EMD-5668 Experiments and Validation

26S proteasome Rpn11AXA Rpn13-delta mutant

Single particle reconstruction
Overview of EMD-5668
Sample name: Yeast Rpn11AXA Rpn13-delta mutant 26S proteasome
Organism: Saccharomyces cerevisiae
Related EM entry by publication: EMD-5669

Map parameters

Recommended contour level: 2.00 (author)
Number of grid points: 240 × 240 × 240
Voxel size: 2.17 × 2.17 × 2.17 Å
Minimum density: -11.61
Maximum density: 15.196
Average density: 0.02
Standard deviation: 0.591

Sample information

Sample name: Yeast Rpn11AXA Rpn13-delta mutant 26S proteasome
Protein: 26S proteasome

Validation information

Experimental information

 

Image processing

Applied symmetry: C2
Software: EMAN2/SPARX, FREALIGN
Number of particles: 80011
Reconstruction protocol: Projection matching
CTF correction: each particle
Details: Final map filtered to local resolution using the blocfilt function in Bsoft. Image processing performed in the Appion processing environment. 3D reconstruction with EMAN2/SPARX libraries; final alignment of particles with FREALIGN. Sharpened with SPIDER; low-pass filtered to local resolution with BSOFT.

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: 100000.0
Defocus max (nm): 2,500.00
Defocus min (nm): 1,200.00
Holder model: GATAN LIQUID NITROGEN
Details: Data acquired with Leginon
Acquisition
Number of images: 4740

Specimen

Preparation
Specimen state: particle
Specimen concentration (mg/mL): 2.00
Buffer: 60mM HEPES, 50mM NaCl, 50mM KCl, 5mM MgCl2, 0.5mM EDTA, 1mM DTT, 2mM ATP, 0.05% NP40
Specimen support: 400-mesh C-flats, 2 um holes with 2 um spacing (Protochips Inc.)
Vitrification
Apparatus: FEI VITROBOT MARK II
Cryogen: ETHANE
Humidity: 50
Protocol: Blot 3 seconds with -1 offset