EMD-2233 Experiments and Validation

Subnanometer structure of the bacterial pKM101 type IV secretion system core complex digested with elastase

Single particle reconstruction
Overview of EMD-2233
Sample name: traN/traO/traF complex encoded by pKM101 digested with 0.02 mg ml-1 of elastase for 40 min at room temperature
Organism: Escherichia coli
Fitted atomic models: 3zbi, 3zbj
Related in-frame EM entry: EMD-2232
Related EM entry by publication: EMD-2232

Map parameters

Minimum density: -3.08
Maximum density: 5.16
Average density: 0.02
Standard deviation: 0.22
Recommended contour level: 0.23 (author)

Sample information

Sample name: traN/traO/traF complex encoded by pKM101 digested with 0.02 mg ml-1 of elastase for 40 min at room temperature
Proteins: traF, traO, traN

Validation information

Experimental information

 

Image processing

Applied symmetry: C14
Software: imagic
Number of particles: 5430
Number of class averages: 1810
Reconstruction protocol: common lines
CTF correction: phase flipping, each CCD image
Details: final map was calculated from 5430 particles Particles picked with BOXER (EMAN)

Fitting

Initial atomic model: 3JQO
Fitting software: Chimera, Situs, Sculptor, Flex-EM
Fitting protocol: flexible
Target criteria: Correlation
Refinement space: REAL
Details: Protocol: Rigid body followed by flexible fitting

Imaging

Session
Microscope model: FEI TECNAI 12
Electron source: FIELD EMISSION GUN
Electron dose (e/A**2): 20.0
Nominal CS (mm): 2.1
Nominal magnification: 66000.0
Defocus max (nm): 3,500
Defocus min (nm): 1,500
Holder model: GATAN LIQUID NITROGEN
Details: 4000x4000 CCD
Acquisition
Number of images: 262
Sampling interval (μm): 15.0

Specimen

Preparation
Specimen state: particle
Specimen concentration (mg/mL): 0.01
Buffer: 50 mM Tris-HCL, 200 mM NaCl, 10 mM LDAO
Specimen support: continuous carbon-coated grids
Vitrification
Apparatus: FEI VITROBOT MARK III
Cryogen: ETHANE
Humidity: 100
Protocol: blot 4 seconds before plunging