3QI5

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

X-RAY DIFFRACTION

Starting Model(s)

Initial Refinement Model(s)
Type	Source	Accession Code	Details
experimental model	PDB	1BNK	PDB entry 1BNK, used as a search model

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 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.