4WIV

Crystal Structure of the first bromodomain of human BRD4 in complex with a novel inhibitor UMB32 (N-TERT-BUTYL-2-[4-(3,5-DIMETHYL-1,2-OXAZOL-4-YL) PHENYL]IMIDAZO[1,2-A]PYRAZIN-3-AMINE)

X-RAY DIFFRACTION

Starting Model(s)

Initial Refinement Model(s)
Type	Source	Accession Code	Details
experimental model	PDB	3MXF

Crystallization

Crystalization Experiments
ID	Method	pH	Temperature	Details
1	VAPOR DIFFUSION, SITTING DROP	7.4	295	Crystals of inhibitor-free BRD4-BD1 were first obtained in a drop with equal volumes of BD1 at 12 mg/ml and a precipitant solution containing 100mM sodium nitrate, 5% ethylene glycol, and 18% (w/v) PEG3350, as precipitant. Rod-like crystals were typically grown in 10 days reaching a maximal size of 0.05x0.05x0.4 mm3. Then, native crystals were transferred and soaked in 1 mM UMB32 in the same crystallization buffer for 7 days.

Crystal Properties
Matthews coefficient	Solvent content
2.2	44.6

Crystal Data

Unit Cell
Length ( Å )	Angle ( ˚ )
a = 43.562	α = 90
b = 49.21	β = 90
c = 60.991	γ = 90

Symmetry
Space Group	P 21 21 21

Diffraction

Diffraction Experiment
ID #	Crystal ID	Scattering Type	Data Collection Temperature	Detector	Detector Type	Details	Collection Date	Monochromator	Protocol
1	1	x-ray	100	CCD	ADSC QUANTUM 315		2014-02-14	M	SINGLE WAVELENGTH

Radiation Source
ID #	Source	Type	Wavelength List	Synchrotron Site	Beamline
1	SYNCHROTRON	APS BEAMLINE 24-ID-E	0.9792	APS	24-ID-E

Data Collection

Overall
ID #	Resolution (High)	Resolution (Low)	Percent Possible (Observed)	Net I Over Average Sigma (I)	Redundancy	Number Reflections (All)	Number Reflections (Observed)	Observed Criterion Sigma (F)	Observed Criterion Sigma (I)	B (Isotropic) From Wilson Plot
1	1.56	35.45	99.58	9.88	3.9		19177			14.04

Highest Resolution Shell
ID #	Resolution (High)	Resolution (Low)	Percent Possible (All)	Percent Possible (Observed)	Mean I Over Sigma (Observed)	Redundancy	Number Unique Reflections (All)
1	1.56	1.616	100		1.62	3.9

Refinement

Statistics
Diffraction ID	Structure Solution Method	Cross Validation method	Starting model	Resolution (High)	Resolution (Low)	Cut-off Sigma (F)	Number Reflections (Observed)	Number Reflections (R-Free)	Percent Reflections (Observed)	R-Factor (Observed)	R-Work	R-Free	R-Free Selection Details	Mean Isotropic B
X-RAY DIFFRACTION	MOLECULAR REPLACEMENT	FREE R-VALUE	3MXF	1.56	35.449	1.34	19175	986	99.57	0.1714	0.1698	0.2013	Random selectio	19.6

Temperature Factor Modeling
Anisotropic B[1][1]	Anisotropic B[1][2]	Anisotropic B[1][3]	Anisotropic B[2][2]	Anisotropic B[2][3]	Anisotropic B[3][3]

RMS Deviations
Key	Refinement Restraint Deviation
f_dihedral_angle_d	11.847
f_angle_d	1.296
f_chiral_restr	0.038
f_bond_d	0.007
f_plane_restr	0.005

Non-Hydrogen Atoms Used in Refinement
Non-Hydrogen Atoms	Number
Protein Atoms	1047
Nucleic Acid Atoms
Solvent Atoms	154
Heterogen Atoms	47

Software

Software
Software Name	Purpose
PHENIX	refinement

RCSB PDB Core Operations are funded by the U.S. National Science Foundation (DBI-2321666), 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 R01GM157729.