6ASP

Structure of Grp94 with methyl 3-chloro-2-(2-(1-(2-ethoxybenzyl)-1 H-imidazol-2-yl)ethyl)-4,6-dihydroxybenzoate, a Grp94-selective inhibitor and promising therapeutic lead for treating myocilin-associated glaucoma

X-RAY DIFFRACTION

Starting Model(s)

Initial Refinement Model(s)
Type	Source	Accession Code	Details
experimental model	PDB	2GFD	PDB entry 2GFD

Crystallization

Crystalization Experiments
ID	Method	pH	Temperature	Details
1	VAPOR DIFFUSION, SITTING DROP	7.8	289.2	1:1 30 mg/mL protein in 100 mM bicine, pH 7.8 against mother liquor (37% PEG400, 5% glycerol, 100 mM bicine, pH 7.8, 75 mM magnesium chloride). Crystals were harvested, and soaked for 48 hours in mother liquor containing 3.69 mM inhibitor. After soaking, glycerol was added to ~25% final concentration.

Crystal Properties
Matthews coefficient	Solvent content
2.55	51.78

Crystal Data

Unit Cell
Length ( Å )	Angle ( ˚ )
a = 65.661	α = 90
b = 84.772	β = 90
c = 96.013	γ = 90

Symmetry
Space Group	P 21 21 21

Diffraction

Diffraction Experiment
ID #	Crystal ID	Scattering Type	Data Collection Temperature	Detector	Detector Type	Details	Collection Date	Monochromator	Protocol
1	1	x-ray	197.5	CCD	MARMOSAIC 225 mm CCD		2016-11-22	M	SINGLE WAVELENGTH

Radiation Source
ID #	Source	Type	Wavelength List	Synchrotron Site	Beamline
1	SYNCHROTRON	APS BEAMLINE 22-BM	1.00000	APS	22-BM

Data Collection

Overall
ID #	Resolution (High)	Resolution (Low)	Percent Possible (Observed)	R Merge 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	2.696	38.78	99	0.1301	22.46	13.8		15293			41.77

Highest Resolution Shell
ID #	Resolution (High)	Resolution (Low)	Percent Possible (All)	Percent Possible (Observed)	R Merge I (Observed)	Mean I Over Sigma (Observed)	Redundancy	Number Unique Reflections (All)
1	2.696	2.792			0.6753

Refinement

Statistics
Diffraction ID	Structure Solution Method	Cross Validation method	Starting model	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	MOLECULAR REPLACEMENT	FREE R-VALUE	PDB entry 2GFD	2.696	38.776	1.35	15264	1527	99.41	0.1836	0.1777	0.2377

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	18.256
f_angle_d	1.244
f_chiral_restr	0.068
f_bond_d	0.01
f_plane_restr	0.007

Non-Hydrogen Atoms Used in Refinement
Non-Hydrogen Atoms	Number
Protein Atoms	3378
Nucleic Acid Atoms
Solvent Atoms	37
Heterogen Atoms	164

Software

Software
Software Name	Purpose
PHENIX	refinement
HKL-3000	data reduction
HKL-3000	data scaling
PHASER	phasing

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.