Cryo EM structure of anti-CRISPRs, AcrF1 and AcrF2, bound to type I-F crRNA-guided CRISPR surveillance complex
ELECTRON MICROSCOPY
Refinement
| RMS Deviations | |
|---|---|
| Key | Refinement Restraint Deviation |
| f_dihedral_angle_d | 6.731 |
| f_angle_d | 0.942 |
| f_chiral_restr | 0.05 |
| f_bond_d | 0.006 |
| f_plane_restr | 0.006 |
| Sample |
|---|
| Anti-CRISPRs AcrF1 and AcrF2 bound P. aeruginosa crRNA-guided CRISPR surveillance(Csy)complex. |
| Specimen Preparation | |
|---|---|
| Sample Aggregation State | PARTICLE |
| Vitrification Instrument | HOMEMADE PLUNGER |
| Cryogen Name | ETHANE |
| Sample Vitrification Details | Sample was applied to poly-L-lysine hydrobromide pre-treated amorphous carbon film coated over a holey grid. Excess sample was blotted with Whatman-1 ... |
| 3D Reconstruction | |
|---|---|
| Reconstruction Method | SINGLE PARTICLE |
| Number of Particles | 51212 |
| Reported Resolution (Å) | 3.4 |
| Resolution Method | FSC 0.143 CUT-OFF |
| Other Details | Per-frame dose weighting was performed using the RELION particle polishing method. A B-factor of -71 square angstrom was applied to sharpen the final ... |
| Refinement Type | |
| Symmetry Type | POINT |
| Point Symmetry | C1 |
| Map-Model Fitting and Refinement | |||||
|---|---|---|---|---|---|
| Id | 1 | 2 | 3 | ||
| Refinement Space | REAL | REAL | REAL | ||
| Refinement Protocol | AB INITIO MODEL | RIGID BODY FIT | RIGID BODY FIT | ||
| Refinement Target | |||||
| Overall B Value | |||||
| Fitting Procedure | |||||
| Details | Top scoring five models out of two hundred models generated using Rosetta were refined using Phenix and deposited as an ensemble atomic model for the ... | The docked Cas6F structure in the "head" region of the complex was reduced to C-alpha backbone. | Initially two copies were fitted using Chimera and then backbones and side chains were fixed using Coot, followed by real space refinement in Phenix. | ||
| Data Acquisition | |||||||||
|---|---|---|---|---|---|---|---|---|---|
| Detector Type | GATAN K2 SUMMIT (4k x 4k) | ||||||||
| Electron Dose (electrons/Å**2) | 46 | ||||||||
| Imaging Experiment | 1 |
|---|---|
| Date of Experiment | |
| Temperature (Kelvin) | |
| Microscope Model | FEI TITAN KRIOS |
| Minimum Defocus (nm) | 1200 |
| Maximum Defocus (nm) | 2500 |
| 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 | 29000 |
| Calibrated Magnification | |
| Source | FIELD EMISSION GUN |
| Acceleration Voltage (kV) | 300 |
| Imaging Details | Objective astigmatism was corrected at nominal magnification of 29000X, using Thon rings visualized with a K2 camera. |
| EM Software | ||
|---|---|---|
| Task | Software Package | Version |
| PARTICLE SELECTION | FindEM | |
| IMAGE ACQUISITION | Leginon | |
| CTF CORRECTION | CTFFIND3 | 3 |
| MODEL FITTING | Coot | 0.8.7 |
| INITIAL EULER ASSIGNMENT | RELION | 1.4 |
| FINAL EULER ASSIGNMENT | RELION | 1.4 |
| CLASSIFICATION | RELION | 1.4 |
| RECONSTRUCTION | RELION | 1.4 |
| MODEL REFINEMENT | PHENIX | 1.11.1 |
| MODEL FITTING | UCSF Chimera | 1.12 |
| MODEL REFINEMENT | PHENIX | 1.11.1 |
| MODEL FITTING | UCSF Chimera | 1.12 |
| MODEL REFINEMENT | PHENIX | 1.11.1 |
| Image Processing | ||||
|---|---|---|---|---|
| CTF Correction Type | CTF Correction Details | Number of Particles Selected | Particle Selection Details | |
| NONE | Particles were ctf-corrected during 2D and 3D processing. | 199348 | Template based particle picking was done using FindEM program implemented in Appion processing package. | |
