5U5T

Crystal structure of EED in complex with H3K27Me3 peptide and 3-(benzo[d][1,3]dioxol-4-ylmethyl)piperidine-1-carboximidamide

Experimental procedure

Experimental method	SINGLE WAVELENGTH
Source type	SYNCHROTRON
Source details	APS BEAMLINE 17-ID
Synchrotron site	APS
Beamline	17-ID
Temperature [K]	270
Detector technology	PIXEL
Collection date	2013-02-22
Detector	DECTRIS PILATUS 300K
Wavelength(s)	0.9200
Spacegroup name	P 21 21 2
Unit cell lengths	92.810, 177.909, 50.340
Unit cell angles	90.00, 90.00, 90.00

Refinement procedure

Resolution	64.220 - 1.600
R-factor	0.17
Rwork	0.168
R-free	0.20000
RMSD bond length	0.010
RMSD bond angle	1.060
Data reduction software	XDS
Data scaling software	SCALA
Phasing software	AMoRE
Refinement software	BUSTER (2.10.2)

Data quality characteristics

	Overall
Low resolution limit [Å]	82.290
High resolution limit [Å]	1.600
Number of reflections	110710
<I/σ(I)>	8
Completeness [%]	97.2
Redundancy	5.8

Crystallization Conditions

crystal ID	method	pH	temperature	details
1	VAPOR DIFFUSION, HANGING DROP		291	Using a PEG/salt combination as a precipitant. Briefly, EED was incubated with 10 mM B-nicotinamide adenine dinucleotide hydrate, 2 mM of a tightly binding proprietary compound, and 0.5 mM of a synthesized peptide which comprises the helix on EZH2 which interacts with EED. The crystals were grown using the vapor diffusion method. One uL of the protein mixture was combined with 1 uL of a precipitant comprised of 20% (w/v) PEG3350, 0.2 M potassium iodide, and 0.1M Tris-HCl, pH 8.5, on a cover slip which was suspended over a reservoir comprised of 0.5 mL of precipitant at 18 Celsius and sealed. The crystals grew in 4-6 days at 18 Celsius and then harvested and soaked in defined drops consisting of 30 uL of precipitant and 2 mM of compound for 24 h. Crystals were cryopreserved for data collection using a cryosolution consisting of 30% PEG 400 (v/v), 20% (w/v) PEG 3350, 0.2 M potassium iodide, and 0.1M Tris-HCl, pH 8.5