R&D for Structural Genomics:

A Fully Automated Crystallization and Observation

Robot System “TERA” and “Sample Auto Changer”

for SPring-8 Structural Genomics Beamlines

In the post-genomic era, there is great hope thatprotein research will reveal the structure andfunction of genome-coded proteins as live nano-robots. Structural genomics is one of the majorpos t -genomic research f ie lds engaged indetermining the number of protein structures at theatomic level based on the information of full-genome sequences from various organisms,including humans from all over the world. AtSPring-8, the Highthroughput Factory (Fig. 1) hasbeen established as an integrated protein X-raycrystallography facility. Its investigations rangefrom protein production to protein structuralanalysis using the synchrotron radiation (SR)source of the RIKEN Structural Genomics Beamlines,BL26B1 and BL26B2. These beamlines are partof the Japanese structural genomics project,

Fig. 1. RIKEN HighthroughputFactory (HTPF) for integratedprotein structure determination.HTPF possesses full-functionalityof SR crystallography, from proteinproduction to crystallographiccalculation. It includes RIKENstructural genomics beamlines withthe in t egr a t ed labor a to r yinformation system (HTPF-DB)developed in collaboration withHITACHI Software Engineering.

“TANPAKU 3000, National Project on ProteinStructural and Analyses of Ministry of Education,Culture, Sports, Science, and Technology (MEXT).”

First, we are trying to overcome the bottlenecksin protein crystallization and data collection.However, in practice there remain many bottlenecksin high-throughput structure determination forstructural genomics.

We designed and developed the full-automatedcrystallization and observation robot system,“TERA,” by the oil-sitting drop method (Fig. 2) [1]based on pilot studies using a commerciallyavailable manual crystallization robot, IMPAX1-5(Douglas Instruments, UK) [2] using the originalinitial crystallization conditions and a standardscoring system to attempt large-scale proteincrystallization. We are currently conducting unique

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BL26 B1/B2

Fig. 2. Full-automatic proteincrystallization and observation robotsystem, “TERA,” jointly developedby Takeda Rika Kogyo, Stec andAdvanSoft. It has four components:(1) a l iquid-handling robot fordispensing the crystallization setupr obo t a t t he r i gh t f ron t , ( 2 )microscopic photo system with CCDcamera and XY plate loader at centerf ron t , ( 3 ) s t o rage f o r 2 , 500crystallization micro-plates and 125crystallization reagent plates in theback, and (4) control software formanaging operations with the data-base at left front. Close-up views:parts of the microscopic photo system(bot tom-cen ter pane l ) and thecrystallization robot (bottom-rightpanel). Upper right panel showsvarious protein crystals.

Figs. 3(a)3(b)

Figs. 3(e) 3(g)

Figs. 3(a) 3(f)crystallization experiments in the three screeningsteps: sparse initial, secondary for optimizing, andtertiary for additives [3]. The RIKEN Structural Genomics Beamlineshave been designed to perform the high-throughputdiffraction data collection for protein crystallography.In order to increase beam time efficiency and theability to handle many crystals, the goal of thisbeamline development is the automated operation.During X-ray diffraction experiments, users wouldhave to mount, dismount, centering and orientatingmany crystals. Therefore, a novel sample changingsystem has been developed to manipulate anumber of crystals with the “Sample Auto Changer”as the core component (the left-hand in and ) [4]. A small crystal of a few hundred microns or lessis mounted on the originally designed plastic screwchips, called “Sample Pin, ” with a turnbuckle-likefigure ( and ). The pin is screwed and

mounted at the tapped hole of the goniostat head(the right-hand side in and ). Contraryto a Hampton-like pin mounted on the gonio-headby a magnet, the orientation and positioning of thesample can be placed automatically at the X-raybeam center repeatedly after being aligned to thecenter by the operator. The mounted crystal isinserted into the tapped long tube the at arm-end ofthe Sample Auto Changer within 5 sec of changingthe sample. We can perform 24-hour X-ray data collection byusing the Sample Auto Changer. This operation iscontrolled by the Beamline Scheduling Software(BBS) with a link vi a a unique crystal and tray ID tothe Highthroughput Factory Database (HTPF-DB)system [5]. Accordingly, t he information of thecrystal sample, from the protein sequence to thestructure analysis process, is obtained in theintegrated and automated manner of TERA withsupervision by HTPF-DB as well.

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Fig. 3. “Sample Auto Changer,” a frozen crystal auto-changing system. (a) Sample Auto Changerand Auto Alignment Goniostat at BL26B2 station. (b) Crystal mounted on a Sample Pin (e and g) ispicked from the tray (d) in liquid-nitrogen Dewer (c) and transferred to the goniostat head bySample Auto Changer from left to right in panel (a), and its close-up view (f). (e) Sample Pin hasleft-handed screw and right-handed screw at each end with small nylon cryo-loop on the top forfishing the crystal. (h) Close-up view of XAFS measurement configuration.

References[1] M. Sugahara and M. Miyano, TanpakushitsuKakusan Koso 47 (2002) 1026.[2] N.E.Chayen e t a l . , J. Appl. Crystallogr. 23(1990) 297.[3] M. Sugahara and M. Miyano - in preparation.[4] M. Yamamoto, G. Ueno, H. Kanda, K. Ida, T.Kumasaka, M. Miyano and T. Ishikawa - inpreparation.[5] H. Taka, R. Yoshida, E. Kanamori, Ishijima andM. Miyano - in preparation.

Koh Ida, Mitsuaki Sugahara, Masaki Yamamoto,and Masashi Miyano

SPring-8 / RIKEN

E-mail: yamamoto@postman.riken.go.jp andmiyano@spring8.or.jp

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d Sample Changer Goniostat

Left-handed screw

Sample loop

Right-handed screw

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