A general module for RNA crystallization.
Crystallization of RNA molecules other than simple oligonucleotide duplexes remains a challenging step in structure determination by X-ray crystallography. Subjecting biochemically, covalently and conformationally homogeneous target molecules to an exhaustive array of crystallization conditions is often insufficient to yield crystals large enough for X-ray data collection. Even when large RNA crystals are obtained, they often do not diffract X-rays to resolutions that would lead to biochemically informative structures. We reasoned that a well-folded RNA molecule would typically present a largely undifferentiated molecular surface dominated by the phosphate backbone. During crystal nucleation and growth, this might result in neighboring molecules packing subtly out of register, leading to premature crystal growth cessation and disorder. To overcome this problem, we have developed a crystallization module consisting of a normally intramolecular RNA-RNA interaction that is recruited to make an intermolecular crystal contact. The target RNA molecule is engineered to contain this module at sites that do not affect biochemical activity. The presence of the crystallization module appears to drive crystal growth, in the course of which other, non-designed contacts are made. We have employed the GAAA tetraloop/tetraloop receptor interaction successfully to crystallize numerous group II intron domain 5-domain 6, and hepatitis delta virus (HDV) ribozyme RNA constructs. The use of the module allows facile growth of large crystals, making it practical to screen a large number of crystal forms for favorable diffraction properties. The method has led to group II intron domain crystals that diffract X-radiation to 3.5 A resolution.
Ferré-D'Amaré
A R
AR
Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT 06520-8814, USA.
Zhou
K
K
Doudna
J A
JA
D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D003460:Crystallization; D018360:Crystallography, X-Ray; D003698:Hepatitis Delta Virus / Q000235:genetics; D007438:Introns / Q000235:genetics; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D009843:Oligoribonucleotides / Q000737:chemistry; D012313:RNA / Q000737:chemistry*; D016337:RNA, Catalytic / Q000737:chemistry; D012542:Scattering, Radiation
D009843:Oligoribonucleotides; D016337:RNA, Catalytic; D012313:RNA