EMD-5679 Experiments and Validation

Electron Microscopy of the Aquaporin-0/Calmodulin Complex

Single particle reconstruction
Overview of EMD-5679
Sample name: Aquaporin-0 bound to Calmodulin
Organisms: Ovis aries, Homo sapiens
Fitted atomic model: 3j41

Map parameters

Minimum density: -3.11
Maximum density: 8.37
Average density: 0
Standard deviation: 1
Recommended contour level: 4.96 (author)

Sample information

Sample name: Aquaporin-0 bound to Calmodulin
Proteins: Aquaporin-0, Calmodulin

Validation information

Experimental information

 

Image processing

Applied symmetry: C2
Software: SPIDER, FREALIGN
Number of particles: 11720
Reconstruction protocol: Random Conical Tilt
CTF correction: CTF-TILT, each micrograph
Details: Final Map with C2 Symmetry and Filtered to 25 Angstrom Particles were selected from a tilted pair dataset at 0 and 50 degree tilt using SPIDER. An initial reconstruction was generated using random conical tilt methods in SPIDER and refined in FREALIGN

Fitting

Initial atomic model: 2B6P
Used chain: A
Fitting software: Chimera
Fitting protocol: rigid body
Target criteria: cross-correlation
Refinement space: REAL

Imaging

Session
Microscope model: FEI TECNAI 12
Electron source: LAB6
Electron dose (e/A**2): 15.0
Nominal CS (mm): 2
Nominal magnification: 52000.0
Defocus max (nm): 2,000
Defocus min (nm): 1,000
Tilt max (degrees): 50
Tilt min (degrees): 0
Holder model: OTHER
Acquisition
Number of images: 200
Scanner model: NIKON SUPER COOLSCAN 9000
Sampling interval (μm): 6.3499999999999996

Specimen

Preparation
Specimen state: particle
Specimen concentration (mg/mL): 0.02
Buffer: 25mM HEPES, 5mM CaCl2, 0.3% decylmaltoside
Staining procedure: 0.75% uranyl formate
Specimen support: 400 mesh carbon coated grid (Ted Pella)
Vitrification
Apparatus: NONE
Cryogen: NONE