A general method for phasing novel complex RNA crystal structures without heavy-atom derivatives.
The crystallographic phase problem [Muirhead & Perutz (1963), Nature (London), 199, 633-638] remains the single major impediment to obtaining a three-dimensional structure of a macromolecule once suitable crystals have been obtained. Recently, it was found that it was possible to solve the structure of a 142-nucleotide L1 ligase ribozyme heterodimer that possesses no noncrystallographic symmetry without heavy-atom derivatives, anomalous scattering atoms or other modifications and without a model of the tertiary structure of the ribozyme [Robertson & Scott (2007), Science, 315, 1549-1553]. Using idealized known RNA secondary-structural fragments such as A-form helices and GNRA tetraloops in an iterative molecular-replacement procedure, it was possible to obtain an estimated phase set that, when subjected to solvent flattening, yielded an interpretable electron-density map with minimized model bias, allowing the tertiary structure of the ribozyme to be solved. This approach has also proven successful with other ribozymes, structured RNAs and RNA-protein complexes.
Department of Chemistry and Biochemistry and The Center for Molecular Biology of RNA, University of California at Santa Cruz, Santa Cruz, CA 95064, USA.
Michael P
MP
Robertson
William G
WG
Scott
D018360:Crystallography, X-Ray / Q000379:methods*; D019281:Dimerization; D008958:Models, Molecular; D009690:Nucleic Acid Conformation; D012313:RNA / Q000737:chemistry*; D016337:RNA, Catalytic / Q000737:chemistry
D016337:RNA, Catalytic; D012313:RNA