Shotgun crystallization strategy for structural genomics: an optimized two-tiered crystallization screen against the Thermotoga maritima proteome.
As the field of structural genomics continues to grow and new technologies are developed, novel strategies are needed to efficiently crystallize large numbers of protein targets, thus increasing output, not just throughput [Chayen & Saridakis (2002). Acta Cryst. D58, 921-927]. One strategy, developed for the high-throughput structure determination of the Thermotoga maritima proteome, is to quickly determine which proteins have a propensity for crystal formation followed by focused SeMet-incorporated protein crystallization attempts. This experimental effort has resulted in over 320 000 individual crystallization experiments. As such, it has provided one of the most extensive systematic data sets of commonly used crystallization conditions against a wide range of proteins to date. Analysis of this data shows that many of the original screening conditions are redundant, as all of the T. maritima proteins that crystallize readily could be identified using just 23% of the original conditions. It also shows that proteins that contain selenomethionine and are more extensively purified often crystallize in distinctly different conditions from those of their native less pure counterparts. Most importantly, it shows that the two-tiered strategy employed here is extremely successful for predicting which proteins will readily crystallize, as greater than 99% of the proteins identified as having a propensity to crystallize under non-optimal native conditions did so again as selenomethionine derivatives during the focused crystallization trials. This crystallization strategy can be adopted for both large-scale genomics programs and individual protein studies with multiple constructs and has the potential to significantly accelerate future crystallographic efforts.
Page
Rebecca
R
Department of Molecular Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA.
Grzechnik
Slawomir K
SK
Canaves
Jaume M
JM
Spraggon
Glen
G
Kreusch
Andreas
A
Kuhn
Peter
P
Stevens
Raymond C
RC
Lesley
Scott A
SA
D001419:Bacteria / Q000737:chemistry*; D001426:Bacterial Proteins / Q000737:chemistry; D003001:Cloning, Molecular; D003460:Crystallization; D018360:Crystallography, X-Ray; D004058:Diffusion; D023281:Genomics / Q000379:methods*; D005990:Glycerol / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D020543:Proteome / Q000737:chemistry* / Q000235:genetics*; D012645:Selenomethionine / Q000737:chemistry; D013696:Temperature; D014961:X-Ray Diffraction
D001426:Bacterial Proteins; D020543:Proteome; D012645:Selenomethionine; D005990:Glycerol