|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 head, motor domain / Myosin tail / Myosin tail / IQ motif, EF-hand binding site / Myosin, N-terminal, SH3-like / Myosin S1 fragment, N-terminal / EF-hand domain pair / EF-Hand 1, calcium-binding site / Myosin IQ motif-containing domain superfamily / P-loop containing nucleoside triphosphate hydrolase ...Myosin head, motor domain / Myosin tail / Myosin tail / IQ motif, EF-hand binding site / Myosin, N-terminal, SH3-like / Myosin S1 fragment, N-terminal / EF-hand domain pair / EF-Hand 1, calcium-binding site / Myosin IQ motif-containing domain superfamily / P-loop containing nucleoside triphosphate hydrolase / EF-hand domain / Kinesin motor domain superfamily / Myosin head (motor domain) / Myosin light chain alkali / Myosin N-terminal SH3-like domain / IQ motif profile. / Translocation of GLUT4 to the plasma membrane / RHO GTPases activate PAKs / Smooth Muscle Contraction / Myosin N-terminal SH3-like domain profile. / EF-hand domain / EF-hand calcium-binding domain profile. / Myosin motor domain profile. / EF-hand calcium-binding domain. / EF-hand domain pair / myosin II filament / regulation of slow-twitch skeletal muscle fiber contraction / myofibril assembly / myosin light chain binding / regulation of the force of skeletal muscle contraction / 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 / microfilament motor activity / skeletal muscle contraction / actomyosin / muscle filament sliding / smooth muscle contraction / myosin complex / myofibril / structural constituent of muscle / myosin heavy chain binding / ATP metabolic process / ventricular cardiac muscle tissue morphogenesis / cardiac muscle contraction / sarcomere / striated muscle contraction / stress fiber / ADP binding / muscle contraction / microtubule motor activity / regulation of heart rate / microtubule-based movement / Z disc / actin filament binding / actin binding / microtubule 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.
Copyright: 2011 Elsevier Ltd. All rights reserved.
#1: Journal: J. Mol. Biol. / Year: 2008
Title: Three-dimensional reconstruction of tarantula myosin filaments suggests how phosphorylation may regulate myosin activity.
Authors: Lorenzo Alamo / Willy Wriggers / Antonio Pinto / Fulvia Bártoli / Leiria Salazar / Fa-Qing Zhao / Roger Craig / Raúl Padrón
Abstract: Muscle contraction involves the interaction of the myosin heads of the thick filaments with actin subunits of the thin filaments. Relaxation occurs when this interaction is blocked by molecular ...Muscle contraction involves the interaction of the myosin heads of the thick filaments with actin subunits of the thin filaments. Relaxation occurs when this interaction is blocked by molecular switches on these filaments. In many muscles, myosin-linked regulation involves phosphorylation of the myosin regulatory light chains (RLCs). Electron microscopy of vertebrate smooth muscle myosin molecules (regulated by phosphorylation) has provided insight into the relaxed structure, revealing that myosin is switched off by intramolecular interactions between its two heads, the free head and the blocked head. Three-dimensional reconstruction of frozen-hydrated specimens revealed that this asymmetric head interaction is also present in native thick filaments of tarantula striated muscle. Our goal in this study was to elucidate the structural features of the tarantula filament involved in phosphorylation-based regulation. A new reconstruction revealed intra- and intermolecular myosin interactions in addition to those seen previously. To help interpret the interactions, we sequenced the tarantula RLC and fitted an atomic model of the myosin head that included the predicted RLC atomic structure and an S2 (subfragment 2) crystal structure to the reconstruction. The fitting suggests one intramolecular interaction, between the cardiomyopathy loop of the free head and its own S2, and two intermolecular interactions, between the cardiac loop of the free head and the essential light chain of the blocked head and between the Leu305-Gln327 interaction loop of the free head and the N-terminal fragment of the RLC of the blocked head. These interactions, added to those previously described, would help switch off the thick filament. Molecular dynamics simulations suggest how phosphorylation could increase the helical content of the RLC N-terminus, weakening these interactions, thus releasing both heads and activating the thick filament.
|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 II / 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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