EMD-5556 Experiments and Validation

Negative stain electron microscopy structure of Nup192

Single particle reconstruction
Overview of EMD-5556
Sample name: Full-length Nup192
Organism: Saccharomyces cerevisiae

Map parameters

Recommended contour level: 0.072 (author)
Number of grid points: 80 × 80 × 80
Voxel size: 2.93 × 2.93 × 2.93 Å
Minimum density: -0.062
Maximum density: 0.153
Average density: 0.002
Standard deviation: 0.017

Sample information

Sample name: Full-length Nup192
Protein: Subunit of the Yeast Nuclear Pore Complex inner ring with weight 192 kDa

Validation information

Experimental information

 

Image processing

Applied symmetry: C1
Software: Spider, Sparx, EMAN2, EMAN
Number of particles: 3883
Number of class averages: 23
Reconstruction protocol: Random Conical Tilt
CTF correction: phase flip each particle
Details: Initial map was calculated by merging 3 maps obtained from random conical tilt. This was then used for 8 rounds of reference-based refinement in SPIDER at a 10-degree angular increment Particles were selected manually using jweb and boxer

Fitting

Initial atomic model: 4IFQ
Used chain: A
Fitting software: Chimera
Fitting protocol: rigid body
Target criteria: Cross-correlation 0.754
Refinement space: REAL
Details: Protocol: Rigid Body. The domains were separately fitted by manual docking followed by "Fit in Map"using Chimera

Imaging

Session 1
Microscope model: JEOL 2100F
Electron source: FIELD EMISSION GUN
Electron dose (e/A**2): 20.0
Nominal CS (mm): 2.00
Nominal magnification: 50000.0
Defocus max (nm): 2,000.00
Defocus min (nm): 1,500.00
Tilt max (degrees): 50.00
Tilt min (degrees): 0.00
Holder model: JEOL
Details: Collected on 2028x2048 CCD, 24 um/pixel
Session 2
Microscope model: JEOL 2100F
Electron source: FIELD EMISSION GUN
Electron dose (e/A**2): 20.0
Nominal CS (mm): 2.00
Nominal magnification: 50000.0
Defocus max (nm): 2,000.00
Defocus min (nm): 1,500.00
Tilt max (degrees): 50.00
Tilt min (degrees): 0.00
Holder model: JEOL
Details: Collected on 2028x2048 CCD, 24 um/pixel. 2 sessions are listed; data collected on multiple days between these dates
Acquisition
Number of images: 400
Sampling interval (μm): 24.0

Specimen

Preparation
Specimen state: particle
Specimen concentration (mg/mL): 0.004
Buffer: 20 mM HEPES, 300 mM NaCl, 2 mM MgCl2, 0.01% Tween 20, 0.01 mM DTT
Staining procedure: 3 ul protein was added to grids. The sample was blotted and 3 uL 1% uranyl formate added. Uranyl formate was blotted and fresh stain added (three times). The sample was allowed to sit 30 seconds before final blot, then air-dried.
Specimen support: 200 mesh copper grid with thin carbon support, glow discharged 20s with Gatan Solarus 20s in H2/O2.
Vitrification
Apparatus: NONE
Cryogen: NONE