EMD-2339 Experiments and Validation

Variable internal flexibility characterizes the helical capsid formed by Agrobacterium VirE2 protein on single-stranded DNA.

Helical reconstruction
Overview of EMD-2339
Sample name: Agrobacterium T-complex
Organism: Agrobacterium fabrum str. C58
Fitted atomic model: 4blf

Map parameters

Recommended contour level: 0.008 (author)
Number of grid points: 64 × 64 × 64
Voxel size: 4.32 × 4.32 × 4.32 Å
Minimum density: -0.045
Maximum density: 0.054
Average density: 0.001
Standard deviation: 0.01

Sample information

Sample name: Agrobacterium T-complex
Nucleic acid: short oligomeric 26mer DNA
Protein: VirE2

Validation information

Experimental information

 

Image processing

Software: Bsoft, EMAN, Xmipp, Spider, IHRSR
CTF correction: Phase-flipping

Fitting

Initial atomic model: 3BTP
Used chain: A
Fitting software: fitPDB2EM
Fitting protocol: flexible
Target criteria: Highest cross-correlation
Refinement space: RECIPROCAL
Details: The N and C terminal domain were fit separately by exhaustive molecular modeling using the fitPDB2EM program. Only the N-terminal domain could be constrained strongly.

Imaging

Session
Microscope model: FEI TECNAI F20
Electron source: FIELD EMISSION GUN
Electron dose (e/A**2): 20.0
Nominal CS (mm): 2.00
Nominal magnification: 50000.0
Defocus max (nm): 3,200.00
Defocus min (nm): 1,000.00
Holder model: GATAN LIQUID NITROGEN
Acquisition
Details: Image data was collected as focal pairs.

Specimen

Preparation
Specimen state: helicalArray
Specimen concentration (mg/mL): 1.00
Buffer: 50 mM Tris, 500 mM NaCl
Specimen support: Quantifoil holey carbon
Crystal grow: Protein was mixed with single-stranded DNA
Aggregation parameters (helical parameters)
Handedness: RIGHT HANDED
Phi increment (degrees): 110.09
Azimuthal (Z) increment (Å): 14.67
Vitrification
Apparatus: HOMEMADE PLUNGER
Cryogen: ETHANE
Humidity: 95