1TUW

Structural and Functional Analysis of Tetracenomycin F2 Cyclase from Streptomyces glaucescens: A Type-II Polyketide Cyclase

X-RAY DIFFRACTION

Crystallization

Crystalization Experiments
ID	Method	pH	Temperature	Details
1	MICROBATCH	7.5	278	Crystals of tetracenomycin F2 cyclase were grown by microbatch from a solution composed of 2.2 M (NH4)2SO4, 3 mg/ml protein, 50 mM HEPES pH 7.5 at 4 C (11). Large crystals were obtained by macroseeding with small crystals (0.06 mm x 0.08 mm) washed in macroseeding 1.4 M (NH4)2SO4, 50 mM HEPES pH 7.5 to slightly dissolve the crystal surfaces. After 3 to 4 weeks the crystals attained a size of 0.3 x 0.3 x 0.6 mm. Crystals of the selenomethionine labeled protein were obtained in a similar manner., Micro batch, temperature 278K

Crystal Properties
Matthews coefficient	Solvent content
2.32	47

Crystal Data

Unit Cell
Length ( Å )	Angle ( ˚ )
a = 46.7	α = 90
b = 46.7	β = 90
c = 188.3	γ = 120

Symmetry
Space Group	P 65 2 2

Diffraction

Diffraction Experiment
ID #	Crystal ID	Scattering Type	Data Collection Temperature	Detector	Detector Type	Details	Collection Date	Monochromator	Protocol
1	1	x-ray	273	AREA DETECTOR	SIEMENS HI-STAR	gobel	2000-03-01	M	MAD

Radiation Source
ID #	Source	Type	Wavelength List	Synchrotron Site	Beamline
1	ROTATING ANODE	RIGAKU RU200

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	1.9	30	92	0.027	23.1	8.8	11605	11355

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)
1	1.9	2	84		0.088	5.4

Refinement

Statistics
Diffraction ID	Structure Solution Method	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	MAD	1.9	30	9599	9599	480		0.199	0.199	0.199	0.245	random

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
t_dihedral_angle_d	19.3
t_angle_deg	2.61
t_bond_d	0.017

Non-Hydrogen Atoms Used in Refinement
Non-Hydrogen Atoms	Number
Protein Atoms	850
Nucleic Acid Atoms
Solvent Atoms	79
Heterogen Atoms	5

Software

Software
Software Name	Purpose
Histar	data collection
XDS	data reduction
SOLVE	phasing
TNT	refinement
HISTAR	data reduction
XDS	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 R01GM133198.