7JHE

Room Temperature Structure of SARS-CoV-2 Nsp10/Nsp16 Methyltransferase in a Complex with 2'-O-methylated m7GpppA Cap-1 and SAH 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	6WQ3

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. Crystals were soaked with m7GpppA (0.5 mM) for 10 minutes before data collection.

Crystal Properties
Matthews coefficient	Solvent content
4.46	72.5

Crystal Data

Unit Cell
Length ( Å )	Angle ( ˚ )
a = 170.859	α = 90
b = 170.859	β = 90
c = 52.797	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 viaPMAC	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, 0.5 mM m7GpppA

Data Reduction
Diffraction ID	Frames Indexed	Crystal Hits	Frames Indexed	Latices Merged
1	7725	7725	7725

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	49.73	100	0.93	2.45	42.94		42145			21.4

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	99.8		0.473	0.66	9.28

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	6WQ3	2.25	49.73	40062	2065	99.93	0.2243	0.2231	0.2485	RANDOM	37.147

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]
-0.01			-0.01		0.02

RMS Deviations
Key	Refinement Restraint Deviation
r_dihedral_angle_2_deg	28.016
r_dihedral_angle_3_deg	10.373
r_dihedral_angle_4_deg	9.598
r_dihedral_angle_1_deg	3.003
r_angle_refined_deg	1.353
r_angle_other_deg	0.356
r_gen_planes_refined	0.053
r_chiral_restr	0.052
r_gen_planes_other	0.05
r_bond_refined_d	0.005

RMS Deviations
Key	Refinement Restraint Deviation
r_dihedral_angle_2_deg	28.016
r_dihedral_angle_3_deg	10.373
r_dihedral_angle_4_deg	9.598
r_dihedral_angle_1_deg	3.003
r_angle_refined_deg	1.353
r_angle_other_deg	0.356
r_gen_planes_refined	0.053
r_chiral_restr	0.052
r_gen_planes_other	0.05
r_bond_refined_d	0.005
r_bond_other_d	0.001

Non-Hydrogen Atoms Used in Refinement
Non-Hydrogen Atoms	Number
Protein Atoms	3193
Nucleic Acid Atoms
Solvent Atoms	160
Heterogen Atoms	114

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.