Combinatorial crystallization of an RNA-protein complex.
One of the most difficult steps in X-ray crystallography of a ribonucleoprotein (RNP) complex is obtaining crystals that diffract to high resolution. This paper describes a procedure for identifying the optimal lengths of the nucleic acid components that provide high-quality crystals of the RNP. Both strands of an RNA duplex were varied in a systematic manner to generate a large number of unique RNPs that were screened for crystallization behavior. As observed in the crystallization of other nucleic acids and their complexes, the exact length of the RNA chains was found to be critical in obtaining diffraction-quality crystals, even though the relative molecular weights of the protein and RNA components were approximately 50 and approximately 10 kDa, respectively. In particular, the helix-loop-helix structure in the mRNA for the Saccharomyces cerevisiae ribosomal protein L30, which functions as an autoregulatory element for L30 expression, was synthesized as two separate RNA chains of variable length (12-14 and 15-17 nucletides). Duplex formation of these RNAs formed the asymmetric, internal loop-binding site for L30. 16 such RNA duplexes, varying by +/-1 residue at the 5' or 3' end of either chain, were used to prepare 16 unique complexes with a maltose-binding protein-L30 fusion protein. The complexes were screened against 48 standard crystallization conditions in 2304 experiments, yielding 30 conditions with single crystals in the initial screen. The most promising of these is being used for structure determination.
Hoggan
Danielle Bodrero
DB
Department of Molecular Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, California 92037, USA.
Chao
Jeffrey A
JA
Prasad
G S
GS
Stout
C David
CD
Williamson
James R
JR
D002352:Carrier Proteins / Q000737:chemistry; D020650:Combinatorial Chemistry Techniques; D003460:Crystallization; D018360:Crystallography, X-Ray; D006860:Hydrogen Bonding; D058619:Maltose-Binding Proteins; D008958:Models, Molecular; D009843:Oligoribonucleotides / Q000737:chemistry; D012335:RNA, Ribosomal / Q000737:chemistry*; D012269:Ribosomal Proteins / Q000737:chemistry*
D002352:Carrier Proteins; D058619:Maltose-Binding Proteins; D009843:Oligoribonucleotides; D012335:RNA, Ribosomal; D012269:Ribosomal Proteins