4PQV

Crystal structure of an Xrn1-resistant RNA from the 3' untranslated region of a flavivirus (Murray Valley Encephalitis virus)

Experimental procedure

Experimental method	SINGLE WAVELENGTH
Source type	SYNCHROTRON
Source details	ALS BEAMLINE 4.2.2
Synchrotron site	ALS
Beamline	4.2.2
Temperature [K]	100
Detector technology	CMOS
Collection date	2013-09-27
Detector	TAURUS-1
Wavelength(s)	1.0972
Spacegroup name	P 32 2 1
Unit cell lengths	76.698, 76.698, 76.448
Unit cell angles	90.00, 90.00, 120.00

Refinement procedure

Resolution	50.140 - 2.463
R-factor	0.2357
Rwork	0.230
R-free	0.28350
Structure solution method	SAD
RMSD bond length	0.006
RMSD bond angle	1.247
Data reduction software	d*TREK
Data scaling software	d*TREK
Phasing software	PHENIX
Refinement software	PHENIX ((phenix.refine: 1.8.4_1496))

Data quality characteristics

	Overall	Outer shell
Low resolution limit [Å]	50.140
High resolution limit [Å]	2.450	2.450
Number of reflections	9499

Crystallization Conditions

crystal ID	method	pH	temperature	details
1	VAPOR DIFFUSION, SITTING DROP	7.5	293	drop: 1 uL 5 mg/mL RNA in 2.5 mM magnesium chloride, 10 mM HEPES-KOH pH 7.5 (heated to 65 C for 3 minutes, cooled at room temperature, 0.5 mM spermidine added, centrifuged for 10 minutes at 13000 x g) + 2 uL 10% MPD, 40 mM sodium cacodylate, pH 6.0, 12 mM spermine, 80 mM sodium chloride, 20 mM magnesium chloride (Nucleic Acid Mini-Screen kit, Hampton Research), well: 20-35% MPD, crystals appeared within 2-3 days and grew to full size over 1-2 weeks. To obtain derivatized crystals for phasing, 5 uL 4 mM iridium (III) hexammine in RNase-free water was added directly to crystallization drops and well solutions <35% MPD were replaced with 35% MPD. Wells were then re-sealed and allowed to re-equilibrate for at least 2 days, VAPOR DIFFUSION, SITTING DROP, temperature 293K