EMD-5490 Experiments and Validation

Reconstruction of the Wild-type Ndc80 Bonsai Decorated Microtubule on CCD for Difference Map Calculation

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

Map parameters

Recommended contour level: 1.67 (author)
Number of grid points: 70 × 70 × 70
Voxel size: 2.74 × 2.74 × 2.74 Å
Minimum density: -5.987
Maximum density: 5.543
Average density: 0.173
Standard deviation: 1.829

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
Reconstruction protocol: IHRSR
CTF correction: Phase-flipping each image
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.

Imaging

Session
Microscope model: FEI TECNAI F20
Electron source: FIELD EMISSION GUN
Electron dose (e/A**2): 20.0
Nominal CS (mm): 2.20
Nominal magnification: 80000.0
Defocus max (nm): 3,200.00
Defocus min (nm): 800.00
Holder model: GATAN LIQUID NITROGEN
Acquisition
Number of images: 202
Sampling interval (μm): 15.0

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): 25.762460000000001
Azimuthal (Z) increment (Å): 9.0401600000000002
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