|Entry||Database: EMDB / ID: 1950|
|Title||3D-Structure of tarantula myosin filament obtained by cryo-electron microscopy|
|Keywords||cryo-EM / thick filament / flexible docking / single particle reconstruction / Iterative Helical Real Space Reconstruction (IHRSR) / Myosin regulation / myosin regulatory light chain / phosphorylation|
|Sample||Myosin filaments from Tarantula striated muscle|
|Source||Aphonopelma sp. / arthropod / Tarantula|
|Map data||This is a density map of tarantula thick filaments, the initial view is from the Z line perspective, if the map is rotated by 90 degress in x direction, the J motif of the interacting heads features and the backbone subfilaments can be seen clearly|
|Method||helical reconstruction, at 20 A resolution|
|Authors||Alamo L / Wriggers W / Pinto A / Bartoli F / Salazar L / Zhao F / Craig R / Padron R|
|Citation||J. Mol. Biol., 2011, 414, 44-61|
primary. J. Mol. Biol., 2011, 414, 44-61 StrPapers
1. J. Mol. Biol., 2008, 384, 780-797 StrPapers
|Date||Deposition: Aug 23, 2011 / Header (metadata) release: Sep 2, 2011 / Map release: Sep 2, 2011 / Last update: Aug 23, 2011|
Downloads & links
|File||emd_1950.map.gz (map file in CCP4 format, 61037 KB)|
|Projections & slices|
Images are generated by Spider package.
(generated in cubic-lattice coordinate)
|Voxel size||X: 2.48 A / Y: 2.48 A / Z: 2.482 A|
CCP4 map header:
-Entire Myosin filaments from Tarantula striated muscle
|Entire||Name: Myosin filaments from Tarantula striated muscle / Number of components: 2|
Oligomeric State: Polymer of a multiple myosin assembled over a paramyosin core
-Component #1: protein, Myosin II
|Protein||Name: Myosin II / a.k.a: Myosin Type II / Oligomeric Details: Polymer / Recombinant expression: No|
|Source||Species: Aphonopelma sp. / arthropod / Tarantula|
|Source (natural)||Location in cell: Sarcomere / Cell: Myofibrils / Organ or tissue: Muscle|
|Helical parameters||Axial symmetry: C4 (4 fold cyclic) / Hand: RIGHT HANDED / Delta z: 100 A / Delta phi: 30 deg.|
|Sample solution||Buffer solution: 100mM NaCl,3mM MgCl2,1mM EGTA, 5mM PIPES, 5mM NaH2PO4,1mM NaN3.|
|Support film||Holey carbon grids 400 mesh|
|Staining||A 6 ul aliquot of native purified tarantula thick filaments suspension (Hidalgo et al. 2001) was applied to a 400 mesh grid coated with a holey carbon film that had been rendered hydrophilic by glow discharge in n-amylamine vapor for 3 minutes before use. After allowing the filaments to adsorb to the grid for 30 seconds, the grid was rinsed with the relaxing rinse, then placed in a humidity chamber (aprox. 80% relative humidity). Blotting was performed from one side of the grid till a thin sample film on it using Whatman No 42 filter paper, then the grid was immediately plunged under gravity into liquid ethane cooled by liquid nitrogen. Grids were stored under liquid nitrogen.|
|Vitrification||Instrument: HOMEMADE PLUNGER / Cryogen name: ETHANE / Temperature: 93 K / Humidity: 80 % / Method: Plunging in a liquid ethane|
Details: Vitrification instrument: Home-made plunger. Blotting was performed from one side of the grid till a thin sample film on it using Whatman No 42 filter paper, then the grid was immediately plunged under gravity into liquid ethane cooled by liquid nitrogen. Grids were stored under liquid nitrogen.
-Electron microscopy imaging
|Imaging||Microscope: FEI/PHILIPS CM120T / Date: Oct 23, 2002|
Details: Holey carbon grids Cryo preserved in Liquid ethane were observed in a Philips CM120 electron microscope under low dose conditions. Only filaments on thin carbon over holes were photographed
|Electron gun||Electron source: LAB6 / Accelerating voltage: 120 kV / Illumination mode: FLOOD BEAM|
|Lens||Magnification: 35000 X (nominal), 35000 X (calibrated) / Cs: 2 mm / Imaging mode: BRIGHT FIELD / Defocus: 1950 nm|
|Specimen Holder||Holder: Eucentric / Model: GATAN LIQUID NITROGEN / Temperature: K ( 88 - 90 K)|
|Camera||Detector: KODAK SO-163 FILM|
|Image acquisition||Number of digital images: 1008 / Scanner: OTHER / Sampling size: 8.47 microns / Bit depth: 14|
|Processing||Method: helical reconstruction|
Details: There are 4 helices of myosin heads, rotated 30 degrees, every 145 Angstroms. The filament segments were selected based on visual judgement of good helical order
|3D reconstruction||Algorithm: Single particle reconstruction with a modification of the IHRSR method|
Details: Three-dimensional single particle reconstruction was carried out by a modification of the IHRSR method, using SPIDER. Low-dose electron micrographs of 1008 frozen-hydrated thick filaments halves ere digitized at 0.248 nm per pixel using a Nikon Super Coolscan 8000 ED scanner. Filaments were aligned with the bare zone at the top, to ensure correct polarity in subsequent steps. A total of 15,504 segments, each 62 nm long, with an overlap of 55.8 nm, and containing aprox. 40,000 unique pairs of interacting myosin heads went into the reconstruction. As an initial reference model we used the tarantula negatively stained 3D-map, which was axially rotated, axially shifted and also out of plane tilted up to plus-minus12deg. for projection matching, giving a total of 4,095 projections (13 tilted projections plus-minus12deg. every 2deg., 45 reference rotated projections (0-90 degrees, 2deg. rotation angle), and 7 image axial shifts of 2.2 nm. The resulting 3D-map combines about 10,700 out of 15,504 filament segments, a yield of 69 percent of included segments.
Resolution: 20 A / Resolution method: FSC 0.5
-Atomic model buiding
|Modeling #1||Software: Situs 2.3 / Refinement protocol: flexible / Target criteria: Correlation / Refinement space: REAL|
Details: Protocol: Flexible Fitting. The flexible docking procedure is based on a connected (motion capture) network of identified features within the atomic model. The atomic model is allowed to move according to displacements tracked by 31 control points defined by the network, in order to find the best match to the cryo-EM map
Input PDB model: 3DTP
Chain ID: 3DTP_A, 3DTP_B, 3DTP_C, 3DTP_D, 3DTP_E, 3DTP_F
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