Capturing the structure of a catalytic RNA intermediate: The hammerhead ribozyme

Science

Volumn 274, Issue 5295, 1996, Pages 2065-2069

  Scott, William G ^a   Murray, James B ^b   Arnold, John R P ^b   Stoddard, Barry L ^c   Klug, Aaron ^a  

^a MRC LABORATORY OF MOLECULAR BIOLOGY   (United Kingdom)

^b UNIVERSITY OF LEEDS   (United Kingdom)

^c Fred Hutchinson Cancer Research Center   (United States)

Author keywords

[No Author keywords available]

Indexed keywords

RIBOZYME;

ANIMAL CELL; ARTICLE; CONFORMATIONAL TRANSITION; CRYSTAL STRUCTURE; CRYSTALLOGRAPHY; DNA FLANKING REGION; ENZYME ACTIVE SITE; ENZYME ACTIVITY; ENZYME MECHANISM; ENZYME SUBSTRATE; NONHUMAN; PRIORITY JOURNAL; RNA STRUCTURE; STRUCTURE ANALYSIS;

BASE SEQUENCE; CRYSTALLIZATION; CRYSTALLOGRAPHY, X-RAY; FREEZING; HYDROGEN-ION CONCENTRATION; MAGNESIUM; MANGANESE; MODELS, MOLECULAR; NUCLEIC ACID CONFORMATION; RNA, CATALYTIC;

ANIMALIA;

EID: 0030476765     PISSN: 00368075     EISSN: None     Source Type: Journal    
DOI: 10.1126/science.274.5295.2065     Document Type: Article

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25. Diffraction data were processed with DENZO [Z. Otwinowski, in Proceedings of the CCP4 Study Weekend, L Sawyer, N. Isaacs, S. Bailey, Eds. [Daresbury, U.K., and SERC, 1993), pp. 56-62]. Further details are described in Table 1.
26. Molecular replacement was done with the AMoRe (automated molecular replacement) software distributed with CCP4 (19) and, as a probe, the dimer structure determined from the previous crystal form, The top translation function solution had an R factor of 53% and a correlation coefficient of 28. However, 10 cycles of rigid-body refinement in AMoRe reduced the R factor to 31%, and the correlation coefficient simultaneously increased to 86. Further rigid-body refinement followed by conventional positional refinement (Powell minimization) in X-PLOR 3.1 [A. T. Brünger, X-PLOR3.1: A System for Crystallography and NMR (Yale Univ. Press, New Haven, CT, 1993)] further reduced the R factor to 26%. The initial model of one hammerhead ribozyme molecule in the crystal asymmetric unit was further refined with a standard simulated annealing slow-cooling molecular dynamics protocol followed by conventional positional and restrained temperature factor refinement in X-PLOR 3.1 with data from 8.0 to 3.0 Å resolution and a modified RNA geometry parameter library [G. Parkinson, J. Vojtechovsky, L. Clowney, A. T. Brünger, H. M. Berman, Acta Crystallogr. D52, 57 (1996)]. Finally, the low resolution data were incorporated, a solvent mask was determined, and partial calculated structure factors were generated to model the bulk solvent contribution to the x-ray scattering amplitudes for further refinement within X-PLOR 3.1. The metal-bound crystal structures were refined identically (Table 1).
27. Molecular replacement was done with the AMoRe (automated molecular replacement) software distributed with CCP4 (19) and, as a probe, the dimer structure determined from the previous crystal form, The top translation function solution had an R factor of 53% and a correlation coefficient of 28. However, 10 cycles of rigid-body refinement in AMoRe reduced the R factor to 31%, and the correlation coefficient simultaneously increased to 86. Further rigid-body refinement followed by conventional positional refinement (Powell minimization) in X-PLOR 3.1 [A. T. Brünger, X-PLOR3.1: A System for Crystallography and NMR (Yale Univ. Press, New Haven, CT, 1993)] further reduced the R factor to 26%. The initial model of one hammerhead ribozyme molecule in the crystal asymmetric unit was further refined with a standard simulated annealing slow-cooling molecular dynamics protocol followed by conventional positional and restrained temperature factor refinement in X-PLOR 3.1 with data from 8.0 to 3.0 Å resolution and a modified RNA geometry parameter library [G. Parkinson, J. Vojtechovsky, L. Clowney, A. T. Brünger, H. M. Berman, Acta Crystallogr. D52, 57 (1996)]. Finally, the low resolution data were incorporated, a solvent mask was determined, and partial calculated structure factors were generated to model the bulk solvent contribution to the x-ray scattering amplitudes for further refinement within X-PLOR 3.1. The metal-bound crystal structures were refined identically (Table 1).
28. Collaborative Computational Project, Number 4 [CCP4], Acta Crystallogr. D50, 760 (1994).
29. We thank P, Stockley, J. Finch, G. Varani, S, Price, K. Nagai, O. Uhlenbeck, and members of their research groups for advice; R. Sweet for help with data collection at Brookhaven synchrotron beamline X12C, and the Daresbury Laboratory for additional data collection; D. McKay and K. Flaherty for discussions and for providing us with an improved set of stereochemical parameters (78) for RNA refinement. Supported by the Medical Research Council of the United Kingdom and the American Cancer Society (W.G.S., PF-3970), and the NIH (B.L.S., GM-49857).