EMD-2077 Experiments and Validation

Electron cryo-microscopy of microtubule-bound human kinesin-5 motor domain in AMPPNP state.

Single particle reconstruction
Overview of EMD-2077
Sample name: 13-protofilament microtubule-bound human kinesin-5 motor domain with AMPPNP
Organisms: Bos taurus, Homo sapiens
Fitted atomic model: 4aqv
Related EM entries by publication: EMD-2078, EMD-2079, EMD-2080, EMD-2081, EMD-2152

Map parameters

Recommended contour level: 1.10 (author)
Number of grid points: 45 × 45 × 45
Voxel size: 2.80 × 2.80 × 2.80 Å
Minimum density: -6.577
Maximum density: 8.063
Average density: 0.08
Standard deviation: 1.81

Sample information

Sample name: 13-protofilament microtubule-bound human kinesin-5 motor domain with AMPPNP
Proteins: alpha-beta tubulin dimer, Kinesin-5 motor domain

Validation information

Experimental information

 

Image processing

Software: SPIDER, FREALIGN
Number of particles: 3587
CTF correction: FREALIGN
Details: Approximately 50,000 asymmetric units were averaged in the final reconstruction. The particles were selected along individual microtubules.

Fitting

Fitting 1
Initial atomic model: 3HQD
Used chain: A
Fitting software: Chimera, FlexEM
Fitting protocol: flexible
Target criteria: cross-correlation
Refinement space: REAL
Details: Protocol: 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.
Fitting 2
Initial atomic model: 1JFF
Used chains: A, B
Fitting software: Chimera
Fitting protocol: rigid body
Target criteria: cross-correlation
Refinement space: REAL
Details: Protocol: rigid body. alpha- and b-tubulin were separately fitted.

Imaging

Session
Microscope model: FEI TECNAI F20
Electron source: FIELD EMISSION GUN
Electron dose (e/A**2): 18.0
Nominal CS (mm): 2.00
Nominal magnification: 50000.0
Defocus max (nm): 2,200.00
Defocus min (nm): 700.00
Holder model: GATAN LIQUID NITROGEN
Acquisition
Number of images: 46
Scanner model: ZEISS SCAI
Sampling interval (μm): 7.0

Specimen

Preparation
Specimen state: particle
Buffer: 80 mM PIPES, 5 mM MgCl2, 1 mM EGTA, 5mM AMPPNP
Specimen support: 400 mesh holey carbon grids
Vitrification
Apparatus: FEI VITROBOT MARK I
Cryogen: ETHANE
Humidity: 100
Protocol: chamber at 24 degrees C, blot 2.5 sec