We are pursuing structure determination of a number of catalytic RNA molecules and catalytic RNA domains by X-ray crystallography. In the past several years we have developed novel methods to obtain diffraction quality crystals of large RNA molecules which employ """"""""RNA crystallization modules"""""""". These are moieties engineered into the primary sequence of the target RNAs to present well-differentiated molecular surfaces that promote inter-molecular contacs, and crystal growth. Progress has been made in two fronts employing the high brilliance X-rays available at CHESS. 1) Crystals of a 70-nucleotide domain of a self-splcing Group II intron were obtained in a hexagonal space group (a=94 A, c=230 A) which diffracted weakly to 4 A resolution with a laboratory X-ray source. Optimization of flash cooling conditions, and data collection at beam-line A-1 of CHESS with the ADSC Quantum-1 area detector produced a complete native data-set to 3.4 A resolution. Preparation of heavy atom derivatives is underway. 2) Crystals of the complete catalytic core of the self-cleaving ribozyme from the genomic RNA of the human Hepatitis Delta Virus (HDV) which diffractedto 3.4 A in the laboratory, were obtained. The structure was solved by multiwavelength anomalous diffraction (MAD) employing synchrotron radiation from a bending magnet at the National Synchrotron Light Source (Brookhaven National Laboratory, NY) where data to 2.7 A resolution were obtained. Improved flash cooling and the high brilliance and collimation of X-rays available at beam-line F1 of CHESS allowed us to collect a superior native data-set which extends to beyond 2.4 A resolution on the ADSC Quantum-4 area detector. Refinement of the structure against this higher resolution data-set is in progress.
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