EMD-2984 Experiments and Validation

2.2 A resolution cryo-EM structure of beta-galactosidase in complex with a cell-permeant inhibitor

Single particle reconstruction
Overview of EMD-2984
Sample name: Escherichia coli beta-galactosidase bound to phenylethyl beta-D-thiogalactopyranoside (PETG)
Organisms: Escherichia coli K-12, unidentified
Fitted atomic model: 5a1a

Map parameters

Recommended contour level: 0.05 (author)
Number of grid points: 292 × 292 × 292
Voxel size: 0.64 × 0.64 × 0.64 Å
Minimum density: -0.121
Maximum density: 0.171
Average density: -0.00
Standard deviation: 0.017

Sample information

Sample name: Escherichia coli beta-galactosidase bound to phenylethyl beta-D-thiogalactopyranoside (PETG)
Ligand: phenylethyl beta-D-thiogalactopyranoside
Protein: beta-galactosidase

Validation information

Experimental information

 

Image processing

Applied symmetry: D2
Software: FREALIGN
Number of particles: 41123
Reconstruction protocol: phase residual minimization
CTF correction: each particle
Details: Final map was obtained from 41,123 particles. D2 symmetry was applied throughout refinement. Map was corrected using a B-factor of -75 A^2.

Imaging

Session
Microscope model: FEI TITAN KRIOS
Electron source: FIELD EMISSION GUN
Electron dose (e/A**2): 45.0
Energy filter: Gatan, Inc.
Energy window (eV): 0.00
Nominal CS (mm): 2.70
Nominal magnification: 215000.0
Defocus max (nm): 2,000.00
Defocus min (nm): 600.00
Holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Details: Parallel beam illumination
Acquisition
Number of images: 1487
Details: Every image is the average of 38 frames recorded by the direct electron detector.

Specimen

Preparation
Specimen state: particle
Specimen concentration (mg/mL): 2.30
Buffer: 25 mM Tris, pH 8.0, 50 mM NaCl, 2 mM MgCl2, 1.0 mM TCEP
Specimen support: 200 mesh Quantifoil R2/2 grids, plasma cleaned
Vitrification
Apparatus: LEICA EM GP
Cryogen: ETHANE
Humidity: 90
Protocol: Blot for 2 seconds before plunging.