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Title:3D-Structure of negatively stained Schistosome myosin filament obtained by low-dose electron microscopy
Authors:Sulbaran G, Alamo L, Pinto A, Marquez G, Mendez F, Padron R, Craig R
Sample:Myosin thick filaments from Schistosoma mansoni smooth muscle
Method:Helical reconstruction (23 angstroms resolution)
Red flagLatest update:2015-11-04
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Sample name: Myosin thick filaments from Schistosoma mansoni smooth muscle
Oligomeric state: polymer of myosin II molecules helically assembled over a paramyosin core
ID Type Name Exp. MW (MDa) Oligomeric details Recombinant expression Synthetic Organism UniProt identifier GO identifier InterPro identifier Virus identifier Details
1proteinMyosin IIpolymer of myosin II molecules helically assembled over a paramyosin corefalseSchistosoma mansoniQ02456GO:0016459, GO:0003774, GO:0005524IPR004009, IPR027417, IPR001609, IPR027401, IPR002928, IPR000048Myosin 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.
Specimen state: Filament
Specimen preparation:
pHSpecimen conc.DetailsStainingSpecimen support details
7.0 mg/mL100 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)One drop of filament suspension was placed on grids and negatively stained with 1% uranyl acetate.400-mesh holey carbon grids. Specimens were imaged on thin carbon extending over the holes.
Helical parameters:
HandPhi incrementAzimuthal (Z) incrementRotational symmetry
Cryogen nameHumidityTemp.Instr.MethodTime resolvedDetails
MicroscopeVoltageIllumination modeImaging modeCsDefocus min.Defocus max.Nominal mag.Calibrated mag.Electron sourceDetectorDetector distanceAstigmatism
FEI/PHILIPS CM120T80 kVFLOOD BEAMBRIGHT FIELD2.0 mm600 nm2400 nm4200042000LAB6TVIPS TEMCAM-F224 (2k x 2k) mmObjective lens astigmatism was corrected at 240,000 times magnification

Specimen holderHolder modelTilt min.Tilt max.Energy filterEnergy windowTemp.Temp. min.Temp. max.Beam tiltElectron doseOther detailsDate
Room temperature holderSIDE ENTRY, EUCENTRIC°° eV K K K mrad10 e/Å21.5 post-magnification, low-dose conditions01-MAR-2013
Protocol:Single particle reconstruction with a modification of the IHRSR method
Software:SPIDER, EMAN2
Resolution by author:23 Å
Resolution method:FSC 0.5, semi-independent
Processing 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. 820 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).
Scanned images:
Num. imagesSampling sizeOD rangeQuant. bit numberOther detailsScanner
263 μm/pixel16Images were acquired with a 2K x 2K CCD TVIPS camera model F224HD at 5.7 A/pixel.
PDBProtocolTarget crit.SoftwareB valueFitting spacePDB chainDetails
3DTP rigid bodyChimeraREAL3DTP was fitted as a rigid body using the "Fit in Map" tool of UCSF Chimera.