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Title:3D-Structure of tarantula myosin filament obtained by cryo-electron microscopy
Authors:Alamo L, Wriggers W, Pinto A, Bartoli F, Salazar L, Zhao F, Craig R, Padron R
Sample:Myosin filaments from Tarantula striated muscle
Method:Helical reconstruction (20 angstroms resolution)
Obsolete entries:EMD-1535
Red flagLatest update:2016-04-20
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Sample
Sample name: Myosin filaments from Tarantula striated muscle
Oligomeric state: Polymer of a multiple myosin 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 IIPolymerfalseAphonopelma sp.
Experiment
Specimen state: Filament
Specimen preparation:
pHSpecimen conc.DetailsStainingSpecimen support details
7.0 mg/mL100mM NaCl,3mM MgCl2,1mM EGTA, 5mM PIPES, 5mM NaH2PO4,1mM NaN3.A 6 ul aliquot of native purified tarantula thick filaments suspension (Hidalgo et al. 2001) was applied to a 400 mesh grid coated with a holey carbon film that had been rendered hydrophilic by glow discharge in n-amylamine vapor for 3 minutes before use. After allowing the filaments to adsorb to the grid for 30 seconds, the grid was rinsed with the relaxing rinse, then placed in a humidity chamber (aprox. 80% relative humidity). 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.Holey carbon grids 400 mesh
Helical parameters:
HandPhi incrementAzimuthal (Z) incrementRotational symmetry
RIGHT HANDED30°100 ÅC4
Vitrification:
Cryogen nameHumidityTemp.Instr.MethodTime resolvedDetails
ETHANE80%93 KHOMEMADE PLUNGERPlunging in a liquid ethane msVitrification instrument: Home-made plunger. 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.
Imaging:
MicroscopeVoltageIllumination modeImaging modeCsDefocus min.Defocus max.Nominal mag.Calibrated mag.Electron sourceDetectorDetector distanceAstigmatism
FEI/PHILIPS CM120T120 kVFLOOD BEAMBRIGHT FIELD2.0 mm1950 nm1950 nm3500035000LAB6KODAK SO-163 FILM mm

Specimen holderHolder modelTilt min.Tilt max.Energy filterEnergy windowTemp.Temp. min.Temp. max.Beam tiltElectron doseOther detailsDate
EucentricGATAN LIQUID NITROGEN°° eV K88 K90 K mrad e/Å2Holey 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 photographed23-OCT-2002
Processing
Protocol:Single particle reconstruction with a modification of the IHRSR method
Software:SPIDER
Resolution by author:20 Å
Resolution method:FSC 0.5
Processing 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 ere 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-minus12deg. every 2deg., 45 reference rotated projections (0-90 degrees, 2deg. 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. There are 4 helices of myosin heads, rotated 30 degrees, every 145 Angstroms. The filament segments were selected based on visual judgement of good helical order.
Scanned images:
Num. imagesSampling sizeOD rangeQuant. bit numberOther detailsScanner
10088.47 μm/pixel14OTHER
Fitting:
PDBProtocolTarget crit.SoftwareB valueFitting spacePDB chainDetails
3DTP flexibleCorrelationSitus 2.3REALProtocol: Flexible Fitting. The flexible docking procedure is based on a connected (motion capture) network of identified features within the atomic model. The atomic model is allowed to move according to displacements tracked by 31 control points defined by the network, in order to find the best match to the cryo-EM map