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- PDB-3dtp: Tarantula heavy meromyosin obtained by flexible docking to Tarant... -

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Entry
Database: PDB / ID: 3dtp
TitleTarantula heavy meromyosin obtained by flexible docking to Tarantula muscle thick filament Cryo-EM 3D-MAP
Components
  • (Myosin 2 heavy chain chimera of smooth and cardiac muscle) x 2
  • Myosin regulatory light chain
  • Smooth muscle myosin essential light chain
KeywordsCONTRACTILE PROTEIN / MUSCLE PROTEIN / SMOOTH MUSCLE / MYOSIN SUBFRAGMENT 2 / HEAVY MEROMYOSIN / ESSENTIAL LIGHT CHAIN / REGULATORY LIGHT CHAIN / MOTOR PROTEIN / COILED-COIL
Function / homologyKinesin motor domain superfamily / Myosin S1 fragment, N-terminal / IQ motif, EF-hand binding site / IQ motif profile. / Myosin head, motor domain / EF-hand domain / EF-hand domain / Myosin N-terminal SH3-like domain / Myosin tail / Myosin head (motor domain) ...Kinesin motor domain superfamily / Myosin S1 fragment, N-terminal / IQ motif, EF-hand binding site / IQ motif profile. / Myosin head, motor domain / EF-hand domain / EF-hand domain / Myosin N-terminal SH3-like domain / Myosin tail / Myosin head (motor domain) / Myosin, N-terminal, SH3-like / EF-hand domain pair / EF-hand domain pair / Myosin tail / EF-hand calcium-binding domain profile. / Myosin motor domain profile. / Myosin N-terminal SH3-like domain profile. / Smooth Muscle Contraction / RHO GTPases activate PAKs / EF-Hand 1, calcium-binding site / Myosin IQ motif-containing domain superfamily / P-loop containing nucleoside triphosphate hydrolase / EF-hand calcium-binding domain. / myosin II filament / regulation of slow-twitch skeletal muscle fiber contraction / regulation of the force of skeletal muscle contraction / myosin light chain binding / myofibril assembly / muscle myosin complex / elastic fiber assembly / adult heart development / actomyosin structure organization / myosin II complex / regulation of the force of heart contraction / transition between fast and slow fiber / myosin II binding / skeletal muscle myosin thick filament assembly / actin-dependent ATPase activity / striated muscle contraction / myosin filament / skeletal muscle contraction / microfilament motor activity / cardiac muscle hypertrophy in response to stress / actomyosin / smooth muscle contraction / cardiac muscle fiber development / muscle filament sliding / myosin complex / structural constituent of muscle / myofibril / myosin heavy chain binding / ventricular cardiac muscle tissue morphogenesis / cardiac muscle contraction / ATP metabolic process / stress fiber / muscle contraction / regulation of heart rate / ADP binding / sarcomere / Z disc / actin filament binding / actin binding / calmodulin binding / ATPase activity / calcium ion binding / protein heterodimerization activity / magnesium ion binding / ATP binding / cytosol / Myosin II regulatory light chain / Myosin light polypeptide 6 / Myosin-11 / Myosin-7
Function and homology information
Specimen sourceGallus gallus (chicken)
Homo sapiens (human)
Avicularia avicularia (pinktoe tarantula)
MethodELECTRON MICROSCOPY / helical reconstruction / cryo EM / 20 Å resolution
AuthorsAlamo, L. / Wriggers, W. / Pinto, A. / Bartoli, F. / Salazar, L. / Zhao, F.Q. / Craig, R. / Padron, R.
CitationJournal: Cell / Year: 1999
Title: Atomic structure of scallop myosin subfragment S1 complexed with MgADP: a novel conformation of the myosin head.
Authors: A Houdusse / V N Kalabokis / D Himmel / A G Szent-Györgyi / C Cohen
Validation Report
SummaryFull reportAbout validation report
DateDeposition: Jul 15, 2008 / Release: Oct 7, 2008
RevisionDateData content typeGroupProviderType
1.0Oct 7, 2008Structure modelrepositoryInitial release
1.1Jul 13, 2011Structure modelVersion format compliance
1.2Jul 22, 2015Structure modelSource and taxonomy
1.3Dec 14, 2016Structure modelOther

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Structure visualization

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  • Biological unit as representative helical assembly
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  • Deposited structure unit
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  • Superimposition on EM map
  • EMDB-1535
  • Imaged by UCSF Chimera
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  • Superimposition on EM map
  • EMDB-1535
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Structure viewerMolecule:
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Assembly

Deposited unit
A: Myosin 2 heavy chain chimera of smooth and cardiac muscle
B: Myosin 2 heavy chain chimera of smooth and cardiac muscle
C: Smooth muscle myosin essential light chain
D: Smooth muscle myosin essential light chain
E: Myosin regulatory light chain
F: Myosin regulatory light chain


Theoretical massNumber of molelcules
Total (without water)301,0676
Polyers301,0676
Non-polymers00
Water0
1
A: Myosin 2 heavy chain chimera of smooth and cardiac muscle
B: Myosin 2 heavy chain chimera of smooth and cardiac muscle
C: Smooth muscle myosin essential light chain
D: Smooth muscle myosin essential light chain
E: Myosin regulatory light chain
F: Myosin regulatory light chain
x 36


Theoretical massNumber of molelcules
Total (without water)10,838,410216
Polyers10,838,410216
Non-polymers00
Water0
TypeNameSymmetry operationNumber
helical symmetry operation36
2


  • idetical with deposited unit in distinct coordinate
  • helical asymmetric unit
TypeNameSymmetry operationNumber
helical symmetry operation1
3


  • idetical with deposited unit in distinct coordinate
  • helical asymmetric unit, std helical frame
TypeNameSymmetry operationNumber
transform to helical frame1
Helical symmetryCircular symmetry: 4 / Dyad axis: no / N subunits divisor: 1 / Number of operations: 36 / Rise per n subunits: 145 / Rotation per n subunits: 3

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Components

#1: Protein/peptide Myosin 2 heavy chain chimera of smooth and cardiac muscle / Myosin heavy chain / gizzard smooth muscle / cardiac muscle beta isoform / MyHC-beta / Myosin heavy chain slow isoform / MyHC-slow


Mass: 111851.070 Da / Num. of mol.: 1 / Fragment: SUBFRAGMENT 1(S1), DELTA-S2 (residues 2-972) / Source: (gene. exp.) Gallus gallus, Homo sapiens / Plasmid name: PVL1392, PET / Cell line (production host): SF9
Production host: Spodoptera frugiperda, Escherichia coli BL21
Strain (production host): BL21 / References: UniProt: P10587, UniProt: P12883
#2: Protein/peptide Myosin 2 heavy chain chimera of smooth and cardiac muscle / Myosin heavy chain / gizzard smooth muscle / cardiac muscle beta isoform / MyHC-beta / Myosin heavy chain slow isoform / MyHC-slow


Mass: 112051.328 Da / Num. of mol.: 1 / Fragment: SUBFRAGMENT 1(S1), DELTA-S2 (residues 2-974) / Source: (gene. exp.) Gallus gallus, Homo sapiens / Plasmid name: PVL1392, PET / Cell line (production host): SF9
Production host: Spodoptera frugiperda, Escherichia coli BL21
Strain (production host): BL21 / References: UniProt: P10587, UniProt: P12883
#3: Protein/peptide Smooth muscle myosin essential light chain / ELC / Myosin light chain alkali smooth-muscle/non-muscle isoforms / G2 catalytic / LC17-GI / LC17-NM


Mass: 16873.025 Da / Num. of mol.: 2 / Source: (gene. exp.) Gallus gallus (chicken) / Plasmid name: PVL1392 / Cell line (production host): SF9 / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: P02607
#4: Protein/peptide Myosin regulatory light chain / RLC


Mass: 21709.250 Da / Num. of mol.: 2 / Source: (natural) Avicularia avicularia (pinktoe tarantula) / Tissue: leg muscle / References: UniProt: B4XT43*PLUS
Sequence detailsTHE CONFLICT LISTED FOR CHAINS A AND B, RESIDUE 2 ARE CONSISTENT WITH PDB ENTRY 1BR1 AND 1I84. THE ...THE CONFLICT LISTED FOR CHAINS A AND B, RESIDUE 2 ARE CONSISTENT WITH PDB ENTRY 1BR1 AND 1I84. THE SEQUENCE OF THE HEAVY CHAIN (CHICKEN) STRUCTURE REPORTED HERE DIFFERS FROM THAT REPORTED IN THE SWISS-PROT DATABASE (ID: P10587), THAT RESIDUE SER 2 WAS CHANGED BY ALA AND DECLARED AS CLONING ARTIFACT IN PDB 1BR1. PEPTIDE CHAIN DESIGNATIONS: THE TERMS "BLOCKED" AND "FREE" REFER TO THE CONFORMATIONS OF THE TWO S1 MYOSIN HEADS. "FREE" MYOSIN HEAD MYOSIN HEAVY CHAIN S1 PLUS S2 FRAGMENT IS CHAIN A ELC IS CHAIN C RLC IS CHAIN E "BLOCKED" MYOSIN HEAD MYOSIN HEAVY CHAIN S1 PLUS S2 FRAGMENT IS CHAIN B ELC IS CHAIN D RLC IS CHAIN F SEQUENCE GAPS IN THE MOLECULAR MODEL: HEAVY CHAIN UNP-P10587 CHAIN A,B: 1, 205-210, 452-457, 635-655, 853-1185 HEAVY CHAIN S2 FRAGMENT UNP-P12883 CHAIN A: 1-841, 962-1935 HEAVY CHAIN S2 FRAGMENT UNP-P12883 CHAIN B: 1-841, 964-1935 ELC UNP-P02607 CHAIN C,D: 1-2

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: FILAMENT / Reconstruction method: helical reconstruction

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Sample preparation

ComponentName: Myosin thick filaments from Tarantula striated muscle / Type: COMPLEX
Details: Polymer of a multiple myosin assembled over a paramyosin core. MODEL BUILDING: the atomic model consists of two s1 heads and a segment of s2. each heavy meromyosin consists of a chimera built by chicken smooth muscle heavy chain (1i84) for s1 plus human cardiac muscle (2fxm) for s2 (chains a,b) and two light chains, the chicken smooth muscle (1i84) for elc (chains c,d) and a homology model based on 1br1 of the tarantula skeletal muscle rlc sequence (chains e,f), this model was flexible fitted to a tarantula 3d map (emd-1535)
Buffer solutionName: 100mM NaCl, 3mM MgCl2, 1mM EGTA, 5mM PIPES, 5mM NaH2PO4, 1mM NaN3
Details: 100mM NaCl, 3mM MgCl2, 1mM EGTA, 5mM PIPES, 5mM NaH2PO4, 1mM NaN3
pH: 7
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: Holey carbon grids, 400 mesh
VitrificationDetails: Plunging in a liquid ethane. 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.

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Electron microscopy imaging

MicroscopyMicroscope model: FEI/PHILIPS CM120T / Date: Sep 19, 2001
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 gunElectron source: LAB6 / Accelerating voltage: 120 / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 35000 / Calibrated magnification: 35000 / Nominal defocus max: 1950 / Nominal defocus min: 1950 / Cs: 2
Specimen holderTemperature: 88
Image scansNumber digital images: 465

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Processing

EM software
IDNameVersionCategory
1Situs3.2model fitting
2X-PLOR3.851model fitting
CTF correctionDetails: no corrected
3D reconstructionMethod: Single particle reconstruction with a modification of the IHRSR method
Resolution: 20 / Number of particles: 15504 / Nominal pixel size: 2.48 / Actual pixel size: 2.48 / Magnification calibration: Tropomyosin paracrystal
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 were 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-minus 12 deg. every 2 deg., 45 reference rotated projections (0-90 deg., every 2 deg. 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.
Symmetry type: HELICAL
Atomic model buildingDetails: METHOD--Flexible Fitting REFINEMENT PROTOCOL--Custom skeleton of 31 positional markers
Ref protocol: FLEXIBLE FIT / Ref space: REAL / Target criteria: Constrained Molecular Dynamics
Atomic model building
IDPDB-ID 3D fitting ID
11I84

1i84

1
22FXM

2fxm

1
31B7T

1b7t

1
Number of atoms included #LASTProtein: 20580 / Nucleic acid: 0 / Ligand: 0 / Solvent: 0 / Total: 20580

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