5UZ9

Cryo EM structure of anti-CRISPRs, AcrF1 and AcrF2, bound to type I-F crRNA-guided CRISPR surveillance complex


ELECTRON MICROSCOPY

Refinement

RMS Deviations
KeyRefinement Restraint Deviation
f_dihedral_angle_d6.731
f_angle_d0.942
f_chiral_restr0.05
f_bond_d0.006
f_plane_restr0.006
Sample
Anti-CRISPRs AcrF1 and AcrF2 bound P. aeruginosa crRNA-guided CRISPR surveillance(Csy)complex.
Specimen Preparation
Sample Aggregation StatePARTICLE
Vitrification InstrumentHOMEMADE PLUNGER
Cryogen NameETHANE
Sample Vitrification DetailsSample was applied to poly-L-lysine hydrobromide pre-treated amorphous carbon film coated over a holey grid. Excess sample was blotted with Whatman-1 ...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 filter paper and plunge frozen. Manual plunge freezing was performed in a cold room.
3D Reconstruction
Reconstruction MethodSINGLE PARTICLE
Number of Particles51212
Reported Resolution (Å)3.4
Resolution MethodFSC 0.143 CUT-OFF
Other DetailsPer-frame dose weighting was performed using the RELION particle polishing method. A B-factor of -71 square angstrom was applied to sharpen the final ...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 map. Signal subtracted focused classification and refinement was performed for the "tail" region of the complex. This lead to a 4 angstrom (Gold standard FSC 0.143) map.
Refinement Type
Symmetry TypePOINT
Point SymmetryC1
Map-Model Fitting and Refinement
Id123
Refinement SpaceREALREALREAL
Refinement ProtocolAB INITIO MODELRIGID BODY FITRIGID BODY FIT
Refinement Target
Overall B Value
Fitting Procedure
DetailsTop scoring five models out of two hundred models generated using Rosetta were refined using Phenix and deposited as an ensemble atomic model for the ...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 final EM map.
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 TypeGATAN K2 SUMMIT (4k x 4k)
Electron Dose (electrons/Å**2)46
Imaging Experiment1
Date of Experiment
Temperature (Kelvin)
Microscope ModelFEI TITAN KRIOS
Minimum Defocus (nm)1200
Maximum Defocus (nm)2500
Minimum Tilt Angle (degrees)
Maximum Tilt Angle (degrees)
Nominal CS2.7
Imaging ModeBRIGHT FIELD
Specimen Holder ModelFEI TITAN KRIOS AUTOGRID HOLDER
Nominal Magnification29000
Calibrated Magnification
SourceFIELD EMISSION GUN
Acceleration Voltage (kV)300
Imaging DetailsObjective astigmatism was corrected at nominal magnification of 29000X, using Thon rings visualized with a K2 camera.
EM Software
TaskSoftware PackageVersion
PARTICLE SELECTIONFindEM
IMAGE ACQUISITIONLeginon
CTF CORRECTIONCTFFIND33
MODEL FITTINGCoot0.8.7
INITIAL EULER ASSIGNMENTRELION1.4
FINAL EULER ASSIGNMENTRELION1.4
CLASSIFICATIONRELION1.4
RECONSTRUCTIONRELION1.4
MODEL REFINEMENTPHENIX1.11.1
MODEL FITTINGUCSF Chimera1.12
MODEL REFINEMENTPHENIX1.11.1
MODEL FITTINGUCSF Chimera1.12
MODEL REFINEMENTPHENIX1.11.1
Image Processing
CTF Correction TypeCTF Correction DetailsNumber of Particles SelectedParticle Selection Details
NONEParticles were ctf-corrected during 2D and 3D processing.199348Template based particle picking was done using FindEM program implemented in Appion processing package.