Experiment: 2OEU

X-RAY DIFFRACTION

Crystallization

Crystalization Experiments
ID	Method	pH	Temperature	Details
1	VAPOR DIFFUSION, HANGING DROP	6.5	301	THE ENZYME AND SUBSTRATE WERE MIXED IN EQUIMOLAR AMOUNTS IN A SOLUTION CONTAINING 50 MM MES PH 5.5, 1.5 MM EDTA. THE COMPLEX WAS FORMED BY INCUBATING THE MIXTURE AT 95C FOR 2 MIN., THEN AT 65C FOR 2 MIN., AND FINALLY AT 27C FOR 5 MIN. 1 MM MGCL2 WAS INCLUDED IN THE MIXTURE BEFORE THE FINAL INCUBATION STEP. A 10 MG/ML CONCENTRATION OF RNA WAS USED FOR THE CRYSTALLIZATION EXPERIMENTS. THE RESERVOIR SOLUTION CONTAINED 0.5M (NH4)2SO4, 100 MM MES PH 6.5, AND 35% PEG 3350. AFTER MIXING THE RESERVOIR SOLUTION, THE SALT AND PEG PHASES WERE ALLOWED TO SEPARATE, AND ONLY THE SALT PHASE WAS USED FOR THE DROPS. THE CRYSTALS GREW IN HANGING DROPS OF 2 MICROLITERS AFTER 12 MONTHS OF INCUBATION AT 28C. THE CRYSTALS WERE WASHED IN THE SALT PHASE OF THE RESERVOIR SOLUTION PRIOR TO CRYO-FREEZING, VAPOR DIFFUSION, HANGING DROP, temperature 301K

Crystal Properties
Matthews coefficient	Solvent content
2.37	48.08

Crystal Data

Unit Cell
Length ( Å )	Angle ( ˚ )
a = 50.312	α = 90
b = 68.981	β = 112.62
c = 60.443	γ = 90

Symmetry
Space Group	C 1 2 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 4		2006-03-15	M	SINGLE WAVELENGTH

Radiation Source
ID #	Source	Type	Wavelength List	Synchrotron Site	Beamline
1	SYNCHROTRON	SSRL BEAMLINE BL9-1	1.37756	SSRL	BL9-1

Data Collection

Overall
ID #	Resolution (High)	Resolution (Low)	Percent Possible (Observed)	R Merge I (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	38.52	97	0.037	23.2	4.2		12527

Highest Resolution Shell
ID #	Resolution (High)	Resolution (Low)	Percent Possible (All)	Percent Possible (Observed)	R Merge I (Observed)	Mean I Over Sigma (Observed)	Redundancy	Number Unique Reflections (All)
	2	2.11	96.3		0.113	10.4	4.2	1814

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 (All)	R-Factor (Observed)	R-Work	R-Free	R-Free Selection Details	Mean Isotropic B
X-RAY DIFFRACTION	same cell as 2goz	THROUGHOUT	2goz	2	55.8	11277	11277	1234	96.55	0.17143	0.17143	0.16586	0.22087	RANDOM	46.082

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]
-0.47		-1.51			-0.69

RMS Deviations
Key	Refinement Restraint Deviation
r_scangle_it	1.933
r_angle_refined_deg	1.728
r_scbond_it	1.44
r_nbtor_refined	0.28
r_symmetry_hbond_refined	0.173
r_nbd_refined	0.168
r_xyhbond_nbd_refined	0.159
r_symmetry_vdw_refined	0.126
r_chiral_restr	0.08
r_metal_ion_refined	0.036

RMS Deviations
Key	Refinement Restraint Deviation
r_scangle_it	1.933
r_angle_refined_deg	1.728
r_scbond_it	1.44
r_nbtor_refined	0.28
r_symmetry_hbond_refined	0.173
r_nbd_refined	0.168
r_xyhbond_nbd_refined	0.159
r_symmetry_vdw_refined	0.126
r_chiral_restr	0.08
r_metal_ion_refined	0.036
r_bond_refined_d	0.01
r_gen_planes_refined	0.009

Non-Hydrogen Atoms Used in Refinement
Non-Hydrogen Atoms	Number
Protein Atoms
Nucleic Acid Atoms	1349
Solvent Atoms	195
Heterogen Atoms	5

Software

Software
Software Name	Purpose
REFMAC	refinement
ADSC	data collection
MOSFLM	data reduction
CCP4	data scaling
Coot	model building

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.