Experiment: 7M31

X-RAY DIFFRACTION

Starting Model(s)

Initial Refinement Model(s)
Type	Source	Accession Code	Details
experimental model	PDB	1H7W

Crystallization

Crystalization Experiments
ID	Method	pH	Temperature	Details
1	VAPOR DIFFUSION, HANGING DROP		293	4.5 mg/mL (40 uM) of DPD variants, C671S in 25 mM HEPES, 2 mM DTT, 10% glycerol at pH 7.5 was prepared and diluted 1:1 with well solution containing 100 mM sodium citrate, 2 mM DTT, 15% PEG 6000 at pH 4.5 to 4.9. Diffraction quality crystals with elongated morphology were obtained by the hanging-drop vapor diffusion method. Incubation was carried out in the dark to eliminate photo-degradation the quasi-labile FMN cofactor. Crystals appeared after 16 hours as both single elongated rectangular hexahedron forms (200 x 50 x 50) or urchin-like clusters. Only the single crystal form were harvested and these were placed in a Plas-Labs 830 series glove box into which a Motic binocular microscope coupled to an Accuscope 1080 P HD camera was placed. Before being placed in the glove box, the well solutions of the selected crystals were made anaerobic with the addition of 10 mM dithionite and resealed with the cover slide. The glove box was then made anaerobic by flushing with pure nitrogen for approximately 10 minutes at which time the fractional dioxygen was 0.1 %, as indicated by a Forensics Detectors oxygen meter. Atmospheric dioxygen was measured throughout the soaking procedure and was held below 1%. C671S DPD crystals were soaked for a minimum of 20 minutes in 25 mM HEPES, 100 mM sodium citrate, 2 mM DTT, 100 uM NADPH, 100 uM thymine, 20% PEG 6000, 20% PEG 400, pH 7.5 prior to submersion in liquid nitrogen. Frozen crystals were then removed from the anaerobic environment and stored under liquid nitrogen.

Crystal Properties
Matthews coefficient	Solvent content
2.38	48.26

Crystal Data

Unit Cell
Length ( ? )	Angle ( ? )
a = 82.04	�� = 90
b = 159.56	�� = 95.75
c = 162.95	�� = 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		2020-12-09	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)	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	1.69	45.35	99	0.19	0.096	0.992	6	4.7		459508			18.44

Highest Resolution Shell
ID #	Resolution (High)	Resolution (Low)	Percent Possible (All)	Percent Possible (Observed)	R Merge I (Observed)	Rpim I (All)	CC (Half)	Mean I Over Sigma (Observed)	Redundancy	Number Unique Reflections (All)
1	1.69	1.73	99.8		1.851	0.944	0.35	0.9	4.8

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	1H7W	1.69	36.15	1.34	459329	22716	98.97	0.1779	0.1768	0.2001	26.8994

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]

Non-Hydrogen Atoms Used in Refinement
Non-Hydrogen Atoms	Number
Protein Atoms	30760
Nucleic Acid Atoms
Solvent Atoms	4306
Heterogen Atoms	691

Software

Software
Software Name	Purpose
xia2	data scaling
PHENIX	refinement
PDB_EXTRACT	data extraction
xia2	data reduction
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.