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- PDB-3jax: Heavy meromyosin from Schistosoma mansoni muscle thick filament b... -

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Basic information

Entry
Database: PDB / ID: 3jax
TitleHeavy meromyosin from Schistosoma mansoni muscle thick filament by negative stain EM
Components
  • myosin 2 heavy chain
  • 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/homologyEF-hand domain / Myosin tail / IQ calmodulin-binding motif / Myosin, N-terminal, SH3-like / Myosin tail / myosin complex / Myosin S1 fragment, N-terminal / microtubule motor activity / Myosin N-terminal SH3-like domain / Myosin head, motor domain ...EF-hand domain / Myosin tail / IQ calmodulin-binding motif / Myosin, N-terminal, SH3-like / Myosin tail / myosin complex / Myosin S1 fragment, N-terminal / microtubule motor activity / Myosin N-terminal SH3-like domain / Myosin head, motor domain / Myosin motor domain profile. / IQ motif profile. / IQ motif, EF-hand binding site / microtubule-based movement / Myosin head (motor domain) / EF-hand domain pair / Kinesin motor domain superfamily / actin filament binding / EF-hand calcium-binding domain profile. / EF-Hand 1, calcium-binding site / EF-hand calcium-binding domain. / EF-hand domain / EF-hand domain pair / microtubule binding / calcium ion binding / P-loop containing nucleoside triphosphate hydrolase / ATP binding / Myosin 2 heavy chain / Smooth muscle myosin essential light chain / Myosin regulatory light chain
Function and homology information
Specimen sourceSchistosoma mansoni / / invertebrata /
MethodElectron microscopy (23 Å resolution / Filament / Helical) / Transmission electron microscopy
AuthorsSulbaran, G. / Alamo, L. / Pinto, A. / Marquez, G. / Mendez, F. / Padron, R. / Craig, 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
Abstract: The crystal structure of a proteolytic subfragment from scallop striated muscle myosin, complexed with MgADP, has been solved at 2.5 A resolution and reveals an unusual conformation of the myosin ...The crystal structure of a proteolytic subfragment from scallop striated muscle myosin, complexed with MgADP, has been solved at 2.5 A resolution and reveals an unusual conformation of the myosin head. The converter and the lever arm are in very different positions from those in either the pre-power stroke or near-rigor state structures; moreover, in contrast to these structures, the SH1 helix is seen to be unwound. Here we compare the overall organization of the myosin head in these three states and show how the conformation of three flexible "joints" produces rearrangements of the four major subdomains in the myosin head with different bound nucleotides. We believe that this novel structure represents one of the prehydrolysis ("ATP") states of the contractile cycle in which the myosin heads stay detached from actin.
Validation Report
SummaryFull reportAbout validation report
DateDeposition: Jul 3, 2015 / Release: Oct 7, 2015
RevisionDateData content typeGroupProviderType
1.0Oct 7, 2015Structure modelrepositoryInitial release
1.1Oct 21, 2015Structure modelDatabase references
1.2Nov 4, 2015Structure modelDatabase references

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Structure visualization

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Assembly

Deposited unit
A: myosin 2 heavy chain
B: myosin 2 heavy chain
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)301,7926
Polyers301,7926
Non-polymers00
Water0
1
A: myosin 2 heavy chain
B: myosin 2 heavy chain
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 20


Theoretical massNumber of molelcules
Total (without water)6,035,839120
Polyers6,035,839120
Non-polymers00
Water0
TypeNameSymmetry operationNumber
helical symmetry operation20
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: 20 / Rise per n subunits: 145 Å / Rotation per n subunits: 3 deg.

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Components

#1: Protein/peptide myosin 2 heavy chain


Mass: 112182.516 Da / Num. of mol.: 2 / Source: (natural) Schistosoma mansoni / / invertebrata / / Cellular location: sarcomere / Organelle: myosin thick filament / Strain: JL / Tissue: smooth muscle / References: UniProt:A0A0R4I956*PLUS
#2: Protein/peptide smooth muscle myosin essential light chain


Mass: 17004.221 Da / Num. of mol.: 2 / Source: (natural) Schistosoma mansoni / / invertebrata / / Cellular location: sarcomere / Organelle: myosin thick filament / Strain: JL / Tissue: smooth muscle / References: UniProt:A0A0R4I957*PLUS
#3: Protein/peptide myosin regulatory light chain


Mass: 21709.250 Da / Num. of mol.: 2 / Source: (natural) Schistosoma mansoni / / invertebrata / / Cellular location: sarcomere / Organelle: myosin thick filament / Strain: JL / Tissue: smooth muscle / References: UniProt:A0A0R4I958*PLUS
Sequence detailsTHE IMAGED FILAMENTS ARE FROM SCHISTOSOMA MANSONI, BUT THE MODELED SEQUENCES ARE FROM CHICKEN/HUMAN ...THE IMAGED FILAMENTS ARE FROM SCHISTOSOMA MANSONI, BUT THE MODELED SEQUENCES ARE FROM CHICKEN/HUMAN CHIMERA (CHAINS A,B), CHICKEN (CHAINS C,D), AND TARANTULA (CHAINS E,F). SEQUENCE CONFLICTS FOR CHAINS A AND B ARE CONSISTENT WITH PDB ENTRIES 1BR1 AND 1I84. THE SEQUENCE OF THE HEAVY CHAIN STRUCTURE REPORTED HERE DIFFERS FROM THAT REPORTED IN UNP P10587. SER 2 TO ALA IS A CLONING ARTIFACT IN PDB ENTRY 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 CHAINS A AND B: 1, 205-210, 452-457, 635-655, 853-1979 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 CHAINS C AND D: 1-3

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: FILAMENT / Reconstruction method: HELICAL

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Sample preparation

Component
IDNameTypeDetailsParent ID
1Myosin thick filaments from Schistosoma mansoni smooth muscleCOMPLEXPolymer of myosin II molecules helically assembled over a paramyosin core0
2Myosin II1
Buffer solutionName: 100 mM NaCl, 3 mM MgCl2, 1 mM EGTA, 5 mM PIPES, 1mM NaN3, 5 mM MgATP, 0.01 mM blebbistatin, protease inhibitor cocktail (Sigma P-8465)
Details: 100 mM NaCl, 3 mM MgCl2, 1 mM EGTA, 5 mM PIPES, 1mM NaN3, 5 mM MgATP, 0.01 mM blebbistatin, protease inhibitor cocktail (Sigma P-8465)
pH: 7
SpecimenDetails: One drop of filament suspension was placed on grids and negatively stained with 1% uranyl acetate.
Embedding applied: NO / Shadowing applied: NO / Staining applied: YES / Vitrification applied: NO
EM stainingType: NEGATIVE / Material: Uranyl Acetate
Specimen supportDetails: 400-mesh holey carbon grids. Specimens were imaged on thin carbon extending over the holes.

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Electron microscopy imaging

MicroscopyMicroscope model: FEI/PHILIPS CM120T / Date: Feb 15, 2013 / Details: 1.5 post-magnification, low-dose conditions
Electron gunElectron source: LAB6 / Accelerating voltage: 80 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 42000 / Calibrated magnification: 42000 / Nominal defocus max: 2400 nm / Nominal defocus min: 600 nm / Cs: 2 mm
Astigmatism: Objective lens astigmatism was corrected at 240,000 times magnification
Specimen holderSpecimen holder model: SIDE ENTRY, EUCENTRIC / Specimen holder type: Room temperature holder
Image recordingElectron dose: 10 e/Å2 / Film or detector model: TVIPS TEMCAM-F224 (2k x 2k)
Image scansNumber digital images: 263
RadiationDiffraction protocol: SINGLE WAVELENGTH / Monochromatic or laue m l: M / Scattering type: x-ray
Radiation wavelengthRelative weight: 1

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Processing

EM software
IDNameVersionCategory
1UCSF ChimeraMODEL FITTING
2EMAN2RECONSTRUCTION
3SPIDERRECONSTRUCTION
Helical symmertyAngular rotation/subunit: 30 deg. / Axial rise/subunit: 145 Å / Axial symmetry: C4
Details: Heavy meromyosin (HMM) is the outermost helical unit of the myosin thick filament formed by two myosin heads that include the motor domains, the proteolytic fragment of myosin heavy chain subfragment 2 (S2), and two associated light chains (ELC and RLC).
3D reconstructionMethod: Single particle reconstruction with a modification of the IHRSR method
Resolution: 23 Å / Resolution method: FSC 0.5 CUT-OFF / Number of particles: 9500 / Nominal pixel size: 5.7 / Actual pixel size: 5.7
Details: For each iteration of reconstruction (30 cycles), filament segment projections were compared with different projections of the reference reconstruction as follows: seven 2.3 nm axial shifts, 2 degree intervals of rotation about the filament axis up to 90 degrees, and 2 degree intervals of out-of-plane tilting from -10 degrees to +10 degrees. The total number of projections was 7 x 45 x 11 = 3465. For the final 19 cycles of the reconstruction, we used only the best-ordered 420 filament halves (those in which >30% of the segments were found good enough to be used by the reconstruction script in the back-projection in previous cycles). From ~17,000 segments, ~9,500 (56%) were included in the final reconstruction. This final 3D-reconstruction was the average of the last 19 reconstructions between cycles 12 - 30. Its resolution, according to the 0.5 Fourier Shell Correlation (FSC) criterion, was 2.3 nm.
Symmetry type: HELICAL
Atomic model buildingDetails: METHOD--rigid docking REFINEMENT PROTOCOL--RIGID BODY DETAILS--3DTP was fitted as a rigid body using the Fit in Map tool of UCSF Chimera.
Ref protocol: RIGID BODY FIT / Ref space: REAL
Atomic model building
IDPDB-IDPdb chain ID 3D fitting ID
13DTPA1
23DTPB1
33DTPC1
43DTPD1
53DTPE1
63DTPF1
Number of atoms included #LASTProtein: 20580 / Nucleic acid: 0 / Ligand: 0 / Solvent: 0 / Total: 20580

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