EMD-20453 Experiments and Validation

Cryo-EM structure of HzTransib/nicked TIR substrate DNA pre-reaction complex (PRC)

Single particle reconstruction
Overview of EMD-20453
Sample name: Pre-reaction complex of HzTransib with nicked TIR substrate DNA
Organism: Helicoverpa zea
Fitted atomic model: 6pqu

Map parameters

Recommended contour level: 0.021 (author)
Number of grid points: 256 × 256 × 256
Voxel size: 1.05 × 1.05 × 1.05 Å
Minimum density: -0.108
Maximum density: 0.166
Average density: -0.00
Standard deviation: 0.004

Sample information

Sample name: Pre-reaction complex of HzTransib with nicked TIR substrate DNA
Ligands: MAGNESIUM ION, ZINC ION, POTASSIUM ION
Nucleic acids: DNA (5'-D(P*AP*TP*CP*TP*GP*GP*CP*CP*TP*AP*GP*AP*TP*CP*T)-3'), DNA (5'-D(P*CP*AP*CP*GP*GP*TP*GP*GP*AP*TP*CP*GP*AP*AP*AP*A)-3'), TIR substrate DNA transferred strand
Proteins: Pre-reaction complex of HzTransib with nicked TIR substrate DNA, DNA-mediated transposase

Validation information

Experimental information

 

Image processing

Applied symmetry: C2
Software: RELION
Number of particles: 59333

Fitting

Initial atomic model: 6PQN
Used chain: A
Fitting protocol: rigid body
Target criteria: Correlation coefficient
Refinement space: REAL
Details: Initial local fitting was done using UCSF Chimera, then manually adjusted and rebuilt in Coot. Final model was refined using Phenix real-space refinement.

Imaging

Session
Microscope model: FEI TITAN KRIOS
Electron source: FIELD EMISSION GUN
Electron dose (e/A**2): 52.200000000000003
Energy filter: GIF Quantum LS
Nominal CS (mm): 2.70
Nominal magnification: 130000.0
Defocus max (nm): 2,400.00
Defocus min (nm): 1,400.00
Holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Details: Preliminary grid screening was performed manually.
Acquisition
Details: Images were collected in movie-mode at 5 frames per second.

Specimen

Preparation
Specimen state: particle
Specimen concentration (mg/mL): 0.30
Buffer: Solutions were made fresh from concentrated and filtered to avoid microbial contamination.
Vitrification
Apparatus: FEI VITROBOT MARK IV
Cryogen: ETHANE
Humidity: 100
Details: Blot for 3 seconds before plunging.