EMD-2232 Experiments and Validation

Full-length structure of the bacterial pKM101 type IV secretion system core complex

Single particle reconstruction
Overview of EMD-2232
Sample name: traN/traO/traF complex encoded by pKM101
Organism: Escherichia coli
Fitted atomic model: 2ypw
Related in-frame EM entry: EMD-2233
Related EM entry by publication: EMD-2233

Map parameters

Minimum density: -3.124
Maximum density: 13.979
Average density: 0.034
Standard deviation: 0.329
Recommended contour level: 0.45 (author)

Sample information

Sample name: traN/traO/traF complex encoded by pKM101
Proteins: traF, traO, traN

Validation information

Experimental information

 

Image processing

Applied symmetry: C14
Software: imagic
Number of particles: 3805
Number of class averages: 800
Reconstruction protocol: common lines
CTF correction: phase flipping, each CCD image
Details: final map were calculated from 3805 particles Particles picked with BOXER (EMAN)

Fitting

Initial atomic model: 3JQO
Fitting software: Chimera, Situs
Fitting protocol: rigid body
Target criteria: Correlation
Refinement space: REAL
Details: Protocol: Rigid body

Imaging

Session
Microscope model: FEI TECNAI F20
Electron source: FIELD EMISSION GUN
Electron dose (e/A**2): 20.0
Nominal CS (mm): 2.10
Nominal magnification: 66000.0
Defocus max (nm): 3,500.00
Defocus min (nm): 1,250.00
Holder model: GATAN LIQUID NITROGEN
Details: 4000x4000 CCD
Acquisition
Number of images: 420
Sampling interval (μm): 15.0

Specimen

Preparation
Specimen state: particle
Specimen concentration (mg/mL): 5.00
Buffer: 50 mM Tris-HCL, 200 mM NaCl, 10 mM LDAO
Specimen support: lacey carbon grids
Vitrification
Apparatus: NONE
Cryogen: ETHANE
Humidity: 60
Protocol: blot 3 seconds before plunging