6XKM

Room Temperature Structure of SARS-CoV-2 NSP10/NSP16 Methyltransferase in a Complex with SAM Determined by Fixed-Target Serial Crystallography

X-RAY DIFFRACTION

Serial Crystallography (SX)

Starting Model(s)

Initial Refinement Model(s)
Type	Source	Accession Code	Details
experimental model	PDB	6W4H

Crystallization

Crystalization Experiments
ID	Method	pH	Temperature	Details
1	BATCH MODE	6.5	295	Protein: 4.0 mg/ml (NSP10/NSP16 1:1), 0.15 M NaCl, 0.01 M Tris-HCl, 2 mM SAM, 1 mM TCEP, 5% Glycerol, pH 7.5. Precipitation buffer: 0.1 M MES pH 6.5, 0.9 M NaF. Batch crystallization: 100 ul of protein mixed with 100 ul of precipitation buffer in 500 ul polypropylene tube.

Crystal Properties
Matthews coefficient	Solvent content
4.49	72.6

Crystal Data

Unit Cell
Length ( Å )	Angle ( ˚ )
a = 170.41	α = 90
b = 170.41	β = 90
c = 52.631	γ = 120

Symmetry
Space Group	P 31 2 1

Diffraction

Diffraction Experiment
ID #	Crystal ID	Scattering Type	Data Collection Temperature	Detector	Detector Type	Details	Collection Date	Monochromator	Protocol
1	1	x-ray	295	PIXEL	DECTRIS PILATUS3 X 6M	Sagittally focusing mono and vertically focusing mirror	2020-06-12	M	SINGLE WAVELENGTH

Radiation Source
ID #	Source	Type	Wavelength List	Synchrotron Site	Beamline
1	SYNCHROTRON	APS BEAMLINE 19-ID	0.97918	APS	19-ID

Serial Crystallography

Sample delivery method
Diffraction ID	Description	Sample Delivery Method
1	Nylon Mesh	fixed target

Fixed Target
Diffraction ID	Description	Sample Holding	Support Base	Motion control	Details	Sample Solvent
1	SBC Mesh Holder	mesh	xyz stage	SmarAct Motors via PMAC	start/stop raster over area	50 mM MES pH 6.5, 0.45 M NaF, 75 mM NaCl, 5 mM Tris-HCl, 1 mM SAM, 0.5 mM TCEP, 2.5% Glycerol

Data Reduction
Diffraction ID	Frames Indexed	Crystal Hits	Frames Indexed	Latices Merged
1	10990	10990	10990

Data Collection

Overall
ID #	Resolution (High)	Resolution (Low)	Percent Possible (Observed)	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	2.25	49.57	100	0.968	2.68	82.41		41770			23.2

Highest Resolution Shell
ID #	Resolution (High)	Resolution (Low)	Percent Possible (All)	Percent Possible (Observed)	CC (Half)	Mean I Over Sigma (Observed)	Redundancy	Number Unique Reflections (All)
1	2.25	2.29	99.7		0.449	0.58	10.24

Refinement

Statistics
Diffraction ID	Structure Solution Method	Cross Validation method	Starting model	Resolution (High)	Resolution (Low)	Number Reflections (Observed)	Number Reflections (R-Free)	Percent Reflections (Observed)	R-Factor (Observed)	R-Work	R-Free	R-Free Selection Details	Mean Isotropic B
X-RAY DIFFRACTION	MOLECULAR REPLACEMENT	THROUGHOUT	6W4H	2.25	49.57	39686	2054	99.89	0.2068	0.2065	0.2126	RANDOM	37.635

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]
-0.29	-0.15		-0.29		0.94

RMS Deviations
Key	Refinement Restraint Deviation
r_dihedral_angle_2_deg	27.727
r_dihedral_angle_3_deg	9.938
r_dihedral_angle_4_deg	7.562
r_dihedral_angle_1_deg	2.98
r_angle_refined_deg	1.217
r_angle_other_deg	0.323
r_gen_planes_refined	0.053
r_chiral_restr	0.049
r_gen_planes_other	0.049
r_bond_refined_d	0.005

RMS Deviations
Key	Refinement Restraint Deviation
r_dihedral_angle_2_deg	27.727
r_dihedral_angle_3_deg	9.938
r_dihedral_angle_4_deg	7.562
r_dihedral_angle_1_deg	2.98
r_angle_refined_deg	1.217
r_angle_other_deg	0.323
r_gen_planes_refined	0.053
r_chiral_restr	0.049
r_gen_planes_other	0.049
r_bond_refined_d	0.005
r_bond_other_d	0.001

Non-Hydrogen Atoms Used in Refinement
Non-Hydrogen Atoms	Number
Protein Atoms	3188
Nucleic Acid Atoms
Solvent Atoms	129
Heterogen Atoms	30

Software

Software
Software Name	Purpose
REFMAC	refinement
PDB_EXTRACT	data extraction
MOLREP	phasing
DIALS	data scaling
DIALS	data reduction

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.