EMD-5489 Experiments and Validation

Improved Reconstruction of the Human Ndc80 Bonsai Decorated Microtubule

Helical reconstruction
Overview of EMD-5489
Sample name: Human Ndc80 bonsai complex bound to the microtubule
Organisms: Bos taurus, Homo sapiens
Related EM entries by publication: EMD-5490, EMD-5491, EMD-5492, EMD-5493

Map parameters

Minimum density: -7.093
Maximum density: 10.645
Average density: 0.00
Standard deviation: 1.00
Recommended contour level: 2.15 (author)

Sample information

Sample name: Human Ndc80 bonsai complex bound to the microtubule
Proteins: tubulin, Ndc80-Spc25 Chimera, Nuf2-Spc24 Chimera

Validation information

Experimental information

 

Image processing

Software: EMAN2/SPARX, FREALIGN
Reconstruction protocol: IHRSR
CTF correction: Each particle
Details: Particles were initially aligned using multi-model IHRSR protocol in EMAN2/SPARX with naked 13 and 14 protofilament microtubules as references. Final reconstruction and CTF correction were performed for 14 protofilament segments with FREALIGN v8.9. A B-factor of -1000 was applied with BFACTOR with a peak at 8.3 Angstrom. FSC was calculated only for MT and Ndc80-NUF2 head, disordered outer head was excluded with soft mask. The particles were aligned using IHRSR in EMAN2/SPARX followed by FREALIGN

Imaging

Session
Microscope model: FEI TECNAI F20
Electron source: FIELD EMISSION GUN
Electron dose (e/A**2): 15.0
Nominal CS (mm): 2.20
Nominal magnification: 50000.0
Defocus max (nm): 2,200.00
Defocus min (nm): 1,200.00
Holder model: GATAN LIQUID NITROGEN
Acquisition
Number of images: 100
Scanner model: NIKON SUPER COOLSCAN 9000
Sampling interval (μm): 6.3499999999999996

Specimen

Preparation
Specimen state: filament
Specimen concentration (mg/mL): 0.25
Buffer: 80mM PIPES, 1mM MgCl2, 1mM EGTA, 1mM DTT, 0.05% Nonidet P-40, 20uM taxol
Specimen support: C-flat 1.2/1.3
Aggregation parameters (helical parameters)
Handedness: LEFT HANDED
Phi increment (degrees): 25.747779999999999
Azimuthal (Z) increment (Å): 8.5960099999999997
Vitrification
Apparatus: FEI VITROBOT MARK II
Cryogen: ETHANE
Humidity: 100
Protocol: 2 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