ELECTRON MICROSCOPY

Sample

ORC-Cdc6 complex of Yarrowia lipolytica bound to a 60bp DNA segment of a mutant of OriA-006 containing the CNNGGNR mutation

Specimen Preperation
Sample Aggregation State PARTICLE
Vitrification Instrument LEICA EM GP
Cryogen Name ETHANE
Sample Vitrification Details Sample was applied to a non-glow discharged Quantifoil R 1.2/1.3 300 mesh copper grid (previously washed with ethyl acetate), incubated for 10 seconds at 25C and 95% humidity, blotted for 2.7 seconds, and plunged into liquid ethane using a Leica Automatic Plunge Freezer EM GP2.
3D Reconstruction
Reconstruction Method SINGLE PARTICLE
Number of Particles 51222
Reported Resolution (Å) 2.56
Resolution Method FSC 0.143 CUT-OFF
Other Details Non-uniform refinement was used for the final reconstruction.
Refinement Type
Symmetry Type POINT
Map-Model Fitting and Refinement
ID 1
Refinement Space REAL
Refinement Protocol FLEXIBLE FIT
Refinement Target ?
Overall B Value ?
Fitting Procedure ?
Details The YlODC60bpOriA-006-WT structure was docked into the map using ChimeraX and used as a starting point, with the DNA sequence altered at the mutated sites and manual refinement in Coot used to refine the model.
Data Acquisition
Detector Type GATAN K3 BIOQUANTUM (6k x 4k)
Electron Dose (electrons/Å2) 54.8
Imaging Experiment
Date of Experiment ?
Temprature (Kelvin)
Microscope Model TFS KRIOS
Minimum Defocus (nm) 600
Maximum Defocus (nm) 2000
Minimum Tilt Angle (degrees) ?
Maximum Tilt Angle (degrees) ?
Nominal CS 2.7
Imaging Mode BRIGHT FIELD
Specimen Holder Model FEI TITAN KRIOS AUTOGRID HOLDER
Nominal Magnification 105000
Calibrated Magnification ?
Source FIELD EMISSION GUN
Acceleration Voltage (kV) 300
Imaging Details ?
Imaging Experiment
Task Software Package Version
PARTICLE SELECTION cryoSPARC 4.7.1
MODEL REFINEMENT PHENIX 2.0_5885
CTF CORRECTION cryoSPARC 4.7.1
INITIAL EULER ASSIGNMENT cryoSPARC 4.7.1
FINAL EULER ASSIGNMENT cryoSPARC 4.7.1
CLASSIFICATION cryoSPARC 4.7.1
RECONSTRUCTION cryoSPARC 4.7.1
Image Processing
CTF Correction Type CTF Correction Details Number of Particles Selected Particle Selection Details
PHASE FLIPPING AND AMPLITUDE CORRECTION Patch CTF correction was carried out in cryoSPARC using the default settings, and was optimized during the refinements/reconstruction of the map in cryoSPARC.
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