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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
Sample name: Myosin thick filaments from Schistosoma mansoni smooth muscle
Oligomeric state: polymer of myosin II molecules helically assembled over a paramyosin core
Components:
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.
Experiment
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
RIGHT HANDED30°145 ÅC4
Vitrification:
Cryogen nameHumidityTemp.Instr.MethodTime resolvedDetails
NONE% KNONE ms
Imaging:
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
Processing
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.
Fitting:
PDBProtocolTarget crit.SoftwareB valueFitting spacePDB chainDetails
3DTP rigid bodyChimeraREAL3DTP was fitted as a rigid body using the "Fit in Map" tool of UCSF Chimera.