3QI5

Crystal structure of human alkyladenine DNA glycosylase in complex with 3,N4-ethenocystosine containing duplex DNA

X-RAY DIFFRACTION

Crystallization

Crystalization Experiments
ID	Method	pH	Temperature	Details
1	VAPOR DIFFUSION, HANGING DROP		295	The 3,N4-ethenocytosine (EDC) containing DNA duplex was prepared by annealing the EDC containing 13-mer crystallization oligonucleotide ('5-GAC ATG (EDC)TT GCC T-3') with its complementary strand that contained G opposite EDC (5'-GGC AAG CAT GTC A-3'). The delta79AAG-EDC complexes were prepared by mixing equimolar ratios of delta79AAG and EDC:G 13-mer DNA duplex at the final protein-DNA complex concentration of 0.3 mM in the complex buffer (20 mM Hepes-NaOH pH 7.5, 100 mM NaCl, 0.1 mM EDTA, 5% v/v glycerol and 1 mM DTT). The complex was incubated on ice for 15 min and used for crystallization. The crystals were obtained upon mixing 1 uL of complex and 1 ul of the reservoir solution (100 mM sodium cacodylate pH 6.0, 200 mM manganese chloride and 20% polyethylene glycol (PEG)-3350) over 0.5 ml of the reservoir solution, followed by incubation for 2 days, VAPOR DIFFUSION, HANGING DROP, temperature 295K

Crystal Properties
Matthews coefficient	Solvent content
2.14	42.41

Crystal Data

Unit Cell
Length ( Å )	Angle ( ˚ )
a = 41.226	α = 81.23
b = 41.22	β = 88.4
c = 82.144	γ = 89.15

Symmetry
Space Group	P 1

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		2006-07-11	M	SINGLE WAVELENGTH

Radiation Source
ID #	Source	Type	Wavelength List	Synchrotron Site	Beamline
1	SYNCHROTRON	ALS BEAMLINE 12.3.1	1.116	ALS	12.3.1

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	2.2	81.11	100			26278	26278

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	2.2	2.252	89.61

Refinement

Statistics
Diffraction ID	Structure Solution Method	Cross Validation method	Starting model	Resolution (High)	Resolution (Low)	Number Reflections (All)	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	THROUGHOUT	PDB entry 1BNK, used as a search model	2.2	81	24947	24947	1331	96.22	0.23881	0.23637	0.28427	RANDOM	15.654

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]
0.62	0.29	0.21	-1.06	0.13	0.39

RMS Deviations
Key	Refinement Restraint Deviation
r_dihedral_angle_2_deg	29.351
r_dihedral_angle_3_deg	15.213
r_dihedral_angle_4_deg	13.988
r_dihedral_angle_1_deg	4.709
r_angle_refined_deg	1.029
r_scangle_it	0.999
r_mcangle_it	0.888
r_scbond_it	0.57
r_mcbond_it	0.486
r_chiral_restr	0.058

RMS Deviations
Key	Refinement Restraint Deviation
r_dihedral_angle_2_deg	29.351
r_dihedral_angle_3_deg	15.213
r_dihedral_angle_4_deg	13.988
r_dihedral_angle_1_deg	4.709
r_angle_refined_deg	1.029
r_scangle_it	0.999
r_mcangle_it	0.888
r_scbond_it	0.57
r_mcbond_it	0.486
r_chiral_restr	0.058
r_bond_refined_d	0.005
r_gen_planes_refined	0.004

Non-Hydrogen Atoms Used in Refinement
Non-Hydrogen Atoms	Number
Protein Atoms	3249
Nucleic Acid Atoms	1024
Solvent Atoms	232
Heterogen Atoms	2

Software

Software
Software Name	Purpose
ADSC	data collection
PHASER	phasing
REFMAC	refinement
DENZO	data reduction
SCALEPACK	data scaling

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.