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Title:Reconstruction of the Wild-type Ndc80 Bonsai Decorated Microtubule on CCD for Difference Map Calculation
Authors:Alushin GM, Musinipally V, Matson D, Tooley J, Stukenberg PT, Nogales E
Sample:Human Ndc80 bonsai complex bound to the microtubule
Method:Helical reconstruction (12.8 angstroms resolution)
Red flagLatest update:2014-03-26
Sample
Sample name: Human Ndc80 bonsai complex bound to the microtubule
Oligomeric state: 2 copies of the Ndc80 bonsai complex bind to each tubulin heterodimer
Theoretical molecular weight of the sample: 0.26
Components:
ID Type Name Exp. MW (MDa) Theo. MW (MDa) Oligomeric details Recombinant expression Synthetic Organism UniProt identifier GO identifier InterPro identifier Virus identifier Details
1proteintubulin0.11heterodimerfalseBos taurus
2proteinNdc80-Spc25 Chimera0.045heterodimertrueHomo sapiensChimera of Ndc80 residues 1-286 and Spc25 residues 118-224
3proteinNuf2-Spc24 Chimera0.029heterodimertrueHomo sapiensChimera of Nuf2 residues 1-169 and Spc24 residues 122-197
Experiment
Specimen state: Filament
Specimen preparation:
pHSpecimen conc.DetailsStainingSpecimen support details
6.80.25 mg/mL80mM PIPES, 1mM MgCl2, 1mM EGTA, 1mM DTT, 0.05% Nonidet P-40, 20uM taxolC-flat 1.2/1.3
Helical parameters:
HandPhi incrementAzimuthal (Z) increment
LEFT HANDED25.76246°9.04016 Å
Vitrification:
Cryogen nameHumidityTemp.Instr.MethodTime resolvedDetails
ETHANE100% KFEI VITROBOT MARK II2 uL of 0.25 mg/mL MTs applied to grid for 1 minute, 4 uL of 0.7 mg/mL Ndc80 bonsai added, manually blot 1 minute, another 4 uL of Ndc80 applied for 1 minute, 2 uL removed with pipettor, blot for 2 seconds before plunging, 0 mm offset ms
Imaging:
MicroscopeVoltageIllumination modeImaging modeCsDefocus min.Defocus max.Nominal mag.Calibrated mag.Electron sourceDetectorDetector distanceAstigmatism
FEI TECNAI F20120 kVFLOOD BEAMBRIGHT FIELD2.2 mm800 nm3200 nm80000FIELD EMISSION GUNGATAN ULTRASCAN 4000 (4k x 4k) mmobjective lens astigmatism corrected at 100Kx mag

Specimen holderHolder modelTilt min.Tilt max.Energy filterEnergy windowTemp.Temp. min.Temp. max.Beam tiltElectron doseOther detailsDate
side-entryGATAN LIQUID NITROGEN°° eV K K K mrad20 e/Å229-OCT-2011
Processing
Protocol:IHRSR
Software:EMAN2/SPARX
CTF correction:Phase-flipping each image
Resolution by author:12.8 Å
Resolution method:FSC 0.5
Processing details:Particles were aligned using multi-model IHRSR protocol in EMAN2/SPARX with naked 13 and 14 protofilament microtubules as references. The deposited map is a segmented region for difference map calculation. No amplitude scaling was applied. The phase-flipped particles were aligned using IHRSR in EMAN2/SPARX.
Scanned images:
Num. imagesSampling sizeOD rangeQuant. bit numberOther detailsScanner
20215 μm/pixel
Fitting:
PDBProtocolTarget crit.SoftwareB valueFitting spacePDB chainDetails