified: 26/05/2011
Buffer
pH: 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
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
Staining
Type:
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 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.
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.
Grid
Details: Carbon holey grids (400 mesh copper)
Vitrification
Cryogen name: NONE
Microscope: FEI/PHILIPS CM120T
Illumination mode: FLOOD BEAM
Imaging mode: BRIGHT FIELD
Electron source: LAB6
Acceleration voltage: 80 kV
Nominal CS: 2.0 mm
Nominal defocus: 0.9 µm - 1.1 µm
Nominal magnification: 42000.0
Calibrated magnification: 42000.0
Specimen holder model: OTHER
Specimen holder details: Eucentric
Alignment procedure: LEGACY (Astigmatism: corrected at 240,000 x, Electron beam tilt params: )
Details: Grids were observed in a Philips CM120 electron microscope (FEI, Hillsboro, OR) under low dose conditions. Only filaments on thin carbon over holes were photographed .
Illumination mode: FLOOD BEAM
Imaging mode: BRIGHT FIELD
Electron source: LAB6
Acceleration voltage: 80 kV
Nominal CS: 2.0 mm
Nominal defocus: 0.9 µm - 1.1 µm
Nominal magnification: 42000.0
Calibrated magnification: 42000.0
Specimen holder model: OTHER
Specimen holder details: Eucentric
Alignment procedure: LEGACY (Astigmatism: corrected at 240,000 x, Electron beam tilt params: )
Details: Grids were observed in a Philips CM120 electron microscope (FEI, Hillsboro, OR) under low dose conditions. Only filaments on thin carbon over holes were photographed .
Image Recording
[1]
Detector category:
CCD
Detector model: TVIPS TEMCAM-F224 (2k x 2k)
Number of real images: 300
Bits per pixel: 8.0
Details: Images were acquired on a CDD camera at 5.7 A/pixel
Detector model: TVIPS TEMCAM-F224 (2k x 2k)
Number of real images: 300
Bits per pixel: 8.0
Details: Images were acquired on a CDD camera at 5.7 A/pixel
Details: The filament shows perturbations from a perfect helical structure.
Final
reconstruction
Resolution: 32.0
Å
(
BY AUTHOR)
Resolution method: FSC 0.5 CUT-OFF
Algorithm: OTHER
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.
Resolution method: FSC 0.5 CUT-OFF
Algorithm: OTHER
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.
⌯ Applied Symmetry
Axial symmetry:
C3
Software
[1]
| Name | Version | Details |
|---|---|---|
| SPIDER | - | - |
Format: CCP4
Data type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotation details: Reconstruction 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.
Details: ::::EMDATABANK.org::::EMD-1465::::
Data type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotation details: Reconstruction 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.
Details: ::::EMDATABANK.org::::EMD-1465::::
⬡ Geometry
| X | Y | Z | |
|---|---|---|---|
| Origin | 0 | 0 | 0 |
| Dimensions (px) | 66 | 153 | 70 |
| Dimensions (Å) | 872.1 | 376.2 | 399 |
| Voxel size (Å) | 5.7 | 5.7 | 5.7 |
Contour list
| Primary | Level | Source |
|---|---|---|
| True | 0.625 | - |