EMD-2387 Experiments and Validation

Structure and substrate induced conformational changes of the secondary citrate/sodium symporter CitS revealed by electron crystallography

Electron crystallography
Overview of EMD-2387
Sample name: CitS dimer
Organism: Klebsiella pneumoniae
Fitted atomic model: 4bpq

Map parameters

Recommended contour level: 46.20 (author)
Number of grid points: 100 × 100 × 100
Voxel size: 1.00 × 1.00 × 1.00 Å
Minimum density: -151.81
Maximum density: 244.378
Average density: 0.381
Standard deviation: 19.64

Sample information

Sample name: CitS dimer
Protein: CitS

Validation information

Experimental information

 

Image processing

Software: 2dx

Imaging

Session
Microscope model: FEI/PHILIPS CM200FEG
Electron source: FIELD EMISSION GUN
Electron dose (e/A**2): 6.0
Nominal CS (mm): 2.00
Nominal magnification: 50000.0
Defocus max (nm): 2,500.00
Defocus min (nm): 300.00
Tilt max (degrees): 45.00
Tilt min (degrees): 0.00
Holder model: GATAN LIQUID NITROGEN
Details: Pre-exposure recorded on TVIPS-F416 of 0.1 ms, to reduce beam-induced resolution loss in subsequent image on film
Acquisition
Number of images: 79
Scanner model: PRIMESCAN
Sampling interval (μm): 5.0

Specimen

Preparation
Specimen state: twoDArray
Specimen concentration (mg/mL): 1.35
Buffer: 20 mM sodium acetate, pH 4.5, 500 mM NaCl, 15 mM MgCl2, 2 mM DTT, 2 mM NaN3
Staining procedure: 20 mM sodium acetate, pH 4.5, 200 mM NaCl, 15 mM MgCl2, 2 mM DTT, 2 mM NaN3
Specimen support: 400 mesh Co grid with 2 micrometer holes from Quantifoil, covered with a very thin additional continuous carbon film
Crystal grow: Dialysis
Aggregation parameters (crystal parameters)
Plane group: P 2 21 21
Unit cell length A (Å): 96.0
Unit cell length B (Å): 106.0
Unit cell angle gamma (degrees): 90.0
Vitrification
Apparatus: FEI VITROBOT MARK IV
Cryogen: ETHANE
Humidity: 95
Protocol: Blot for 4.5 seconds