A systematic study of 50S ribosomal subunit purification enabling robust crystallization.
A systematic analysis was undertaken to seek correlations between the integrity, purity and activity of 50S ribosomal subunit preparations from Deinococcus radiodurans and their ability to crystallize. Conditions of fermentation, purification and crystallization were varied in a search for crystals that could reliably supply an industrial X-ray crystallography program for the structure-based design of ribosomal antibiotics. A robust protocol was obtained to routinely obtain crystals that gave diffraction patterns extending to 2.9 A resolution and that were large enough to yield a complete data set from a single crystal. To our knowledge, this is the most systematic study of this challenging area so far undertaken. Ribosome crystallization is a complex multi-factorial problem and although a clear correlation of crystallization with subunit properties was not obtained, the search for key factors that potentiate crystallization has been greatly narrowed and promising areas for further inquiry are suggested.
McLellan
Thomas J
TJ
Pfizer Inc., Department of Exploratory Medicinal Sciences, Pfizer Global Research and Development, Groton, CT 06340, USA.
Marr
Eric S
ES
Wondrack
Lillian M
LM
Subashi
Timothy A
TA
Aeed
Paul A
PA
Han
Seungil
S
Xu
Zuoyu
Z
Wang
Ing-Kae
IK
Maguire
Bruce A
BA
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry* / Q000302:isolation & purification; D002458:Cell Fractionation; D018360:Crystallography, X-Ray; D034301:Deinococcus / Q000737:chemistry* / Q000235:genetics / Q000254:growth & development; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D020836:Protein Structure, Quaternary; D012329:RNA, Bacterial / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D012269:Ribosomal Proteins / Q000737:chemistry* / Q000302:isolation & purification; D054681:Ribosome Subunits, Large, Bacterial / Q000737:chemistry* / Q000235:genetics
D001426:Bacterial Proteins; D012329:RNA, Bacterial; D012269:Ribosomal Proteins