The high-throughput protein-to-structure pipeline at SECSG.
Using a high degree of automation, the crystallography core at the Southeast Collaboratory for Structural Genomics (SECSG) has developed a high-throughput protein-to-structure pipeline. Various robots and automation procedures have been adopted and integrated into a pipeline that is capable of screening 40 proteins for crystallization and solving four protein structures per week. This pipeline is composed of three major units: crystallization, structure determination/validation and crystallomics. Coupled with the protein-production cores at SECSG, the protein-to-structure pipeline provides a two-tiered approach for protein production at SECSG. In tier 1, all protein samples supplied by the protein-production cores pass through the pipeline using standard crystallization screening and optimization procedures. The protein targets that failed to yield diffraction-quality crystals (resolution better than 3.0 A) become tier 2 or salvaging targets. The goal of tier 2 target salvaging, carried out by the crystallomics core, is to produce the target proteins with increased purity and homogeneity, which would render them more likely to yield well diffracting crystals. This is performed by alternative purification procedures and/or the introduction of chemical modifications to the proteins (such as tag removal, methylation, surface mutagenesis, selenomethionine labelling etc.). Details of the various procedures in the pipeline for protein crystallization, target salvaging, data collection/processing and high-throughput structure determination/validation, as well as some examples, are described.
Liu
Zhi-Jie
ZJ
Southeast Collaboratory for Structural Genomics, Department of Biochechemistry and Molecular Biology, University of Georgia, Athens, GA 30602, USA.
Tempel
Wolfram
W
Ng
Joseph D
JD
Lin
Dawei
D
Shah
Ashit K
AK
Chen
Lirong
L
Horanyi
Peter S
PS
Habel
Jeff E
JE
Kataeva
Irina A
IA
Xu
Hao
H
Yang
Hua
H
Chang
Jessie C
JC
Huang
Lei
L
Chang
Shu-Huey
SH
Zhou
Weihong
W
Lee
Doowon
D
Praissman
Jeremy L
JL
Zhang
Hua
H
Newton
M Gary
MG
Rose
John P
JP
Richardson
Jane S
JS
Richardson
David C
DC
Wang
Bi-Cheng
BC
D003460:Crystallization; D018360:Crystallography, X-Ray / Q000379:methods*; D011506:Proteins / Q000737:chemistry* / Q000302:isolation & purification
D011506:Proteins