ELECTRON MICROSCOPY

Sample

ribonuclease P RNA

Specimen Preperation
Sample Aggregation State PARTICLE
Vitrification Instrument FEI VITROBOT MARK IV
Cryogen Name ETHANE
Sample Vitrification Details Grids were glow-discharged on both sides prior to vitrification. Vitrification was performed using a Vitrobot Mark IV with a 10s wait time, followed by a 3s blot and blot force 3 to 10 in air
3D Reconstruction
Reconstruction Method SINGLE PARTICLE
Number of Particles 82634
Reported Resolution (Å) 3.30
Resolution Method FSC 0.143 CUT-OFF
Other Details ?
Refinement Type
Symmetry Type POINT
Map-Model Fitting and Refinement
ID 1
Refinement Space REAL
Refinement Protocol FLEXIBLE FIT
Refinement Target maximization of map-to-model correlation
Overall B Value 57
Fitting Procedure ?
Details ?
Data Acquisition
Detector Type FEI FALCON IV (4k x 4k)
Electron Dose (electrons/Å2) 53
Imaging Experiment
Date of Experiment ?
Temprature (Kelvin)
Microscope Model TFS KRIOS
Minimum Defocus (nm) 500
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 165000
Calibrated Magnification ?
Source FIELD EMISSION GUN
Acceleration Voltage (kV) 300
Imaging Details The alignment procedure was performed using the EPU software utilizing its autofocus and auto-tuning functions for initial microscope alignment. This was followed by manual fine-tuning adjustments of the beam shift, stigmation, and focus controls to ensure optimal image quality. Grid squares were screened systematically, ensuring the selection of areas with appropriate ice thickness and particle distribution. Image shift calibration and eucentric height adjustment were performed within EPU to maintain beam alignment during data acquisition
Imaging Experiment
Task Software Package Version
PARTICLE SELECTION cryoSPARC 4.7
MODEL REFINEMENT PHENIX 1.21.2_5419
RECONSTRUCTION cryoSPARC 4,7
Image Processing
CTF Correction Type CTF Correction Details Number of Particles Selected Particle Selection Details
PHASE FLIPPING AND AMPLITUDE CORRECTION CTF correction was performed in cryoSPARC by patch-based motion correction followed by patch-based CTF estimation, providing per-particle local CTF parameters that were applied during 3D refinement for phase and amplitude correction of all particles
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