3ZD5

THE 2.2 A STRUCTURE OF A FULL-LENGTH CATALYTICALLY ACTIVE HAMMERHEAD RIBOZYME

Replaces:  2GOZ

Experimental procedure

Experimental method	SINGLE WAVELENGTH
Source type	SYNCHROTRON
Source details	SSRL BEAMLINE BL9-1
Synchrotron site	SSRL
Beamline	BL9-1
Temperature [K]	100
Detector technology	CCD
Collection date	2006-02-27
Detector	ADSC QUANTUM 315
Wavelength(s)	0.919, 0.920, 0.9266
Spacegroup name	C 1 2 1
Unit cell lengths	49.886, 69.210, 60.076
Unit cell angles	90.00, 111.83, 90.00

Refinement procedure

Resolution	20.220 - 2.200
R-factor	0.1805
Rwork	0.177
R-free	0.21170
Structure solution method	MAD
RMSD bond length	0.005
RMSD bond angle	0.937
Data reduction software	ADSC
Data scaling software	MOSFLM
Phasing software	CNS
Refinement software	PHENIX ((PHENIX.REFINE))

Data quality characteristics

	Overall	Outer shell
Low resolution limit [Å]	55.730	2.320
High resolution limit [Å]	2.200	2.200
Rmerge	0.040	0.220
Number of reflections	9712
<I/σ(I)>	16.9	4.9
Completeness [%]	99.2	100
Redundancy	4	4.1

Crystallization Conditions

crystal ID	method	pH	temperature	details
1	VAPOR DIFFUSION, HANGING DROP		301	THE ENZYME AND SUBSTRATE WERE MIXED IN EQUIMOLAR AMOUNTS IN A SOLUTION CONTAINING 50 MM MES PH 5.5, 1.5 MM EDTA. THE COMPLEX WAS FORMED BY INCUBATING THE MIXTURE AT 95C FOR 2 MIN., THEN AT 65C FOR 2 MIN., AND FINALLY AT 27C FOR 5 MIN. 1 MM MGCL2 WAS INCLUDED IN THE MIXTURE BEFORE THE FINAL INCUBATION STEP. A 10 MG/ML CONCENTRATION OF RNA WAS USED FOR THE CRYSTALLIZATION EXPERIMENTS. THE RESERVOIR SOLUTION CONTAINED 0.5M (NH4)2SO4, 100 MM MES PH 6.5, AND 35% PEG 3350. AFTER MIXING THE RESERVOIR SOLUTION, THE SALT AND PEG PHASES WERE ALLOWED TO SEPARATE, AND ONLY THE SALT PHASE WAS USED FOR THE DROPS. THE CRYSTALS GREW IN HANGING DROPS OF 2 MICROLITERS AFTER 12 MONTHS OF INCUBATION AT 28C. THE CRYSTALS WERE WASHED IN THE SALT PHASE OF THE RESERVOIR SOLUTION PRIOR TO CRYO-FREEZING.