|Entry||Database: EMDB / ID: 1950|
|Title||3D-Structure of tarantula myosin filament obtained by cryo-electron microscopy|
|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|
|Sample||Myosin filaments from Tarantula striated muscle:|
|Keywords||cryo-EM / thick filament / flexible docking / single particle reconstruction / Iterative Helical Real Space Reconstruction (IHRSR) / Myosin regulation / myosin regulatory light chain / phosphorylation|
|Function / homology||Myosin S1 fragment, N-terminal / Myosin N-terminal SH3-like domain / Myosin, N-terminal, SH3-like / Myosin tail / EF-hand domain / Myosin head, motor domain / IQ motif, EF-hand binding site / EF-Hand 1, calcium-binding site / Myosin IQ motif-containing domain superfamily / P-loop containing nucleoside triphosphate hydrolase ...Myosin S1 fragment, N-terminal / Myosin N-terminal SH3-like domain / Myosin, N-terminal, SH3-like / Myosin tail / EF-hand domain / Myosin head, motor domain / IQ motif, EF-hand binding site / EF-Hand 1, calcium-binding site / Myosin IQ motif-containing domain superfamily / P-loop containing nucleoside triphosphate hydrolase / Myosin light chain alkali / Kinesin motor domain superfamily / EF-hand domain pair / Myosin tail / Myosin head (motor domain) / EF-hand calcium-binding domain profile. / EF-hand domain pair / Myosin motor domain profile. / Myosin N-terminal SH3-like domain profile. / EF-hand domain / Smooth Muscle Contraction / EF-hand calcium-binding domain. / IQ motif profile. / RHO GTPases activate PAKs / myosin II filament / regulation of slow-twitch skeletal muscle fiber contraction / regulation of the force of skeletal muscle contraction / myofibril assembly / myosin light chain binding / elastic fiber assembly / adult heart development / actomyosin structure organization / skeletal muscle myosin thick filament assembly / regulation of the force of heart contraction / myosin II complex / cardiac muscle hypertrophy in response to stress / transition between fast and slow fiber / myosin II binding / muscle myosin complex / cardiac muscle fiber development / actin-dependent ATPase activity / myosin filament / skeletal muscle contraction / microfilament motor activity / striated muscle contraction / actomyosin / muscle filament sliding / myosin complex / smooth muscle contraction / structural constituent of muscle / myofibril / myosin heavy chain binding / ventricular cardiac muscle tissue morphogenesis / ATP metabolic process / cardiac muscle contraction / motor activity / stress fiber / ADP binding / sarcomere / regulation of heart rate / muscle contraction / Z disc / actin filament binding / actin binding / ATPase activity / calmodulin binding / magnesium ion binding / protein heterodimerization activity / calcium ion binding / ATP binding / cytosol / Myosin 2 heavy chain striated muscle / Myosin 2 essential light chain striated muscle / Myosin 2 regulatory light chain striated muscle / Myosin II regulatory light chain / Myosin light polypeptide 6 / Myosin-11 / Myosin-7|
Function and homology information
|Source||Aphonopelma sp. (spider)|
|Method||helical reconstruction / cryo EM / negative staining / 20 Å resolution|
|Authors||Alamo L / Wriggers W / Pinto A / Bartoli F / Salazar L / Zhao F / Craig R / Padron R|
Journal: J. Mol. Biol. / Year: 2011
Title: A molecular model of phosphorylation-based activation and potentiation of tarantula muscle thick filaments.
Authors: Reicy Brito / Lorenzo Alamo / Ulf Lundberg / José R Guerrero / Antonio Pinto / Guidenn Sulbarán / Mary Ann Gawinowicz / Roger Craig / Raúl Padrón
Abstract: Myosin filaments from many muscles are activated by phosphorylation of their regulatory light chains (RLCs). To elucidate the structural mechanism of activation, we have studied RLC phosphorylation ...Myosin filaments from many muscles are activated by phosphorylation of their regulatory light chains (RLCs). To elucidate the structural mechanism of activation, we have studied RLC phosphorylation in tarantula thick filaments, whose high-resolution structure is known. In the relaxed state, tarantula RLCs are ~50% non-phosphorylated and 50% mono-phosphorylated, while on activation, mono-phosphorylation increases, and some RLCs become bi-phosphorylated. Mass spectrometry shows that relaxed-state mono-phosphorylation occurs on Ser35, while Ca(2+)-activated phosphorylation is on Ser45, both located near the RLC N-terminus. The sequences around these serines suggest that they are the targets for protein kinase C and myosin light chain kinase (MLCK), respectively. The atomic model of the tarantula filament shows that the two myosin heads ("free" and "blocked") are in different environments, with only the free head serines readily accessible to kinases. Thus, protein kinase C Ser35 mono-phosphorylation in relaxed filaments would occur only on the free heads. Structural considerations suggest that these heads are less strongly bound to the filament backbone and may oscillate occasionally between attached and detached states ("swaying" heads). These heads would be available for immediate actin interaction upon Ca(2)(+) activation of the thin filaments. Once MLCK becomes activated, it phosphorylates free heads on Ser45. These heads become fully mobile, exposing blocked head Ser45 to MLCK. This would release the blocked heads, allowing their interaction with actin. On this model, twitch force would be produced by rapid interaction of swaying free heads with activated thin filaments, while prolonged exposure to Ca(2+) on tetanus would recruit new MLCK-activated heads, resulting in force potentiation.
|Validation Report||PDB-ID: 3dtp|
About validation report
|Date||Deposition: Aug 23, 2011 / Header (metadata) release: Sep 2, 2011 / Map release: Sep 2, 2011 / Last update: Apr 20, 2016|
|Structure viewer||EM map: |
Downloads & links
|File||emd_1950.map.gz (map file in CCP4 format, 61037 KB)|
|Projections & slices|
Images are generated by Spider.
(generated in cubic-lattice coordinate)
|Voxel size||X: 2.48 Å / Y: 2.48 Å / Z: 2.482 Å|
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 IIMyosin / a.k.a: Myosin Type II / Oligomeric Details: Polymer / Recombinant expression: No|
|Source||Species: Aphonopelma sp. (spider)|
|Source (natural)||Location in cell: Sarcomere / Cell: Myofibrils / Organ or tissue: Muscle|
|Specimen||Specimen state: filament / Method: negative staining, cryo EM|
|Helical parameters||Axial symmetry: C4 (4 fold cyclic) / Hand: RIGHT HANDED / Delta z: 100 Å / 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 Å / 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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