6EXL

The Transcriptional Regulator PrfA from Listeria Monocytogenes in complex with a ring-fused 2-pyridone (MK206) - folded HTH motif

X-RAY DIFFRACTION

Starting Model(s)

Initial Refinement Model(s)
Type	Source	Accession Code	Details
experimental model	PDB	5F1R

Crystallization

Crystalization Experiments
ID	Method	pH	Temperature	Details
1	VAPOR DIFFUSION, HANGING DROP	5.5	291	Following cell lysis, the protein was extracted by Ni-NTA Superflow FF, followed by TEV protease cleavage in buffer (200 mM NaCl, 1 mM BME, 20 mM NaP (NaH2PO4+Na2HPO4, pH 7.1). Further purification included Ni-NTA, MonoS and Gelfiltration (in 200 mM NaCl and 20 mM NaP buffer pH 6.5). Purified PrfA was co-crystallized with complex (5 mol excess) using the hanging-drop vapor-diffusion technique. Crystals grew in 5 days after 2 microL of the protein solution (3.2-3.5 mg per ml PrfA, 200 mM NaCl, 20 mM NaP buffer, pH 6.5) was mixed with an equal volume of precipitant solution containing 20-24% PEG-4000, 17% isopropanol, 100 mM Na citrate pH 5.5 and allowed to equilibrate over a 1 ml solution of the precipitant in a Linbro plate (Hampton Research).

Crystal Properties
Matthews coefficient	Solvent content
2.21	44.37

Crystal Data

Unit Cell
Length ( Å )	Angle ( ˚ )
a = 47.749	α = 90
b = 87.795	β = 90
c = 116.183	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	100	PIXEL	DECTRIS PILATUS 6M-F		2015-12-15	M	SINGLE WAVELENGTH

Radiation Source
ID #	Source	Type	Wavelength List	Synchrotron Site	Beamline
1	SYNCHROTRON	ESRF BEAMLINE ID23-1	0.975	ESRF	ID23-1

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	1.9	43.897	99.4	0.089	13.4	7.2		38994

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	1.97	97.9		0.783	2.5	6.9

Refinement

Statistics
Diffraction ID	Structure Solution Method	Cross Validation method	Starting model	Resolution (High)	Resolution (Low)	Cut-off Sigma (F)	Number Reflections (Observed)	Number Reflections (R-Free)	Percent Reflections (Observed)	R-Factor (Observed)	R-Work	R-Free	Mean Isotropic B
X-RAY DIFFRACTION	MOLECULAR REPLACEMENT	FREE R-VALUE	5F1R	1.9	43.9	1.34	38991	1930	99.2	0.175	0.173	0.225

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
f_dihedral_angle_d	13.303
f_angle_d	1.154
f_chiral_restr	0.064
f_bond_d	0.021
f_plane_restr	0.007

Non-Hydrogen Atoms Used in Refinement
Non-Hydrogen Atoms	Number
Protein Atoms	3816
Nucleic Acid Atoms
Solvent Atoms	302
Heterogen Atoms	67

Software

Software
Software Name	Purpose
PHENIX	refinement
XDS	data reduction
Aimless	data scaling
PHASER	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 R01GM157729.