4XEJ

IRES bound to bacterial Ribosome

This is a non-PDB format compatible entry.

Experimental procedure

Experimental method	SINGLE WAVELENGTH
Source type	SYNCHROTRON
Source details	APS BEAMLINE 23-ID-B
Synchrotron site	APS
Beamline	23-ID-B
Temperature [K]	100
Detector technology	CCD
Collection date	2010-10-20
Detector	MARMOSAIC 300 mm CCD
Wavelength(s)	1.033
Spacegroup name	P 21 21 21
Unit cell lengths	209.050, 447.220, 608.960
Unit cell angles	90.00, 90.00, 90.00

Refinement procedure

Resolution	60.000 - 3.800
Rwork	0.246
R-free	0.28400
Structure solution method	MOLECULAR REPLACEMENT
Starting model (for MR)	4v83
Data reduction software	XDS
Data scaling software	XSCALE
Phasing software	PHENIX
Refinement software	PHENIX

Data quality characteristics

	Overall	Outer shell
Low resolution limit [Å]	60.000	4.000
High resolution limit [Å]	3.800	3.800
Rmeas	0.200	1.600
Number of reflections	555726
<I/σ(I)>	8.28	1.2
Completeness [%]	99.9	99.8
Redundancy	4.8	3.4
CC(1/2)	0.995	0.998

Crystallization Conditions

crystal ID	method	pH	temperature	details
1	VAPOR DIFFUSION, HANGING DROP	7	295.5	Initial crystallization screening was performed around conditions previously reported (31, 48) by dispensing 0.2 + 0.2 mL sitting drops with a Phoenix robotic liquid handling system (Art Robbins) on 96-well plates. Once optimal crystallization conditions were determined, crystals were grown by the sitting-drop vapor-diffusion method using drops dispensed by the Phoenix with 1- to 2-mL ribosome complexes mixed with 1-2 mL of reservoir solution [100 mM Tris-OAc, pH 7.0, 200 mM potassium thiocyanate (KSCN), 3.6-5% PEG 20,000, 6-14% 2-methyl-2,4-pentanediol (MPD)] at 22.5C. Crystals emerged after 5-7 d and matured between 2-3 wk. Crystals were then subjected to cryoprotection by gradually replacing the mother liquor with cryoprotection buffer I (100 mM Tris-OAc, pH 7.0, 200 mM KSCN, 5% PEG 20,000, 25% MPD, 14% PEG 200, and 10 mM Mg(OAc)2). The crystals then were flash-frozen by plunging into liquid nitrogen.