EMD-2317 Experiments and Validation

Single particle cryo-microscopy reconstruction of the isolated Manduca sexta V1 domain in complex with subunit C

Single particle reconstruction
Overview of EMD-2317
Sample name: M. sexta isolated V1 domain reconstituted with subunit C
Organism: Manduca sexta
Related in-frame EM entry: EMD-2318
Related EM entry by publication: EMD-2318

Map parameters

Minimum density: -2.422
Maximum density: 8.655
Average density: 0.00
Standard deviation: 1.00
Recommended contour level: 2.00 (author)

Sample information

Sample name: M. sexta isolated V1 domain reconstituted with subunit C
Proteins: Vacuolar ATPase isolated V1 domain, Subunit C

Validation information

Experimental information

 

Image processing

Applied symmetry: C1
Software: Relion
Number of particles: 10746
Reconstruction protocol: Maximum Likelihood
CTF correction: Relion
Details: Maximum likelihood in Relion using the MLF3D protocol The particles were handpicked in BOXER

Fitting

Initial atomic model: 3A5D
Used chains: A, B, C, D, E, F, G, H
Fitting software: Chimera
Fitting protocol: rigid body
Target criteria: best fit
Refinement space: REAL
Details: The domains were fitted using Chimera

Imaging

Session
Microscope model: FEI TECNAI F20
Electron source: FIELD EMISSION GUN
Electron dose (e/A**2): 16.0
Nominal CS (mm): 2.00
Nominal magnification: 50000.0
Defocus max (nm): 4.00
Defocus min (nm): 1.00
Holder model: GATAN LIQUID NITROGEN
Details: FEI Low dose mode
Acquisition
Number of images: 232
Details: All collected on Gatan 4K x 4K CCD

Specimen

Preparation
Specimen state: particle
Specimen concentration (mg/mL): 1.00
Buffer: 150mM NaCl, 20mM Tris-HCL, 0.01% C12E10
Staining procedure: negative stain data was collected using 1% w/v uranyl acetate for 1 minute.
Specimen support: UV glow discharged grids, 400 mesh
Vitrification
Apparatus: FEI VITROBOT MARK IV
Cryogen: ETHANE
Humidity: 100
Protocol: Grids were blotted for 6 seconds with a 6 second drain time