3FYP

Crystal structure of the quadruple mutant (N23C/C246S/D247E/P249A) of 3-deoxy-D-manno-octulosonate 8-phosphate synthase (KDO8PS) from Neisseria meningitidis

X-RAY DIFFRACTION

Crystallization

Crystalization Experiments
ID	Method	pH	Temperature	Details
1	VAPOR DIFFUSION, HANGING DROP	5.4	297	27.7 mg/mL protein mixed 1:1 with reservoir liquor containing 100 mM NaOAc (pH 5.0), 2 mM PEP, 850 microM MnSO4 and 2.0 M NaCl. Immediately prior to data collection, crystals were harvested and soaked briefly in cryoprotectant solution, comprising 20% glycerol and the resevoir solution, vapor diffusion, hanging drop, temperature 297K, pH 5.4

Crystal Properties
Matthews coefficient	Solvent content
2.35	47.74

Crystal Data

Unit Cell
Length ( Å )	Angle ( ˚ )
a = 81.781	α = 90
b = 85.679	β = 90
c = 163.828	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	110	IMAGE PLATE	RIGAKU RAXIS IV++	Osmic blue optic	2007-01-01		SINGLE WAVELENGTH

Radiation Source
ID #	Source	Type	Wavelength List	Synchrotron Site	Beamline
1	ROTATING ANODE	RIGAKU MICROMAX-007	1.5418

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	1.85	37.96	99.7	0.046	14.1	4.88	98477	98477			31.8

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	1.85	1.92	99.9		0.343	3.4	4.7	9740

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	MOLECULAR REPLACEMENT	THROUGHOUT	PDB entry 2QKF	1.85	37.96	98469	98469	4964	99.65	0.207	0.207	0.205	0.247	RANDOM	25.785

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			0.3		-0.77

RMS Deviations
Key	Refinement Restraint Deviation
r_dihedral_angle_2_deg	35.812
r_dihedral_angle_4_deg	21.756
r_dihedral_angle_3_deg	15.417
r_dihedral_angle_1_deg	6.844
r_mcangle_it	3.968
r_scangle_it	3.668
r_mcbond_it	2.873
r_scbond_it	2.443
r_angle_other_deg	1.628
r_mcbond_other	1.313

RMS Deviations
Key	Refinement Restraint Deviation
r_dihedral_angle_2_deg	35.812
r_dihedral_angle_4_deg	21.756
r_dihedral_angle_3_deg	15.417
r_dihedral_angle_1_deg	6.844
r_mcangle_it	3.968
r_scangle_it	3.668
r_mcbond_it	2.873
r_scbond_it	2.443
r_angle_other_deg	1.628
r_mcbond_other	1.313
r_angle_refined_deg	1.257
r_chiral_restr	0.081
r_gen_planes_refined	0.015
r_bond_refined_d	0.013
r_gen_planes_other	0.006
r_bond_other_d	0.001

Non-Hydrogen Atoms Used in Refinement
Non-Hydrogen Atoms	Number
Protein Atoms	7786
Nucleic Acid Atoms
Solvent Atoms	590
Heterogen Atoms	27

Software

Software
Software Name	Purpose
d*TREK	data scaling
REFMAC	refinement
PDB_EXTRACT	data extraction
CrystalClear	data collection
d*TREK	data reduction
AMoRE	phasing

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.