5M1S

Cryo-EM structure of the E. coli replicative DNA polymerase-clamp-exonuclase-theta complex bound to DNA in the editing mode


ELECTRON MICROSCOPY
Sample
DNA polymerase III alpha, beta, epsilon, theta complex with mismatched DNA duplex
Sample Components
DNA polymerase III alpha, beta, epsilon, theta complex with mismatched DNA duplex
mismatched DNA duplex
Specimen Preparation
Sample Aggregation StatePARTICLE
Vitrification InstrumentFEI VITROBOT MARK IV
Cryogen NameETHANE
Sample Vitrification DetailsPrior to sample preparation 0.1 volumes of 0.05% Tween 20 were added to the sample 3 microliters were pipetted onto the grid and blotted for 4 seconds ...Prior to sample preparation 0.1 volumes of 0.05% Tween 20 were added to the sample 3 microliters were pipetted onto the grid and blotted for 4 seconds
3D Reconstruction
Reconstruction MethodSINGLE PARTICLE
Number of Particles15616
Reported Resolution (Å)6.7
Resolution MethodFSC 0.143 CUT-OFF
Other Details
Refinement Type
Symmetry TypePOINT
Point SymmetryC1
Map-Model Fitting and Refinement
Id1
Refinement Space
Refinement ProtocolOTHER
Refinement Target
Overall B Value
Fitting Procedure
DetailsThe cryo-EM structure of the PolIIIalpha-clamp-exonuclease complex in the polymerase mode (PDB code: 5FKW) was used as a starting model, and the NMR s ...The cryo-EM structure of the PolIIIalpha-clamp-exonuclease complex in the polymerase mode (PDB code: 5FKW) was used as a starting model, and the NMR structure of theta bound to the exonuclease catalytic domain (PDB code: 2XY8) was used to place theta into the cryo-EM map. The model was manually adjusted in Coot and geometry of the protein optimized in Refmac5 using DNA-specific restraints generated in LibG
Data Acquisition
Detector TypeGATAN K2 SUMMIT (4k x 4k)
Electron Dose (electrons/Å**2)2
Imaging Experiment1
Date of Experiment
Temperature (Kelvin)
Microscope ModelFEI TITAN KRIOS
Minimum Defocus (nm)1800
Maximum Defocus (nm)3500
Minimum Tilt Angle (degrees)
Maximum Tilt Angle (degrees)
Nominal CS2.7
Imaging ModeBRIGHT FIELD
Specimen Holder ModelFEI TITAN KRIOS AUTOGRID HOLDER
Nominal Magnification64000
Calibrated Magnification79545
SourceFIELD EMISSION GUN
Acceleration Voltage (kV)300
Imaging Details
EM Software
TaskSoftware PackageVersion
PARTICLE SELECTIONRELION2
IMAGE ACQUISITIONDigitalMicrograph2.3
IMAGE ACQUISITIONTitan User Interface
CTF CORRECTIONGctf
MODEL FITTINGCoot0.8.2
INITIAL EULER ASSIGNMENTRELION2
FINAL EULER ASSIGNMENTRELION2
CLASSIFICATIONRELION2
RECONSTRUCTIONRELION2
MODEL REFINEMENTREFMAC5.8
MODEL REFINEMENTLIBG
Image Processing
CTF Correction TypeCTF Correction DetailsNumber of Particles SelectedParticle Selection Details
NONE150000