5KGF

Structural model of 53BP1 bound to a ubiquitylated and methylated nucleosome, at 4.5 A resolution

ELECTRON MICROSCOPY

Refinement

RMS Deviations
Key	Refinement Restraint Deviation
f_dihedral_angle_d	20.874
f_angle_d	0.963
f_chiral_restr	0.063
f_bond_d	0.009
f_plane_restr	0.006

Sample
NCP-ubme/GST-53BP1 complex
Sample Components
NCP-ubme
Widom-601 DNA
GST-53BP1
Ubiquitylated methylated histone octamer
Histone H4kc20Me2
Histone H3
Histone H2B.1
Histone H2A.1 K13RK36R
Ubiquitin

Specimen Preparation
Sample Aggregation State	PARTICLE
Vitrification Instrument	FEI VITROBOT MARK III
Cryogen Name	ETHANE-PROPANE
Sample Vitrification Details	Plunged into liquid ethane-propane (FEI VITROBOT MARK III)

3D Reconstruction
Reconstruction Method	SINGLE PARTICLE
Number of Particles	45361
Reported Resolution (Å)	4.54
Resolution Method	FSC 0.143 CUT-OFF
Other Details
Refinement Type
Symmetry Type	POINT
Point Symmetry	C2

Map-Model Fitting and Refinement
Id	1
Refinement Space	REAL
Refinement Protocol	RIGID BODY FIT
Refinement Target
Overall B Value	207.5
Fitting Procedure
Details	The atomic models of Widom-601 DNA (PDB ID 3LZ0), octameric histones (PDB ID 1KX5), ubiquitin (PDB ID 1UBI), and H4K20me2/53BP1 tandem Tudor domain (P ...	The atomic models of Widom-601 DNA (PDB ID 3LZ0), octameric histones (PDB ID 1KX5), ubiquitin (PDB ID 1UBI), and H4K20me2/53BP1 tandem Tudor domain (PDB ID 2IG0) were fitted without allowing flexibility into the 3D maps using UCSF Chimera. Segmentation was performed in UCSF Chimera. For the NCP-ubme structure the ubiquitin segmentation was further modified to remove obvious NCP density from the ubiquitin segment. The H2A/H2B sequence was mutated to the human H2AK13R/K36R and H2B manually in UCSF Chimera. A polyalanine model of the UDR was built within the UDR density in Coot, which compared well to predicted structures generated by Rosetta. The UDR model was mutated and fitted using UCSF Chimera, followed by iterative rounds of real-space refinement in PHENIX and model optimization in Coot. All figures were prepared in UCSF Chimera.

Data Acquisition
Detector Type	GATAN K2 SUMMIT (4k x 4k)
Electron Dose (electrons/Å**2)	36

Imaging Experiment	1
Date of Experiment
Temperature (Kelvin)
Microscope Model	FEI TECNAI F20
Minimum Defocus (nm)
Maximum Defocus (nm)
Minimum Tilt Angle (degrees)
Maximum Tilt Angle (degrees)
Nominal CS	2
Imaging Mode	BRIGHT FIELD
Specimen Holder Model	GATAN 626 SINGLE TILT LIQUID NITROGEN CRYO TRANSFER HOLDER
Nominal Magnification	25000
Calibrated Magnification	34483
Source	FIELD EMISSION GUN
Acceleration Voltage (kV)	200
Imaging Details

EM Software
Task	Software Package	Version
IMAGE ACQUISITION	DigitalMicrograph	2.31.691.0
CTF CORRECTION	CTFFIND	3
MODEL FITTING	UCSF Chimera	1.10.1
MODEL REFINEMENT	PHENIX	1.10.1.2115
INITIAL EULER ASSIGNMENT	RELION	1.3
FINAL EULER ASSIGNMENT	RELION	1.3
CLASSIFICATION	RELION	1.3
RECONSTRUCTION	RELION	1.3

Image Processing
CTF Correction Type	CTF Correction Details	Number of Particles Selected	Particle Selection Details
PHASE FLIPPING AND AMPLITUDE CORRECTION		174185	Automatically picked from roughly 3000 particles using a manually picked template

RCSB PDB Core Operations are funded by the National Science Foundation (DBI-1832184), the US Department of Energy (DE-SC0019749), and the National Cancer Institute, National Institute of Allergy and Infectious Diseases, and National Institute of General Medical Sciences of the National Institutes of Health under grant R01GM133198.