7JPE

Room Temperature Structure of SARS-CoV-2 Nsp10/Nsp16 Methyltransferase in a Complex with m7GpppA Cap-0 and 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	6WQ3

Crystallization

Crystalization Experiments
ID	Method	pH	Temperature	Details
1	BATCH MODE	6.5	297	Protein: 4.0 mg/ml (NSP10/NSP16 1:1), 0.15 M CaCl, 0.01 M Tris-HCl, 2 mM SAM, 1 mM TCEP, 5% Glycerol, pH 7.5. Precipitation buffer: 0.1M MES pH 6.5, 0.9 M NaF. Batch crystallization: 100 ul of protein mixed with 100 ul of precipitation buffer in 500 ul polypropylane tube. Two days before data collection 1 mM EDTA was added to batch crystallization. Crystal were soaked with m7GpppA (0.5 mM) for 10 minutes before data collection.

Crystal Properties
Matthews coefficient	Solvent content
4.49	72.6

Crystal Data

Unit Cell
Length ( Å )	Angle ( ˚ )
a = 170.93	α = 90
b = 170.93	β = 90
c = 52.786	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	293	PIXEL	DECTRIS PILATUS3 X 6M	Sagittally focusing mono and vertically focusing mirror	2020-08-02	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, 1 mM EDTA, 0.5 mM TCEP, 2.5% Glycerol, 0.5 mM m7GpppA

Data Reduction
Diffraction ID	Frames Indexed	Crystal Hits	Frames Indexed	Latices Merged
1	5734	5734	5734

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	2.18	49.72	100	0.95	2.55	50.51		46273			22.8

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	2.18	2.22	99.7		0.398	0.53	11.42

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	2.18	49.72	44016	2215	99.87	0.2179	0.217	0.237	RANDOM	37.222

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]
-0.23	-0.12		-0.23		0.75

RMS Deviations
Key	Refinement Restraint Deviation
r_dihedral_angle_2_deg	26.054
r_dihedral_angle_3_deg	9.069
r_dihedral_angle_4_deg	4.736
r_dihedral_angle_1_deg	2.273
r_angle_refined_deg	1.351
r_angle_other_deg	0.37
r_chiral_restr	0.059
r_gen_planes_refined	0.053
r_gen_planes_other	0.049
r_bond_refined_d	0.005

RMS Deviations
Key	Refinement Restraint Deviation
r_dihedral_angle_2_deg	26.054
r_dihedral_angle_3_deg	9.069
r_dihedral_angle_4_deg	4.736
r_dihedral_angle_1_deg	2.273
r_angle_refined_deg	1.351
r_angle_other_deg	0.37
r_chiral_restr	0.059
r_gen_planes_refined	0.053
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	3193
Nucleic Acid Atoms
Solvent Atoms	131
Heterogen Atoms	109

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.