8H3K

Crystal Structure of SARS-CoV-2 Main Protease (Mpro) Double Mutant (L50F and E166V) in Complex with Inhibitor Enstrelvir

X-RAY DIFFRACTION

Crystallization

Crystalization Experiments
ID	Method	pH	Temperature	Details
1	VAPOR DIFFUSION, HANGING DROP		293.15	0.3% w/v Sodium-L ascorbate, 0.3% w/v Choline Chloride, 0.3% v/v D-Panthenol, 0.3% w/v Pyridoxine hydrochloride, 0.3% w/v Thiamine hydrochloride 20% v/v PEG 500* MME; 10% w/v PEG 20000; 0.1 M 0.1 M Buffer System 2 pH 7.5 (sodium HEPES, MOPS acid)

Crystal Properties
Matthews coefficient	Solvent content
2.28	45.98

Crystal Data

Unit Cell
Length ( ? )	Angle ( ? )
a = 55.543	�� = 90
b = 98.875	�� = 108
c = 58.991	�� = 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 EIGER2 S 9M		2022-08-17	M	SINGLE WAVELENGTH

Radiation Source
ID #	Source	Type	Wavelength List	Synchrotron Site	Beamline
1	SYNCHROTRON	SSRF BEAMLINE BL02U1	0.978642	SSRF	BL02U1

Data Collection

Overall
ID #	Resolution (High)	Resolution (Low)	Percent Possible (Observed)	R Merge I (Observed)	Rrim 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	1.8	52.83	97.4	0.079	0.085	0.999	15.29	6.912		54655			25.55

Highest Resolution Shell
ID #	Resolution (High)	Resolution (Low)	Percent Possible (All)	Percent Possible (Observed)	R Merge I (Observed)	Rrim I (All)	CC (Half)	Mean I Over Sigma (Observed)	Redundancy	Number Unique Reflections (All)
1	1.8	1.91	95.8		0.788	0.854	0.833	2.35	6.451

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	7VU6	1.8	52.83	1.36	54633	1995	97.48	0.1693	0.1681	0.2013	30.5487

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	4617
Nucleic Acid Atoms
Solvent Atoms	474
Heterogen Atoms	97

Software

Software
Software Name	Purpose
XDS	data scaling
PHENIX	refinement
PHENIX	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.