Experiment: 8F6N

X-RAY DIFFRACTION

Starting Model(s)

Initial Refinement Model(s)
Type	Source	Accession Code	Details
experimental model	PDB	7M31

Crystallization

Crystalization Experiments
ID	Method	pH	Temperature	Details
1	VAPOR DIFFUSION, HANGING DROP		293	DPD variant C671S (39.2 uM) in 25 mM HEPES, 2 mM DTT, 10% glycerol at pH 7.5 was mixed 1:1 with well solution containing 100 mM sodium citrate, 2 mM DTT, 19% PEG 6000 at pH 4.7 to yield a 6 uL drop. Crystallization was carried out in the dark to prevent photo-degradation of the somewhat dissociable FMN cofactor. Under these conditions DPD crystals appeared within 16 hours and were left to grow for additional 24 hours. Ligands were combined with the crystals under near anaerobic conditions as follows: before being placed in a Plas-Labs 830 series glove box, the well solution beneath selected crystallization drops were made anaerobic with the addition of 10 mM dithionite and resealed with the cover slide. Crystals trays were placed in the glove box that contained a Motic binocular microscope coupled to an Accuscope 1080p high-definition camera. The glove box was sealed and made quasi-anaerobic by flushing with high-purity nitrogen gas for approximately 10 minutes at which time fractional dioxygen was recorded as 0.1 %, by a Forensics Detectors oxygen meter. Atmospheric dioxygen was measured throughout the soaking procedure and was held <1%. C671S DPD crystals were soaked for a minimum of 20 minutes with thymine (100 uM), each dissolved in the following cryo-solution: 20 mM sodium citrate, 0.4 mM DTT, 20% PEG 6000, 20% PEG 400, pH 7.5. The crystals were then submerged in liquid nitrogen, removed from the anaerobic environment, and stored under liquid nitrogen.

Crystal Properties
Matthews coefficient	Solvent content
2.4	48.82

Crystal Data

Unit Cell
Length ( Å )	Angle ( ˚ )
a = 82.445	α = 90
b = 158.144	β = 96.78
c = 165.707	γ = 90

Symmetry
Space Group	P 1 21 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	PIXEL	DECTRIS EIGER X 9M		2022-06-08	M	SINGLE WAVELENGTH

Radiation Source
ID #	Source	Type	Wavelength List	Synchrotron Site	Beamline
1	SYNCHROTRON	APS BEAMLINE 21-ID-D	1.1272	APS	21-ID-D

Data Collection

Overall
ID #	Resolution (High)	Resolution (Low)	Percent Possible (Observed)	R Merge I (Observed)	Rrim I (All)	Rpim I (All)	CC (Half)	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	2.12	114.022	88	0.179	0.202	0.091	0.99	7.3	4.9		146043			29.82

Highest Resolution Shell
ID #	Resolution (High)	Resolution (Low)	Percent Possible (All)	Percent Possible (Observed)	R Merge I (Observed)	Rrim I (All)	Rpim I (All)	CC (Half)	Mean I Over Sigma (Observed)	Redundancy	Number Unique Reflections (All)
1	2.121	2.197			1.175	1.3	0.779	0.433	1.6

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	THROUGHOUT	7M31	2.12	26.98	1.34	145954	7333	61.43	0.1855	0.1824	0.2442

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	11.265
f_angle_d	2.67
f_chiral_restr	2.018
f_bond_d	0.038
f_plane_restr	0.022

Non-Hydrogen Atoms Used in Refinement
Non-Hydrogen Atoms	Number
Protein Atoms	30952
Nucleic Acid Atoms
Solvent Atoms	593
Heterogen Atoms

Software

Software
Software Name	Purpose
PHENIX	refinement
autoPROC	data reduction
Aimless	data scaling
PHASER	phasing
PDB_EXTRACT	data extraction

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.