5D9F

Crystal structure of TrmD, a M1G37 tRNA Methyltransferase with SAM-competitive compounds

X-RAY DIFFRACTION

Crystallization

Crystalization Experiments
ID	Method	pH	Temperature	Details
1	VAPOR DIFFUSION, SITTING DROP	7.5	295	Protein solution:( 12/mg/mL in 100mM HEPES pH 7.5, 150mM NaCl, 10mM MgCl2 2mM DTT) Well solution: (20% PEG3,350 and 0.2M potassium citrate tribasic monohydrate). 4uL of S-adenosyl methionine in water were added to 100uL of protein and allowed to incubate on ice for 1 hour before protein was mixed with well at 1:1 ratio. Seeding used to improve crystals. Compound stock solutions (either 100mM or 1M stocks) were added up to a final drop concentration of 4.8% DMSO. Crystals were soaked for 4-6 hours. 20% glycerol in well solution was used as cryoprotectant for a quick dip of crystal in liquid N2

Crystal Properties
Matthews coefficient	Solvent content
2.71	54.66

Crystal Data

Unit Cell
Length ( Å )	Angle ( ˚ )
a = 94.784	α = 90
b = 94.784	β = 90
c = 178.164	γ = 120

Symmetry
Space Group	H 3 2

Diffraction

Diffraction Experiment
ID #	Crystal ID	Scattering Type	Data Collection Temperature	Detector	Detector Type	Details	Collection Date	Monochromator	Protocol
1	1	x-ray	100	CCD	MARMOSAIC 300 mm CCD		2010-04-27	M	SINGLE WAVELENGTH

Radiation Source
ID #	Source	Type	Wavelength List	Synchrotron Site	Beamline
1	SYNCHROTRON	APS BEAMLINE 21-ID-G	0.97856	APS	21-ID-G

Data Collection

Overall
ID #	Resolution (High)	Resolution (Low)	Percent Possible (Observed)	R Merge I (Observed)	R Sym I (Observed)	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	1.91	74.553	96.3	0.093	0.093	13	6.3		23203			28.03

Highest Resolution Shell
ID #	Resolution (High)	Resolution (Low)	Percent Possible (All)	Percent Possible (Observed)	R Merge I (Observed)	R-Sym I (Observed)	Mean I Over Sigma (Observed)	Redundancy	Number Unique Reflections (All)
1	1.91	2.01	90.5		0.916	0.916	0.8	6.3	3158

Refinement

Statistics
Diffraction ID	Structure Solution Method	Cross Validation method	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		THROUGHOUT	1.91	47.392	1.35	23175	2000	95.7	0.191	0.1883	0.2195	29.87

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]

RMS Deviations
Key	Refinement Restraint Deviation
f_dihedral_angle_d	13.186
f_angle_d	1.064
f_chiral_restr	0.067
f_bond_d	0.006
f_plane_restr	0.005

Non-Hydrogen Atoms Used in Refinement
Non-Hydrogen Atoms	Number
Protein Atoms	1868
Nucleic Acid Atoms
Solvent Atoms	125
Heterogen Atoms	21

Software

Software
Software Name	Purpose
SCALA	data scaling
PHENIX	refinement
PDB_EXTRACT	data extraction
PHASER	phasing

RCSB PDB Core Operations are funded by the National Science Foundation (DBI-1832184), 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 R01GM133198.