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- EMDB-6370: 3D-Structure of negatively stained Schistosome myosin filament ob... -

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Basic information

Entry
Database: EMDB / ID: EMD-6370
Title3D-Structure of negatively stained Schistosome myosin filament obtained by low-dose electron microscopy
Map dataMap of Schistosome thick filaments. Initial view is from the Z-line perspective. If the map is rotated by 90 degrees in x direction, the J motif of the interacting heads is featured and the backbone subfilaments can be seen clearly.
Sample
  • Sample: Myosin thick filaments from Schistosoma mansoni smooth muscle
  • Protein or peptide: Myosin IIMyosin
KeywordsSchistosoma mansoni / rigid docking / single particle reconstruction / Iterative Helical Real Space Reconstruction (IHRSR) / negative stain / thick filament / smooth muscle
Function / homology
Function and homology information


myosin complex / myofibril / cytoskeletal motor activity / actin filament binding / calcium ion binding / ATP binding
Similarity search - Function
: / : / EF-hand domain / Myosin tail / Myosin tail / Myosin tail / Myosin N-terminal SH3-like domain / IQ calmodulin-binding motif / Myosin S1 fragment, N-terminal / Myosin, N-terminal, SH3-like ...: / : / EF-hand domain / Myosin tail / Myosin tail / Myosin tail / Myosin N-terminal SH3-like domain / IQ calmodulin-binding motif / Myosin S1 fragment, N-terminal / Myosin, N-terminal, SH3-like / Myosin, N-terminal, SH3-like / Myosin N-terminal SH3-like domain profile. / Short calmodulin-binding motif containing conserved Ile and Gln residues. / Myosin head, motor domain / Myosin head, motor domain / Myosin head (motor domain) / Myosin motor domain profile. / Myosin. Large ATPases. / IQ motif profile. / IQ motif, EF-hand binding site / IQ motif, EF-hand binding site / Kinesin motor domain superfamily / EF-hand, calcium binding motif / EF-Hand 1, calcium-binding site / EF-hand calcium-binding domain. / EF-hand calcium-binding domain profile. / EF-hand domain / EF-hand domain pair / P-loop containing nucleoside triphosphate hydrolase / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
Myosin 2 heavy chain / Smooth muscle myosin essential light chain / Myosin regulatory light chain / Myosin heavy chain
Similarity search - Component
Biological speciesSchistosoma mansoni (invertebrata)
Methodhelical reconstruction / negative staining / Resolution: 23.0 Å
AuthorsSulbaran G / Alamo L / Pinto A / Marquez G / Mendez F / Padron R / Craig R
Citation
Journal: Proc Natl Acad Sci U S A / Year: 2015
Title: An invertebrate smooth muscle with striated muscle myosin filaments.
Authors: Guidenn Sulbarán / Lorenzo Alamo / Antonio Pinto / Gustavo Márquez / Franklin Méndez / Raúl Padrón / Roger Craig /
Abstract: Muscle tissues are classically divided into two major types, depending on the presence or absence of striations. In striated muscles, the actin filaments are anchored at Z-lines and the myosin and ...Muscle tissues are classically divided into two major types, depending on the presence or absence of striations. In striated muscles, the actin filaments are anchored at Z-lines and the myosin and actin filaments are in register, whereas in smooth muscles, the actin filaments are attached to dense bodies and the myosin and actin filaments are out of register. The structure of the filaments in smooth muscles is also different from that in striated muscles. Here we have studied the structure of myosin filaments from the smooth muscles of the human parasite Schistosoma mansoni. We find, surprisingly, that they are indistinguishable from those in an arthropod striated muscle. This structural similarity is supported by sequence comparison between the schistosome myosin II heavy chain and known striated muscle myosins. In contrast, the actin filaments of schistosomes are similar to those of smooth muscles, lacking troponin-dependent regulation. We conclude that schistosome muscles are hybrids, containing striated muscle-like myosin filaments and smooth muscle-like actin filaments in a smooth muscle architecture. This surprising finding has broad significance for understanding how muscles are built and how they evolved, and challenges the paradigm that smooth and striated muscles always have distinctly different components.
#1: Journal: BIOPHYS.J. / Year: 2014
Title: Schistosome Muscles Contain Striated Muscle-Like Myosin Filaments in a Smooth Muscle-Like Architecture
Authors: Sulbaran G / Alamo L / Pinto A / Marquez G / Mendez F / Padron R / Craig R
History
DepositionJul 2, 2015-
Header (metadata) releaseAug 19, 2015-
Map releaseOct 7, 2015-
UpdateNov 4, 2015-
Current statusNov 4, 2015Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 6
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 6
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-3jax
  • Surface level: 6
  • Imaged by UCSF Chimera
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  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-3jax
  • Imaged by Jmol
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_6370.tif

emd_6370_1.tif

emd_6370_2.tif

EMD-6370_1.tif

EMD-6370_2.tif

Downloads & links

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Map

FileDownload / File: emd_6370.map.gz / Format: CCP4 / Size: 8.4 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationMap of Schistosome thick filaments. Initial view is from the Z-line perspective. If the map is rotated by 90 degrees in x direction, the J motif of the interacting heads is featured and the backbone subfilaments can be seen clearly.
Voxel sizeX=Y=Z: 5.7 Å
Density
Contour LevelBy AUTHOR: 6.0 / Movie #1: 6
Minimum - Maximum-0.16773927 - 36.991428380000002
Average (Standard dev.)1.01146114 (±3.85783195)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-65-650
Dimensions131131131
Spacing131131131
CellA=B=C: 746.69995 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z5.75.75.7
M x/y/z131131131
origin x/y/z0.0000.0000.000
length x/y/z746.700746.700746.700
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS-65-650
NC/NR/NS131131131
D min/max/mean-0.16836.9911.011

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Supplemental data

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Sample components

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Entire : Myosin thick filaments from Schistosoma mansoni smooth muscle

EntireName: Myosin thick filaments from Schistosoma mansoni smooth muscle
Components
  • Sample: Myosin thick filaments from Schistosoma mansoni smooth muscle
  • Protein or peptide: Myosin IIMyosin

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Supramolecule #1000: Myosin thick filaments from Schistosoma mansoni smooth muscle

SupramoleculeName: Myosin thick filaments from Schistosoma mansoni smooth muscle
type: sample / ID: 1000
Details: S. mansoni relaxed myosin thick filaments were isolated by permeabilizing and homogenizing whole animals in relaxing solution, centrifuged, and resuspended in blebbistatin.
Oligomeric state: polymer of myosin II molecules helically assembled over a paramyosin core
Number unique components: 1

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Macromolecule #1: Myosin II

MacromoleculeName: Myosin II / type: protein_or_peptide / ID: 1 / Name.synonym: Myosin Type II
Details: Myosin II is a protein complex formed by two heavy chains and two associated light chains (for each myosin head), plus additional proteins such as paramyosin. Using ATP hydrolysis, myosin ...Details: Myosin II is a protein complex formed by two heavy chains and two associated light chains (for each myosin head), plus additional proteins such as paramyosin. Using ATP hydrolysis, myosin functions as a molecular motor, producing movement by causing actin filaments to slide.
Number of copies: 1
Oligomeric state: polymer of myosin II molecules helically assembled over a paramyosin core
Recombinant expression: No / Database: NCBI
Source (natural)Organism: Schistosoma mansoni (invertebrata) / Strain: JL / synonym: Blood fluke / Tissue: smooth muscle / Organelle: myosin thick filament / Location in cell: sarcomere
SequenceUniProtKB: Myosin heavy chain
GO: myosin complex, cytoskeletal motor activity, ATP binding
InterPro: Myosin, N-terminal, SH3-like, P-loop containing nucleoside triphosphate hydrolase, Myosin head, motor domain, INTERPRO: IPR027401, Myosin tail, IQ motif, EF-hand binding site

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Experimental details

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Structure determination

Methodnegative staining
Processinghelical reconstruction
Aggregation statefilament

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Sample preparation

BufferpH: 7
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)
StainingType: NEGATIVE
Details: One drop of filament suspension was placed on grids and negatively stained with 1% uranyl acetate.
GridDetails: 400-mesh holey carbon grids. Specimens were imaged on thin carbon extending over the holes.
VitrificationCryogen name: NONE / Instrument: OTHER

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Electron microscopy

MicroscopeFEI/PHILIPS CM120T
Electron beamAcceleration voltage: 80 kV / Electron source: LAB6
Electron opticsCalibrated magnification: 42000 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.0 mm / Nominal defocus max: 2.4 µm / Nominal defocus min: 0.6 µm / Nominal magnification: 42000
Sample stageSpecimen holder: Room temperature holder / Specimen holder model: SIDE ENTRY, EUCENTRIC
Alignment procedureLegacy - Astigmatism: Objective lens astigmatism was corrected at 240,000 times magnification
Details1.5 post-magnification, low-dose conditions
DateMar 1, 2013
Image recordingCategory: CCD / Film or detector model: TVIPS TEMCAM-F224 (2k x 2k) / Number real images: 263 / Average electron dose: 10 e/Å2
Details: Images were acquired with a 2K x 2K CCD TVIPS camera model F224HD at 5.7 A/pixel.
Bits/pixel: 16

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Image processing

Final reconstructionApplied symmetry - Helical parameters - Δz: 145 Å
Applied symmetry - Helical parameters - Δ&Phi: 30 °
Applied symmetry - Helical parameters - Axial symmetry: C4 (4 fold cyclic)
Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 23.0 Å / Resolution method: OTHER / Software - Name: SPIDER, EMAN2
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, ...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.
Details820 thick filament halves were selected from micrographs and stored in SPIDER format. 131 x 131 pixel segments were cut from these filaments, corresponding to a window of 74.7 nm (~five 14.5 nm-spaced crowns of heads).
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

3dtp


Chain - #0 - Chain ID: A / Chain - #1 - Chain ID: B / Chain - #2 - Chain ID: C / Chain - #3 - Chain ID: D / Chain - #4 - Chain ID: E / Chain - #5 - Chain ID: F
SoftwareName: Chimera
Details3DTP was fitted as a rigid body using the "Fit in Map" tool of UCSF Chimera.
RefinementSpace: REAL / Protocol: RIGID BODY FIT
Output model

PDB-3jax:
Heavy meromyosin from Schistosoma mansoni muscle thick filament by negative stain EM

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