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Title:Electron cryo-microscopy of microtubule-bound human kinesin-5 motor domain in AMPPNP state.
Authors:Goulet A, Behnke-Parks WM, Sindelar C, Rosenfeld S, Moores C
Sample:13-protofilament microtubule-bound human kinesin-5 motor domain with AMPPNP
Method:Single particle reconstruction (9.7 angstroms resolution)
Red flagLatest update:2013-01-09
Sample
Sample name: 13-protofilament microtubule-bound human kinesin-5 motor domain with AMPPNP
Oligomeric state: 13-protofilament microtubule with one kineisn-5 motor domain bound every tubulin heterodimers
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
1proteinalpha-beta tubulin dimerheterodimerfalseBos taurus
2proteinKinesin-5 motor domainmonomertrueHomo sapiens
Experiment
Specimen state: Particle
Specimen preparation:
pHSpecimen conc.DetailsStainingSpecimen support details
6.8 mg/mL80 mM PIPES, 5 mM MgCl2, 1 mM EGTA, 5mM AMPPNP400 mesh holey carbon grids
Vitrification:
Cryogen nameHumidityTemp.Instr.MethodTime resolvedDetails
ETHANE100% KFEI VITROBOT MARK Ichamber at 24 degrees C, blot 2.5 sec ms
Imaging:
MicroscopeVoltageIllumination modeImaging modeCsDefocus min.Defocus max.Nominal mag.Calibrated mag.Electron sourceDetectorDetector distanceAstigmatism
FEI TECNAI F20200 kVFLOOD BEAMBRIGHT FIELD2.0 mm700 nm2200 nm50000FIELD EMISSION GUNKODAK SO-163 FILM mmObjective lens astigmatism was corrected at 150,000 times magnification

Specimen holderHolder modelTilt min.Tilt max.Energy filterEnergy windowTemp.Temp. min.Temp. max.Beam tiltElectron doseOther detailsDate
GATAN LIQUID NITROGEN°° eV90 K K K mrad18 e/Å210-JAN-2011
Processing
Software:SPIDER, FREALIGN
CTF correction:FREALIGN
Number of particles:3587
Resolution by author:9.7 Å
Resolution method:FSC 0.5
Processing details:Approximately 50,000 asymmetric units were averaged in the final reconstruction. The particles were selected along individual microtubules.
Scanned images:
Num. imagesSampling sizeOD rangeQuant. bit numberOther detailsScanner
467 μm/pixel8ZEISS SCAI
Fitting:
PDBProtocolTarget crit.SoftwareB valueFitting spacePDB chainDetails
3HQD flexiblecross-correlationChimera, FlexEMREALProtocol: rigid body then flexible fitting. The domain was fitted as a rigid body. The N-terminal residues 6 to 16 were built in the EM map and the final model was refined by flexible fitting.
1JFF rigid bodycross-correlationChimeraREALProtocol: rigid body. alpha- and b-tubulin were separately fitted.