7Z4S

Crystal structure of SARS-CoV-2 Mpro in complex with cyclic peptide GM4 including unnatural amino acids.

Experimental procedure

Experimental method	SINGLE WAVELENGTH
Source type	SYNCHROTRON
Source details	DIAMOND BEAMLINE I03
Synchrotron site	Diamond
Beamline	I03
Temperature [K]	100
Detector technology	PIXEL
Collection date	2021-12-11
Detector	DECTRIS EIGER X 16M
Wavelength(s)	0.9762
Spacegroup name	P 1
Unit cell lengths	54.515, 56.112, 63.238
Unit cell angles	113.87, 110.53, 90.92

Refinement procedure

Resolution	53.230 - 1.700
R-factor	0.19985
Rwork	0.198
R-free	0.23379
Structure solution method	MOLECULAR REPLACEMENT
Starting model (for MR)	6yb7
RMSD bond length	0.008
RMSD bond angle	1.322
Data reduction software	xia2
Data scaling software	Aimless
Phasing software	MOLREP
Refinement software	REFMAC (5.8.0267)

Data quality characteristics

	Overall	Inner shell	Outer shell
Low resolution limit [Å]	53.230	53.230	1.730
High resolution limit [Å]	1.700	8.990	1.700
Rmerge	0.129	0.027	2.454
Rmeas		0.030	3.059
Rpim	0.058	0.012
Number of reflections	407784	2867	19306
<I/σ(I)>	6.6	29	0.6
Completeness [%]	99.9	99.6	99.2
Redundancy	5.9	6.1
CC(1/2)	0.998	0.999	0.298

Crystallization Conditions

crystal ID	method	pH	temperature	details
1	VAPOR DIFFUSION, SITTING DROP	6	293	Mpro was thawed and diluted to 6 mg/ml using 20 mM Hepes pH 7.5, 50 mM NaCl. GM4 was diluted into the protein solution to a final concentration of 10 mM and allowed to incubate for two hours at room temperature prior to dispensing plates. The drop composition was 0.15 ul protein ligand solution, 0.3 ul 11% (v/v) PEG 4K, 0.1 M MES pH 6.5, and 0.05 ul Mpro crystal seed stock. The Mpro crystal seed stock was prepared by crushing Mpro crystals with a pipette tip, suspending them in 30% PEG 4K, 5% (v/v) DMSO, 0.1 M MES pH 6.5, and vortexing for 60 s with approximately 10 glass beads (1.0 mm diameter, BioSpec products). Reservoir solution was 11% (v/v) PEG 4K, 5% (v/v) DMSO, 0.1 M MES pH 6.5. Crystals were grown using the sitting drop vapor diffusion method at 20 C and appeared within 24 hours, reaching full size within 36 hours. Crystals were looped after one week.