EMD-2538 Experiments and Validation

cryo-electron microscopy of microtubule-bound human kinesin-5 motor domain in the ADP state with a gold cluster attached to the loop5 (T126C)

Single particle reconstruction
Overview of EMD-2538
Sample name: 13-protofilament microtubule-bound human kinesin-5 motor domain in presence of ADP
Organisms: Bos taurus, Homo sapiens

Map parameters

Recommended contour level: 1.00 (author)
Number of grid points: 50 × 50 × 50
Voxel size: 2.20 × 2.20 × 2.20 Å
Minimum density: -6.866
Maximum density: 6.469
Average density: 0.045
Standard deviation: 1.901

Sample information

Sample name: 13-protofilament microtubule-bound human kinesin-5 motor domain in presence of ADP
Proteins: alpha tubulin, beta tubulin, Kinesin-5 motor domain

Validation information

Experimental information

 

Image processing

Applied symmetry: C1
Software: SPIDER, FREALIGN
Number of particles: 1000
CTF correction: FREALIGN
Details: Approximately 135,000 asymmetric units were averaged in the final reconstruction. The particles were selected along individual microtubules.

Imaging

Session
Microscope model: FEI TECNAI F20
Electron source: FIELD EMISSION GUN
Electron dose (e/A**2): 18.0
Nominal CS (mm): 2.00
Defocus max (nm): 2,600.00
Defocus min (nm): 1,100.00
Holder model: GATAN LIQUID NITROGEN
Acquisition
Number of images: 20

Specimen

Preparation
Specimen state: particle
Buffer: 20 mM PIPES, 5 mM MgCl2, 1 mM EGTA, 10 mM ADP, 2% glycerol
Specimen support: 400 mesh holey carbon grids
Vitrification
Apparatus: FEI VITROBOT MARK I
Cryogen: ETHANE
Humidity: 100
Protocol: chamber at 24 degrees C, blot 3.5 sec