Crystallization screening test for the whole-cell project on Thermus thermophilus HB8.
It was essential for the structural genomics of Thermus thermophilus HB8 to efficiently crystallize a number of proteins. To this end, three conventional robots, an HTS-80 (sitting-drop vapour diffusion), a Crystal Finder (hanging-drop vapour diffusion) and a TERA (modified microbatch) robot, were subjected to a crystallization condition screening test involving 18 proteins from T. thermophilus HB8. In addition, a TOPAZ (microfluidic free-interface diffusion) designed specifically for initial screening was also briefly examined. The number of diffraction-quality crystals and the time of appearance of crystals increased in the order HTS-80, Crystal Finder, TERA. With the HTS-80 and Crystal Finder, the time of appearance was short and the rate of salt crystallization was low. With the TERA, the number of diffraction-quality crystals was high, while the time of appearance was long and the rate of salt crystallization was relatively high. For the protein samples exhibiting low crystallization success rates, there were few crystallization conditions that were common to the robots used. In some cases, the success rate depended greatly on the robot used. The TOPAZ showed the shortest time of appearance and the highest success rate, although the crystals obtained were too small for diffraction studies. These results showed that the combined use of different robots significantly increases the chance of obtaining crystals, especially for proteins exhibiting low crystallization success rates. The structures of 360 of 944 purified proteins have been successfully determined through the combined use of an HTS-80 and a TERA.
Iino
Hitoshi
H
RIKEN SPring-8 Center, Harima Institute, 1-1-1 Kouto, Sayo-cho, Sayo-gun, Hyogo 679-5148, Japan.
Naitow
Hisashi
H
Nakamura
Yuki
Y
Nakagawa
Noriko
N
Agari
Yoshihiro
Y
Kanagawa
Mayumi
M
Ebihara
Akio
A
Shinkai
Akeo
A
Sugahara
Mitsuaki
M
Miyano
Masashi
M
Kamiya
Nobuo
N
Yokoyama
Shigeyuki
S
Hirotsu
Ken
K
Kuramitsu
Seiki
S
D001426:Bacterial Proteins / Q000737:chemistry* / Q000302:isolation & purification; D003460:Crystallization; D018360:Crystallography, X-Ray; D004926:Escherichia coli / Q000235:genetics; D044085:Microfluidics; D011487:Protein Conformation; D016963:Thermus thermophilus / Q000737:chemistry*; D013997:Time Factors; D014961:X-Ray Diffraction
D001426:Bacterial Proteins