R & D f o r S t r u c t u r a l G e n o m i c s : A F u l l y A u t o m a t e d C r y s t a l l i z a t i o n a n d O b s e r v a t i o n R o b o t S y s t e m “ T E R A ” a n d “ S a m p l e A u t o C h a n g e r ” f o r S P r i n g - 8 S t r u c t u r a l G e n o m i c s B e a m l i n e s I n t h e p o s t - g e n o m i c e r a , t h e r e i s g r e a t h o p e t h a t p r o t e i n r e s e a r c h w i l l r e v e a l t h e s t r u c t u r e a n d f u n c t i o n o f g e n o m e - c o d e d p r o t e i n s a s l i v e n a n o - r o b o t s . S t r u c t u r a l g e n o m i c s i s o n e o f t h e m a j o r p o s t - g e n o m i c r e s e a r c h f i e l d s e n g a g e d i n d e t e r m i n i n g t h e n u m b e r o f p r o t e i n s t r u c t u r e s a t t h e a t o m i c l e v e l b a s e d o n t h e i n f o r m a t i o n o f f u l l - g e n o m e s e q u e n c e s f r o m v a r i o u s o r g a n i s m s , i n c l u d i n g h u m a n s f r o m a l l o v e r t h e w o r l d . A t S P r i n g - 8 , t h e H i g h t h r o u g h p u t F a c t o r y ( F i g . 1 ) h a s b e e n e s t a b l i s h e d a s a n i n t e g r a t e d p r o t e i n X - r a y c r y s t a l l o g r a p h y f a c i l i t y . I t s i n v e s t i g a t i o n s r a n g e f r o m p r o t e i n p r o d u c t i o n t o p r o t e i n s t r u c t u r a l a n a l y s i s u s i n g t h e s y n c h r o t r o n r a d i a t i o n ( S R ) s o u r c e o f t h e R I K E N S t r u c t u r a l G e n o m i c s B e a m l i n e s , B L 2 6 B 1 a n d B L 2 6 B 2 . T h e s e b e a m l i n e s a r e p a r t o f t h e J a p a n e s e s t r u c t u r a l g e n o m i c s p r o j e c t , F i g . 1 . R I K E N H i g h t h r o u g h p u t F a c t o r y ( H T P F ) f o r i n t e g r a t e d p r o t e i n s t r u c t u r e d e t e r m i n a t i o n . H T P F p o s s e s s e s f u l l - f u n c t i o n a l i t y o f S R c r y s t a l l o g r a p h y , f r o m p r o t e i n p r o d u c t i o n t o c r y s t a l l o g r a p h i c c a l c u l a t i o n . I t i n c l u d e s R I K E N s t r u c t u r a l g e n o m i c s b e a m l i n e s w i t h t h e i n t e g r a t e d l a b o r a t o r y i n f o r m a t i o n s y s t e m ( H T P F - D B ) d e v e l o p e d i n c o l l a b o r a t i o n w i t h H I T A C H I S o f t w a r e E n g i n e e r i n g . “ T A N P A K U 3 0 0 0 , N a t i o n a l P r o j e c t o n P r o t e i n S t r u c t u r a l a n d A n a l y s e s o f M i n i s t r y o f E d u c a t i o n , C u l t u r e , S p o r t s , S c i e n c e , a n d T e c h n o l o g y ( M E X T ) . ” F i r s t , w e a r e t r y i n g t o o v e r c o m e t h e b o t t l e n e c k s i n p r o t e i n c r y s t a l l i z a t i o n a n d d a t a c o l l e c t i o n . H o w e v e r , i n p r a c t i c e t h e r e r e m a i n m a n y b o t t l e n e c k s i n h i g h - t h r o u g h p u t s t r u c t u r e d e t e r m i n a t i o n f o r s t r u c t u r a l g e n o m i c s . W e d e s i g n e d a n d d e v e l o p e d t h e f u l l - a u t o m a t e d c r y s t a l l i z a t i o n a n d o b s e r v a t i o n r o b o t s y s t e m , “ T E R A , ” b y t h e o i l - s i t t i n g d r o p m e t h o d ( F i g . 2 ) [ 1 ] b a s e d o n p i l o t s t u d i e s u s i n g a c o m m e r c i a l l y a v a i l a b l e m a n u a l c r y s t a l l i z a t i o n r o b o t , I M P A X 1 - 5 ( D o u g l a s I n s t r u m e n t s , U K ) [ 2 ] u s i n g t h e o r i g i n a l i n i t i a l c r y s t a l l i z a t i o n c o n d i t i o n s a n d a s t a n d a r d s c o r i n g s y s t e m t o a t t e m p t l a r g e - s c a l e p r o t e i n c r y s t a l l i z a t i o n . W e a r e c u r r e n t l y c o n d u c t i n g u n i q u e 118 BL26 B1/B2 Fig. 2. Full-automatic protei n crystallization and observation robot system, “TERA,” jointly d evelope d by Taked a Rika Kogyo, Stec and AdvanSoft. It has four components : (1) a liquid-handling r obot for dispensing the crystallization s etu p r o b o t a t t h e r i g h t f r o n t , ( 2 ) microscopic photo system with CCD camera and XY plate loader at cente r f r o n t , ( 3 ) s t o r a g e f o r 2 , 5 0 0 crystallization micro-plates and 125 crystallization reagent plates in th e back, and (4) c ontrol s oftware for m anaging operations w ith the data- base at left front. Close-up views: parts of the microscopic photo system ( b o t t o m - c e n t e r p a n e l ) a n d t h e crystallization r obot (bottom-right panel). U pper r ight panel s hows various protein crystals . Figs. 3(a) 3(b) Figs. 3(e) 3(g) Figs. 3(a) 3(f ) crystallization experiments in t he three screening steps : s parse initial, secondary for optimizing, and tertiary for additives [3]. T he RIKEN Structural G enomics B eamlines have been designed to perform the high - throughput diffraction data collection for protein crystallography . In order to increase beam time efficiency and th e ability to handle m any crystals, t he goal of this beamline development is the automated operation. During X-ray diffraction experiment s, users woul d have to mount, dismount, centering and orientating many crystals. Therefore, a novel sample changing system has been developed to manipulate a number of crystals with the “Sample Auto Changer” as the core component (the left-hand in and ) [4]. A small crystal of a few hundred micron s or less is mounted on the originally designed plastic screw chips, called “Sample Pin , ” with a turnbuckle-lik e figure ( and ). T he pin is screwed and mounted at the tapped hole of the goniostat head (the right-hand side in and ). Contrary to a Hampton-like pin mounted on the gonio-head by a magnet, the orientation and positioning of th e sample c an be placed automatically at t he X -ra y beam center repeatedly after being aligned to th e c enter by t he operator. T he m ounted crystal is inserted into the tapped long tube the at arm-end of th e Sample Auto Changer w ithin 5 sec of changin g th e sa mple . We can perform 24-hour X-ray data collection by using the Sample Auto Changer. This operation is c ontrolled by th e B eamline Scheduling Softwar e ( BBS) with a lin k vi a a unique crystal and tray ID to the Highthroughput Factory Database (HTPF-DB ) system [ 5]. A ccordingly, t he information of th e crystal s ampl e, from t he protein s equence to th e st ructure analysis proces s, is obtained in th e integrated and automated m anner of TERA wit h supervision by HTPF-DB as well. 119 F i g . 3 . “ S a m p l e A u t o C h a n g e r , ” a f r o z e n c r y s t a l a u t o - c h a n g i n g s y s t e m . ( a ) S a m p l e A u t o C h a n g e r a n d A u t o A l i g n m e n t G o n i o s t a t a t B L 2 6 B 2 s t a t i o n . ( b ) C r y s t a l m o u n t e d o n a S a m p l e P i n ( e a n d g ) i s p i c k e d f r o m t h e t r a y ( d ) i n l i q u i d - n i t r o g e n D e w e r ( c ) a n d t r a n s f e r r e d t o t h e g o n i o s t a t h e a d b y S a m p l e A u t o C h a n g e r f r o m l e f t t o r i g h t i n p a n e l ( a ) , a n d i t s c l o s e - u p v i e w ( f ) . ( e ) S a m p l e P i n h a s l e f t - h a n d e d s c r e w a n d r i g h t - h a n d e d s c r e w a t e a c h e n d w i t h s m a l l n y l o n c r y o - l o o p o n t h e t o p f o r f i s h i n g t h e c r y s t a l . ( h ) C l o s e - u p v i e w o f X A F S m e a s u r e m e n t c o n f i g u r a t i o n . R e f e r e n c e s [ 1 ] M . S u g a h a r a a n d M . M i y a n o , T a n p a k u s h i t s u K a k u s a n K o s o 4 7 ( 2 0 0 2 ) 1 0 2 6 . [ 2 ] N . E . C h a y e n e t a l . , J . A p p l . C r y s t a l l o g r . 2 3 ( 1 9 9 0 ) 2 9 7 . [ 3 ] M . S u g a h a r a a n d M . M i y a n o - i n p r e p a r a t i o n . [ 4 ] M . Y a m a m o t o , G . U e n o , H . K a n d a , K . I d a , T . K u m a s a k a , M . M i y a n o a n d T . I s h i k a w a - i n p r e p a r a t i o n . [ 5 ] H . T a k a , R . Y o s h i d a , E . K a n a m o r i , I s h i j i m a a n d M . M i y a n o - i n p r e p a r a t i o n . K o h I d a , M i t s u a k i S u g a h a r a , M a s a k i Y a m a m o t o , a n d M a s a s h i M i y a n o S P r i n g - 8 / R I K E N E - m a i l : y a m a m o t o @ p o s t m a n . r i k e n . g o . j p a n d m i y a n o @ s p r i n g 8 . o r . j p a e f g h b c d Sample Changer Goniostat Left-handed screw Sample loop Right-handed screw 120
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