MANUFACTURED BY WELDING INDUSTRIES HEAD O F F I C E AND EXPORT D I V I S I O N 2-20 A u s t a r A u e n u e 5 c ThorrIac.town, ERUI PMEbIT MANUFACTURING D I U 1 SION 2 C ) ~ O J S. Rclad Edwards t own SCIIJt h

OF AUSTRALIA PTY LTD

V i c e p h 1 A A Facsimile G861 2
SALES RRANC:HES M e l t u u r n e Sydney Er Newca5.t 1 e E r9 WCo n g u n g G 4 25' 12 Bric.bane 844

6A A 2655 A A 3447 1371

Mackay Adel aide

Perth Launceston

7088 53822

AA82856 AA72936

MELDPIAT I C 0 S.
PLEASE Et4SURE THAT T H I S FOLDER I S KEPT I N A SAFE PLACE REFERENCE WHEN REQUIRED.

REAlD'Y

CONTENT8

SECT1 ON

CONTENTS SFEC I F I CAT I O N $ RECE I O 1NG I NSTALLAT ION CONTROLS WELDING SET-UP GENERAL MAINTENANCE TROUBLE SHClOTING CONTROL P R I N T E D I R C U I T C SFARE PARTS LISTS

BOARDS

F 1 GUHE 5

P 18 11

WELDMATIC 188s

MANUAL

PAGE 1

L SPEC1 F1CAT1 ONS

TECHNI CkL
W e l d i n gO u t p u t O u t p u t us1 tagE. Duty C y c l e.
- O v e r 1 clad P r c t t e c t ior1
W i r eS p e e dR a n g e In5.u 1 a t i or1 - Encl osure
- ~ I T I ~ C. 21 - 38 VDC 1 E;@ ArrIF'E., 58% 8 Amps, 35X R e c t i f i er Therrrlcts.tats (Thermal j G1 - 16 m e t r e w ' m i n C : ~ ~ E. C'H'' - 8 C; r i s e a t 48 C a m t l i e n t. D r ip - p r o o - f

STANDARD PACKAGE

AM132-8/'? AM132-0/15
N i n e M e t r e Accec.c.ory Lead Kit F i f t e e n M e t r e A c c e s s o r y Lead K it

- RECEIVING 2.

In genera.1 i t i a g o o d r a c t i c e s p o fi n s t a l l a t i o n before unpackinq t cl t h e i t ems en c 1 o s e d.
t o mc~ue the quipmerlt t o t h es i t e Use c a r e i no r d e r t o a v o i d damage
damage haE. c l c c u r r e di n If any t r a ni s r t r r l e d i a tn urt i f ). ril el r We1 d i n g I n d u s t r i e s cl+ A u s t r a l i a o t h e i r a u t h o r i E. e d a g e n t.

WELDMATIC 180s

PAGE 2

I NSTALLAT I ON

3.1 MACHINE LOCATION
Where p o c , c. i t t l e , in chctclsirrg a s i t e o a n y e l d i n g f r w c o n s i d e r a t i o ns h o u l d be g i v e n t u t h e f c l ll o w i n gp o i n t s : pOWer-50UrCe,
A v a i l a h i l i t y o f e l e c t r i c a la n dc. h i e l d i n g

g a s c.upp1iec.

A c c e s s i b i l i tf o r y t h e replacement o f consumables.e.elding i, w w i r e a n d gas b o t t l e s , a n d f o r a n y f u t u r er r l a i n t e r l a r l c er e q u i r e m e n t s.
power-source cclcll i n q a i r t h r o u g h
m u s t be p o s i ticsned so t h a t t h e the m a c h i n e i s not i m p e d e d.

f 1 ow

- P r c l t e c t i o nf r o mw i n da n dd r i v e n r a i n - Breezes bluwing hrough t an o p e n o o r w a y a ne c. u l d c r t in d e g r a d a t i o n f o the w e l d. h i e 1 d i n q c gas c o v e r a gw h i c h e, ca.n c a u wee pd r o s i t y. s lo I f such a s i t e i s u n a u o i d a t ~ l e ,c o n s i d e r a t i o ns h o u l d tle g i v e n tcl t h e u s e o fp o r t a t ~ l e a s u i t a b l em a t e r i a l. The nut+low o f cooling a i r w e l d i r ~ gs c r e e n so f f r o m t h e w 1 d i n g p o w e r - ~. c ~ u r c e c a n a1 5.0 c a u s e s i m i l a r prcatl1 ems., a n d e
t h e m a c h i n es h o u l d
tle pc1s.i t i o n e d w i t h

t h i s in mind.

A i r h o rd u s tr t i c l e~s1 i 1 l np a e 1 a l w a y s a c c u m uu i n e e d le n t at e q u i p m e n t , p a r t i c u l a r l y that w i t h f o r c e d a i r c o o l i n g a n d e x c e s s i v e tluildupan ften c o tle the c a u s e of premature m a c h i n e a i l u r e. f Ai: at i m e se n d e a v o u r t o p o s i t i o n t h e m a c h i n et om i n i m i s e the i n t a. k e cl+ contaminated a i r , particularly that c o n t a i r r i r l q metal 1 i c a s f r o m g r i n d io p e r a t i o n s. ng isee a1 5. 0 particles h suc MA1NTENANC:E SECT ION of t h i c. M a n u a l , plage }

- SUITABLE 3.2

M A I N S VOLTAGES

T h e WELDtIATIC: 1861s.

i s d e s i g n e d f o r c o n n e c t i c ~ nt u a s. i n g l e phase! e l e c t r i c am a i n c. l power s u p p l > .

WELDMATIC 8 s

PAGE 3
3. 3 CONNECTION TA ELECTRICAL M A I N S POWER SUPPLY
The r e c o m m e n d e dS u p p l yF u s eR a t i n g

i s 28 A m p s.

W e m a k i n g corrnec t i o n t o t h e t 4 a c h i n eS u p p l yt e r m i n a l hn s, ensure that l o cc o d e s al are adhered t o , a n d t h a t t h e EARTH c o n n e c t i o n i s

seCurE.1 y

T h e b i n s. Power pply Su Cab1 e c-.hould o n l y e q u i v a l e n t s i z e cattle as that f i t t e d.

replaced

Tighten the two s.crews o f the i n t e r n a b l a 1 ep. v i n g c l em s c s u f f i c i e 'n t. 1 a cik ' 5 n t h e S u p p l y F l e x i b l e Cat11e C t e r m i n a t e d w i r e c. a r e n o t i nt e n s i o n.

. U C ~

F1 GURE

1 CONNECT1O N.

SUPPLY FLEX1 BLE CABLE

WELDMATIC: 186s

PAGE 4
3.4 CABLE AND HOSE CONNECTIONS
A I 1 w e l d i n gc u r r e n t I c o n t r o la n ds h i e l d i n g ac. c o n n e c t i o n c. a r e c.hown in f i g u r e 3. The c o m p l e t e s e t on e c e s s a r c a b l e z.n d f y a h o s e s are c o n t a i n e di n t h e r.tar1dar.d accec-.sory k i t AP1132.

-SHIELDING

HOSES.
T h e a c c e s s o r y k i t CctntainC-. two gas. h o s e s. T h E 4 - 1 o r t e r 1 en y t h i E. u s e d t o j o i n the o u p u t o f t h e s h i e l d i n g gas r e g u l a t c l r. - f l o w m e t e r t o t h e g a s n i p p l e on the r e a r panel cl+ the P o w e r - ~. c ~ u r c e. The c.ec und hose is. used t o m a k e t h e gas c o n n e c t i o n f r o m t h e ~ C I O s o u~r c ef r o n t J ~ panel t o the r e a r of the L521 w i r e f e e d e r.

-CONTROL

Engage

CABLE,

the W i r e f e e d e rc o n t r c l l panel on the p o w e rs o u r c ef r o n t the w i r e f e e d e r a r e c.huwn
the m a r k e d s o c k e t c a t l l e l u gn t o p i T h e con trcll cab1 e c o n n e c t i u n s f o r t h e a d j a c ed ita g r a m , n
-I.IELD I N G CURRENT CABLES.
G.t.1.k. we1 d i n g i s c o n d u c t e dw i t h t h e el e c r c l d e p o l a r i t y pc1c.i t i v e , t and the workp i e c e n e q a t i v e. T h e p o s i t i v e ou t p u t of the p o w e r s c ~ u r c e is. c o n n e c t e d o t the w e l d i n g gun via an a d a p t c t m o u n t e d r with t h e mire d r i v e rnechanic,m. Ensure t h a t t h e p o s i t i v e eldin ding cat11 e iz, E.ecure1 y bo1 t e d t a t h i 5 a d a p t o r.

n e g a t i u eo u t p u t of t h e power s o u r c ei sc o n n e c t e d t o t h e welding c a t liln c c l r p o r a t i n g e the work clamp. Bckth l e a d s. c. e c u r e d a.t t h e puwer s o u r c e by i n s e r t i n g t h e p l u gi n t oi t 5a p p r o p r i a t es o c k e t.

PAGE 5

CONTROL CONNECTIONS

"

W - 2 W l R E FEEDER

" "

POWER SOU=

OR W H I T E

CONTROL
F1 GURE 2. CABLE AND HOSE CONNECT1O N S

PAGE 6

SPnr T t M E

WELDMATI C 8 s

PAGE 7

-INCH/PURGE

SWITCH:
T h i s i s. a d u a l+ u n c t i o ns w i t c h.
M o v i n gi e c h / p u r y e i t c h thn sw f e e d i n t h w i r ie t t h g u c a b l 5 y s t e m. g e n o e n e d u r i nn c h i g i s determined contra1
up i n i t i a t e E. w i r e the feed mcltor, T h e w i r ee e d f speed tly the ttiny se cl+ thJ i r e - c , p e e d Ie

-LOtTACiE ,SW ITCH :

O u t p u t v01 t a g e w 1 i l

-W I HE SPEED C:CNTHOL :

increasewithincreasingposition

numbers.

T I ME CONTROL :
- I NTERVAL C O N T R O L :

WELDMATIC 0 s

MANUAL PAGE

- MELDING 5.

SET-UP
ELECTRODE W1 RE/SHI ELDING
- Se1 e c t t h e d e s i r e d o n s u m a b l c e el e c t r o d e w i r e n d a wirefeeder. E n s u r e that t h e w i r e f e e d e ircf. i t t e d i t h w d r i v e rollers - 5 e e p a g e 2 3.

to the cctrrect

- Conrrect t o t h e gac. r e g u l a t o r / f lo w m e t e r , a p p r o p r i a t e t o t h e w e l d i n gc o n s u m a b l e.

MI L6, STEEL

STAINLESS STEEL
a s u p p l y of c. h i e l d i n g y a s

TYFICAL HIELDING S

ARGON o r ARGON /' HEL I UP1 P1 I XTURES

S I L 1 CON BRONZE

Sethe as loo~ ate g f r
t o s u i t the w e l d i n g r o c e d u r e , p the r a n g e o f t o 1 i t r e s. / m i n.

usually

N O T E :x c e s s i v e Eg f lso w a t h e we1 d i n g r e s u l t.

c o c. ta y d ln

a d v e r s e lay f e c t f

WELDING CURRENT(Arnps)

0 50,604 ~

70,85,100,120#140#t60,iB

WIRESPEED DIAL SETTING 6mm VOLTS WIRE SPEED DIAL SETTING 8mm VOLTS

1-4 2.l

3.1.6 3

7.2.2 6

5 1.3 2

8 2.7 1.2 6

WIRE SPEED DIAL SETTING 9mm VOLTS WlRE SPEED DIAL SETTING 1.2mm VOLTS

.81.2 1

0 2.5 9

1.5 1.8

SETTINGS GIVEN FOR C SHIELDING GAS.-FLOW RATE 8-1OLITRES/MINlJTE. USE THIS CHART AS A PRELIMINARY SETTING GUIDE. TO'TUNE'EACH WELD SETTING, ADJUST WIRE SPEED ONLY

WELD SETTING CHART

WELDMATIC 18Bs

PAGE 10

7.1 POOR WELDING RESULT
- C h e c k f o r gclud w i r ef e e dc o n d i t i o n s , i.e. s p r ~ o l h o l d e r b r a k e not o u e r - t i g h t e n e d ,d r i v e rollers not lcqged c w i t h d u s tb u i l d - u p ,d r i u e rcll l e r s a r e the c o r r e c t s i z e a n d t y p e t o s u i t t h e w e l d i n gw i r e 5a n d the d r i u e o l l r pressure is a d e q u a t e o r o n s i s t e n. f e e d i n g. f c t Chec k & l 5.0 t h a t the guncab1 e 1 i n e r i 5. o f c c f r r e c t r. i r e a n d n o t b l c l c k e d a n d t h a t t h e w e l d i n g t i p is i n g o o dc o n d i t i o na n dn u te x c e s s i v e l y worn. Aof the a h u e c a n c a u s e e r r a t i c el d i n g cclrrdi t i o n s a n d I T I S THE AREA WHICH HAS THE HIGHEST P R O B A B I L I T Y OF FAILURE I N ALL GAS METAL ARC WELDING a n d , h e r e f c l r r ,. h c ~ u l d t ~ be t h e i r 5. p c l i n c h e c k e d f t t whef-1 p r o b l emE. o c c u r.
Check t h a t t h e s h i e l d i n q gas. ic. c c ~ r r e c tf o r the e l e c t r o d ew i r e i n a n d f o r c o r r e c t gas f l o w r a t e a t t h e e l d i n g o r c h o z z l e. w t n E n s u r e t h e r e a r e n o gas 1 e a k c. , as. t h i s wi 1 l r e s u l t i n w 1 d p c l r o s i t ; ~ ,. e The gas nuzzle m u s t be f r e e from s p a t t e r a n df i r m l ya t t a c h e d to t h e w e l d i n g n o z z l e t o e n s u r e that atmctspheric as. g i s n c ~ t dra,wn intct the s h i e l d i n g gas.
- E s t a t l l i s h that the a r c { J O I t a g e i s c o r r e c t 1 y 5.et A n a r c I , J C I ~t a g e w h i c h i 5 t o o 1 ow wi c a u s e e x c e c. c , i {le c r a c k 1 e I s h o r t i n g a n d s p a t t e r wh i 1 e an a r c u u l t a g et o oh i g h may r e s u l t i rl el d u n d e r c u ta n d / o r A r c l e n g t hi sp r o p o r t i o n a l t o arc I,JOIa g e. t l o s so fa r c d i r e c t i o n . -

l.pJctr k -p i e c e 5.u r f ac e c on tam i n a t i on by w t er a cti 1 , grease, g a l u a n i s i np a i n t , g, or o x i d e l a y e r s c a n s e v e r e l y d i s t u rtb 1 he w 1 d i rig a r c e r e c. u l t i n 9i n a poor. we1 d d e p o s i t Shou 1 d th iS c o n d i t i o n c usru r f a c e e a n i n g oc , l c?f the w o r k p i e c e wibe n e c esc,ar Y.

WELDMATIC

PAGE i l

7.2 NO WELDING CURRENT

Power Check t h a t M a i n s S u p p l y is. a u a i l a t l l e a t t h e WELDMATIC 188s i s on. t h ei n d i c a t o r is r u n n i n ga n d i. e. , t h a t t h ef a n light Source,
- Check c o n t i n u i t y o f the w e l d i n gc u r r e n tc i r c u i t ,i. e. y w o r k c l a m p a n d gun c a b l ec o n n e c t i o n s. - Inspect,
a nrd p l a c e e inecessaryuses f f Power S o u r c ef r o n t panel, Replaceuses nly f o (3AGi) and c o r r e c tc u r r e n t rating.

work lead,

i86zs type
on the WELDMATIC with the c o r r e c t
- C h eok e r a t i o n cp of t h ee l d i n g n i t c ih ,. w gu w c.e , wir~?-teed m o t o r r u n s , a n d g a s v a l u e CIFIerates w h e n - gun s u i t c h c 1 o s e d. I f not r e p l a c e t h e gun c a b l e assembly, a n d re-test. If t h e f o r g o i n g c h e c k s h a u e tleerl made. m d n o t e v e a l e d r the n possible t h a t a. f a i l u r e has o c c u r rie d it is condition e l e c t r o n i cc o n t r o l c i r c u i t so ft h e WELDMATIC 1538s

Saul t

In thisnstance, i Q U A L I F I E D S E R V I C E PERSONNEL s h o u l d r e f e r t s t h e i n - f o r m a t i o nc o n t a i n e di nt h ef c c ~ ~ i n qS e c t i o n.

PAGE 1 2

- CONTROL 8,
PRINTED C I R C U I T BOARDS
CONTROL BOARD W 1 A - 2 1
n c o n j u n c t i o n w i t h PCB W i d - i perfctrmc, t h e f o l l c l w i n q

- WIRE-FEED 8.2

T h e PCB W16-ZI f uric t i onc. :
- C l o s e d - l c ~ a p speed c c l n t r o l f u r the w i r e f e e d m o t c l r.
Run, a n d b r a k i n g c o n t r o l o f t h e w i r e f e e d m a t o r. D r i v e r for t h e gas c. c t 1 e n c ~ i d a l v e. v " B u r n - b a c k " o f the e l e c t r o d e w i r e a t end a f w e l d.
I t a1 5.0 p r a v i d e s t h e el e c t r i e a l E.cIcket5. t o ac ~ w p c t s t - f i t o f v d r i c I u 5 W14 wire-feeder ' O p t i o n s ' s u c h a5 :

3 O Y. AC UPPLY S

UTILUX H I P P 6
F 1 GURE - W1 RE-FEEDER MOTHER BOARD pcS OUERLAY W 1 6-21 7.

WELDMATIC 18Gis

PAGE 13

FIGURE

3 WIRE-FEEDER
SPEED BOARD PCB OIJERLAY W - 2 0

WELDMATIC i805

PAGE 1 4
T h i s tmard d e t e c t s w e l d i n g c u r r e n t i n i t i a t i o n and times Welding a n d C y c l e A r c o p e r a t i o n.

-1-1-1- l>

PAGE 15

eSPAREEm3LL.i

9.1 POWER SOURCE ASSEMBLY
DESCRIPTION BASE ASSEMBLY TRANSFORMER ASSEMBLY L I M I T I N G BC WELDING INDUCTANCE ASSEMBLY R E C T I F 1ER ASSEMBLY WIRE-FEEDER MOTHER BOARD PCB W1 RE-FEEDER SPEED BOARD PCE SPOT TIMER/CYOLE ARC BOARD PCB CURRENT RELAY CO1 L REED RELAY CONTROL TRANSFORMER 8 - V F U A 2 CONTACTOR VOLTAGE SWITCH FUSE HOLDER FAN BLADE FAN MOTOR 6H z CONTROL PLUG TYPE bIO./SERIAL NO. NAMEPLATE KNOB - bJ1 RE SPEED KNOB - SP@T/CYCLE PCB PlOUNT I NGS GAS VALVE HOSE GARB GAS VALVE CONNECT1 O N GAS HOSE N I P P L E NUT FRONT PANEL BACK PANEL BAFFLE BC COWONENT MOUNT I NG PANEL TOP COUER S I DE PANEL LOOM ASSEMBLY - POWER SOURCE OFF/ON SWITCH LOOM A s s E r I B w - W I RE FEEDER I r m I CATOR L I GHT INCH/PURGE SWITCH W 1 RE SPEED POTENTI OtIETER SPOT/CYCLE SELECTION SI*!ITCH GAS HOSE OUTPUT T E R t l I N A L ASSEMBLY CABLE ENTRY BUSH CABLE ENTRY BUSH CASTOR WHEELS F I X E D WHEELS HOSE PLUG SUPPLY FLEX CAPAC I TOR BOTTLE CRADLE ASSEMBLY HANDLE

L 8.16

CP17-29 CP17-25 CP17-28 CP17-11 Wid-21 W 6-Ard 8 - W 1-28 1

Ar113-14

CP1 7-8/Y

CPi7-6/16

CPl7-6/11 W 1 -23 CP15-61/4 CP17-B/14 CP15-6/14 W I rd W-8/i 6 k15-16/19 CP28- 1 4/ W 1- / 1 W 1 1-W1 7-TC262 TC262N C:P17-CPL 7 - CP17-14 CP17-16 CP17-17 CP17-6/38 w1-19 CPf 7 - 8 / Id- 8 / J1 7-6, I

22 Lb 26 27

, l 31 31. 1 31.2 31.3 31.33 34

W1 1-82/15

AA35 (MC- 6 / < M C- 8 ~ 91 CP17-8/36 GP l 7 - 8 / C P 1 7-8/38
W1 1-8/13 AM97 CP17-@1/4i

PAGE 16

WELDMATI C 118s

9. 3 TWO-ROLL-DRIVE

ITEM# PART
ASSEMBLY DESCRIPTION 2 R O L L DRIVE BODY
W2-44/1 w2-44/2 b52-44/25
2 ROLL DRIUE ROLLER TOP HOUSING
SHAFT SOCKET SET SCREW 11 x 1 8.5 WASHER M 5 STAR LOCK WASKER CAP HEAD SOCKET SCREW r m X SOCKET HEAD SHOULDER SCREW M 8 x 26 B E L L E L L E WASHER JW417 TENS1 ON ARM ' SPRING NUT TOP R O L L SHAFT 11 x 48 S. H. SHOULDER SCREW.8 BEAR I N G 2 BEARING FL6C-rEZZ M 6 HEXAGONAL DOME NUT DRIUEN GEAR WHEEL TOP ROLLER BUSH 0 aL.REW NO.8-32 X 1/ 2 " LING: S0C.K , CAP HEAD D R I w r d G GEAR WHEEL SLOTTED PAN HEAD SCREW rl3 x e; CAP HEAD SOCKET SCREM M5 >: CAP HEAD SOCKET SCREW 1.16 x 12 C/SUNK HEAD SOCKET SCREW r.16 X DRIVE ROLLERS - SEE TABLE - SEE TABLE WIRE INPUT GUIDE ADAPTER BUSH LOCKNUT P124 x 1 CAP HEAD SOCKET SCREW 11 X 2s.8 SPRING WASHER 5'1 6" CAP HEAD SUCKET SCREW r w >: 1s FLAT WASHER 5 / " I NSLILAT IbiG R I N G FLAT MASHER 24mm I.D. x 34mrr1 O. D. BERNARD Q U I Cl< CONNECT BERNARD CABLE ADAPTER GLlI DE TUBE

14 1'; 18

15' 34
W2-44/&. W2-44/'13 w2-44/7 W2-44/8
w2-15 w2-44/9 03r356ZP W 2- 16
WZ-, W2-44/12. W2-44/2X W2-44.>'22
LJ2-44/5 w2-44/W2-44/.W2-44./26

FIGURE i l.

TWO R O L L D R I V E ASSEMBLY

WELDMAT I C 8 s

PAGE 15'

FLAT ROLLER

-" I

PLAIN VEE ROLLER

t+$"/

1S 2 -0

w2-20 PLAIN VEE ROLLER w2-22

W24/12- 1

ROLLER

I W244h2-1

W 2 -24

3.2 4-0 5.0

-26 W2 W2 - 27 W2 -28

1W2-27 W2 -28

w242-2

W24411 2-3

w w / l 2 -4

W /24 1

W/l22N

w2- 50

1.6 24

w2-22 W2-23

W2-23 k t I 2 -3N DRAWING NtW2-52.tSSUE N t z

PAGE 2 6

1.WELDMATIC 4

CIRCUIT IAGRAM D

PRACTICES

WELDING

EQUIPMENT
Produced by Welding I n d u s t r i e s o f A u s t r a l i a P t y. L t d. i n t h e interests o f improving0perato.rsafety. These notesshouldbeconsideredonlyas a basic guide t o S a f e Working Habits. A f u l l l i s t o fS t a n d a r d sp e r t a i n i n gt oi n d u s t r y i s a v a i l a b l e from t h e S t a n d a r d s A s s o c i a t i o n o f A u s t r a l i a , a l s o v a r i o u s S t a t e E l e c t r i c i t y A u t h o r i t i e s , DepartmentsofLabourand I n d u s t r y o r Mines Departmay have ment and other Local H e a l t h o r S a f e t y I n s p e c t i o n A u t h o r i t i e s a d d i t i o n a lr e q u i r e m e n t s.
1. A n e a t u n c l u t t e r e d work a r e a makes f o r s a f e working h a b i t s.

Burn P r e v e n t i o n

The w e l d i n ga r c i s i n t e n s e and v i s i b l y b r i g h t. I t s r a d i a t i o n can damage e y e s , penetratelightweightclothing,reflect from l i g h t - c o l o u r e d s u r f a c e s , andburn t h e s k i n and eyes.Skinburnsresembleacutesunburn,those from gas-shielded a r c s a r e more s e v e r e and p a i n f u l. and Wear p r o t e c t i v e c l o t h i n g - l e a t h e r ( o r a s b e s t o s ) g a u n t l e t g l o v e s , h a t , safety-toeboots.Buttonshirtcollar andpocketflaps,and wear c u f f l e s s trouserstoavoidentryofsparks and s l a g.

NEVER LOOK AT A N ARC WITHOUT PROTECTION.
Wear h e l m e t w i t h s a f e t y g o g g l e s or glasses with side shields underneath, a p p r o p r i a t ef i l t e rl e n s e s or p l a t e s( p r o t e c t e d by c l e a rc o v e rg l a s s ).T h i s i s a M S forweldingorcutting,(andchipping)toprotecttheeyes UT from r a d i a n te n e r g y and f l y i n gm e t a l.R e p l a c ec o v e rg l a s s when b r o k e n ,p i t t e d , o r spattered.
Avoid oily o rg r e a s yc l o t h i n g. A s p a r k may i g n i t e them. Hot metalsuchas he handled without gloves. e l e c t r o d e stubs andworkpiecesshouldnever Ear plugs should be worn when welding i n o v e r h e a d p o s i t i o n s o r i n a confined worn when o t h e r s work overhead. s p a c e. A hardhatshouldbe
Flammable hair preparations should not be used or c u t.
by p e r s o n s i n t e n d i n g t o w e l d
Toxic Fume P r e v e n t i o n
Adequate v e n t i l a t i o n i s e s s e n t i a l.S e v e r ed i s c o m f o r t ,i l l n e s so rd e a t hc a n r e s u l t fromfumes,vapors,heat,or oxygen e n r i c h m e n t o r d e p l e t i o n t h a t w e l d i n g (or c u t t i n g ) may produce. Prevent them w i t ha d e q u a t ev e n t i l a t i o n. NEVER v e n t i l a t e w i t h oxygen. Lead,cadium, z i n c , mercury,andberylliumbearingand similar m a t e r i a l s when welded ( o r c u t ) may produce harmful concentrations toxic of fumes. Adequate l o c a l e x h a u s t v e n t i l a t i o n must b e u s e d , o r e a c h p e r s o n i n t h e a r e a a s w e l l a s t h e o p e r a t o r must wear an a i r - s u p p l i e d r e s p i r a t o r. Forberyllium,both must be u s e d.M e t a l sc o a t e dw i t ho rc o n t a i n i n gm a t e r i a l st h a t emit fumes should i s removed from t h e work s u r f a c e , t h e a r e a is notbeheatedunlesscoating wellventilated,ortheoperator w e a r sa na i r - s u p p l i e dr e s p i r a t o r. Work i n a confinedspaceonlywhile while wearing air-supplied respirator.
it i s beingventilatedand,ifnecessary,
Vaporsfrom c h l o r i n a t e d s o l v e n t s c a n b e decomposed by t h e h e a t o f t h e a r c ( o r flame) t o form PHOSGENE, a h i g h l y t o x i c g a s , and lung' andeye i r r i t a t i n g p r o d u c t s. The u l t r a - v i o l e t( r a d i a n t )e n e r g yo ft h ea r cc a n a l s o decompose form phosgene. DO n o t WELD t r i c h l o r e t h y l e n e and p e r c h l o r e t h y l e n ev a p o r st o o r c u t where solvent vapors can be drawn i n t o t h e w e l d i n g or cutting atmosphere o r where t h e r a d i a n t e n e r g y can p e n e t r a t e t o a t m o s p h e r e s c o n t a i n i n g evenminute amounts o f t r i c h l o r e t h y l e n e o r p e r c h o l o r e t h y l e n e.

F i r e and Explosion Prevention
by t h ea r c ,f l a m e ,f l y Causes of f i r e andexplosionare:-Combustiblesreached ingsparks,hotslag,orheatedmaterial;misuse of compressed g a s e s and c y l i n d e r s ; and s h o r t c i r c u i t s. Be aware t h a t f l y i n g s p a r k s o r f a l l i n g s l a g canpassthroughcracks,alongpipes, ox f l o o r o p e n i n g s , o u t of s i g h t o f through windows o r d o o r s , andthroughwall t h eg o g g l e do p e r a t o r.S p a r k sa n ds l a gc a nf l y1 0m e t r e s.
To p r e v e n tf i r e s and explosions:Keep equipment c l e a n and o p e r a b l e ,f r e eo f o i l , g r e a s e , and ( i n e l e c t r i c a l p a r t s ) of m e t a l l i c p a r t i c l e s t h a t c a n c a u s e s h o r t circuits.
I f combustibles are i n a r e a , do NOT weld o r c u t. Move t h e work if p r a c t i c a b l e , t o a na r e af r e eo fc o m b u s t i b l e s. Avoid p a i n ts p r a y rooms, d i pt a n k s ,s t o r a g e areas, v e n t i l a t o r s. If t h e work can notbe moved, move combustibles a t l e a s t and h e a t ; o r p r o t e c t a g a i n s t i g n i t i o n w i t h 10 metres away o u t of reach of sparks suitable and snug-fitting fire-resistant covers or shields. Walls touching combustibles on o p p o s i t e s i d e s s b o u l d n o t b e welded on ( o r c u t ). W a l l s , c e i l i n g s , and f l o o r n e a r work should be protected by h e a t - r e s i s t a n t c o v e r s or shields. F i r e w a t c h e r m u s t be standing by w i t h s u i t a b l e f i r e e x t i n g u i s h i n g equipment if: d u r i n g a n d f o r some t i m e a f t e r w e l d i n g o r c u t t i n g
(1) c o m b u s t i b l e s i n c l u d i n g u i l d i n g o n s t r u c t i o n ) r e i t h i n ( b c a w LO metres. (11) c o m b u s t i b l e sa r ef u r t h e rt h a n 10 metresbutcanbeignited by s p a r k s. (111) openings(concealed or v i s i b l e ) i n floors o r w a l l s w i t h i n 10 metres may exposecombustiblestosparks. (1V) c o m b u s t i b l e sa d j a c e n tt o walls, c e i l i n g s ,r o o f s ,o rm e t a lp a r t i t i o n s c a n b e i g n i t e d by r a d i a n t ox conducted heat.

A f t e r work i s done,check

thatarea

is freeofsparks,
glowing embers, andflames.
when h e a t e d , must neverbewelded on o r c u t , u n l e s s c o n t a i n e r h a s f i r s t been c l e a n e d a sd e s c r i b e d i n AS.1674-1974, t h e S.A.A. C u t t i n g and Welding S a f e t y Code. T h i s i n c l u d e s : a thoroughsteam o r c a u s t i c c l e a n i n g ( o r a s o l v e n to rw a t e rw a s h i n g , depending on t h e c o m b u s t i b l e ' s s o l u b i l i t y ) f o l l o w e d b y ' p u r g i n g a n d i n e r t i n g w i t h n i t r o g e n o r carbondioxide,andusingprotectiveequipment as recommended i n working l e v e l may s u b s t i t u t e f o r i n e r t i n g. AS.1674-1974. W a t e r - f i l l i n g j u s t below Hollow c a s t i n g s o r c o n t a i n e r s must beventedbeforewelding orcutting. They. can explode. Never weld o r c u t where t h e a i r may c o n t a i n flammable d u s t , g a s , o r Liquidvaporssuchaspetrol). 5. Shock P r e v e n t i o n
An empty C o n t a i n e r t h a t hel-d combustibles, or can produce- flamable vapors
Exposed c o n d u c t o r s o r o t h e r b a r e m e t a l i n t h e w e l d i n g c i r c u i t , o r ungrounded e l e c t r i c a l l y a l i v e equipmentcan f a t a l l y shock a person whose bodybecomes a cond u c t o r.E n s u r et h a tt h e machine i s c o r r e c t l yc o n n e c t e d and e a r t h e d.I fu n s u r e On mobile o rp o r t a b l ee q u i p m e n t , havemachine i n s t a l l e d by a q u a l i f i e d e l e c t r i c i a n. r e g u l a r l yi n s p e c tc o n d i t i o n of t r a i l i n g power l e a d s andconnectingplugs.Repair o r r e p l a c e damaged l e a d s. 6. Electrode Holders Connectors and F u l l yi n s u l a t e de l e c t r o d eh o l d e r ss h o u l db eu s e d. s c r e w s.F u l l yi n s u l a t e dl o c k - t y p ec o n n e c t o r ss h o u l d lengths.
Do n o tu s eh o l d e r s

be used

w i t h protruding t o j o i nw e l d i n gc a b l e

Terminals knobs o r

Texminalsand o t h e r exposed p a r t s o f e l e c t r i c a l units should have insulated coverssecuredbeforeoperation.

RNA (1999), 5:13841395+ Cambridge University Press+ Printed in the USA+ Copyright 1999 RNA Society+
A sulfate pocket formed by three GoU pairs in the 0.97 resolution X-ray structure of a nonameric RNA
BENOT MASQUIDA, CLAUDE SAUTER, and ERIC WESTHOF
Institut de Biologie Molculaire et CellulaireCentre National de la Recherche Scientifique, UPR 9002 Structure des Macromolcules Biologiques et Mcanismes de Reconnaissance, 67084 Strasbourg, France
ABSTRACT The crystal structure of the RNA duplex [r(CGUGAUCG)dC] 2 has been solved at a resolution of 0.97. The model has been refined to R-work and R-free of 14.88% and 19.54% for 23,838 independent reflections. The base-pairing scheme forces the 59-rC to be excluded from the helix and to be disordered. In the crystals, the sequence promotes the formation of two GoU wobble pairs that cluster around a crystallographic threefold axis in two different ways. In the first contact type, the GoU pairs are exclusively surrounded by water molecules, whereas in the other contact type, the three amino groups of the guanine residues of the symmetry-related GoU pairs trap a sulfate ion. This work provides the first example of the interaction of a GoU pair with a sulfate ion in a helical context. Despite the negative charge on the polynucleotide backbone, the guanine amino N2 is able to attract negatively charged groups that could, in the folding of complex RNA molecules, belong to a negative phosphodiester group from a neighboring strand and, in a RNAprotein complex, to a negative carboxyl group of an aspartate or glutamate side chain. Keywords: GoU pair; sulfate ion; synchrotron; X-ray crystallography
INTRODUCTION Several structures of RNA helices containing GoU pairs have been obtained [for a review, see Auffinger & Westhof (1998)]+ The GoU pairs observed in crystals of helices are often accompanied by non-Watson Crick pairs, like U{U (Baeyens et al+, 1995) or U{C pairs (Holbrook et al+, 1991)+ G{A and A{A mismatches have also been observed (Baeyens et al+, 1996)+ Another kind of unexpected feature consists in the slippage in the 59 direction of one strand of the helix upon crystallization (Biswas & Sundaralingam, 1997)+ Interestingly, those crystal structures of RNA helices incorporating mismatches were obtained upon crystallization of sequences designed with the hope of observing tetraloops at high resolution+ Such small single-stranded hairpin motifs are in dynamic equilibrium with intermolecular double-stranded helices incorporating necessarily base mismatches+ Apparently, despite the noticeable thermal stability of the closing tetraloops
Reprint requests to: Eric Westhof, Institut de Biologie Molculaire et CellulaireCentre National de la Recherche Scientifique, UPR 9002 Structure des Macromolcules Biologiques et Mcanismes de Reconnaissance, 15 rue Ren Descartes, 67084 Strasbourg, France; e-mail: westhof@ibmc+u-strasbg+fr+
(Antao & Tinoco, 1992), crystal assembly and packing drive the equilibrium toward double-stranded helices and non-WatsonCrick base pairing instead of hairpin motifs+ A rearrangement of the base-pair scheme due to the slippage of one strand in the 59 direction led to the formation of a WatsonCrick paired double helix with two GoU pairs in the RNA structure presented in this paper+ Crystals were obtained while attempting to crystallize the 4-nt sequence 59-UGAU-39, responsible for the most determining structural feature of the 39untranslated region of eukaryotic selenoprotein messengers (Walczak et al+, 1996, 1998)+ The crystal structure of the resulting nonameric RNA 59-rCGUGAUCGdC has been solved and refined at a resolution of 0+97 + GoU pairs have a prominent role in RNARNA recognition [e+g+, the wobble interaction in the anticodon codon triplets (Crick, 1966)] and in RNAprotein complex formation [e+g+, tRNAAla (Park et al+, 1989)]+ Besides, in some catalytic RNA [group I intron (Cech et al+, 1981) or the hepatitis delta virus ribozyme (Sharmeen et al+, 1988)], a GoU pair is present at the cleavage site+ For group I introns, specific contacts in the shallow groove of the GoU pair have been proposed (Michel & Westhof, 1990) and experimentally determined (Strobel &

backbone interactions taking place between symmetryrelated duplexes (see below)+ At the step between two stacked duplexes, the twist angle is low (16+958), as observed in the two crystal structures of 8-bp duplexes solved in R3 (Wahl et al+, 1996; Biswas et al+, 1997)+ Strikingly, the 59-rC is not observed in the electronic density; however, its presence has been assessed by mass spectrometry (see Materials & Methods)+ Moreover, the phosphate group of G2 and G11 are clearly seen indicating that the 59-cytidine is not lost+ Alkaline hydrolysis would have produced 59-hydroxyl G instead of phosphorylated ones+ The 59-cytidine is thus disordered (Fig+ 2A)+ Each wobble pair participates in the packing by clustering around the threefold axis in two distinct manners+ In the first contact (G17oU3), the N2 amino group of the G contacts one oxygen atom of a sulfate ion+ The sulfur atom is located on the threefold axis, but is not oriented so as to satisfy the threefold symmetry, that is, with an oxygen atom along the axis+ In fact, two oxygen atoms belong to the plane perpendicular to the threefold axis with the remaining oxygen atoms placed above and below the plane at 1+2 of the axis+ Three conformers of the sulfate are, thus, present on the threefold axis (Fig+ 2b)+ The sulfate ion is bonded to three water molecules (W103, W105; W145, not located in
the plane of the GoU pair, is only depicted in Fig+ 3A)+ Packing interactions are strengthened through van der Waals contacts between the ribose moieties of the threefold related GoU pairs (Fig+ 3A)+ In the second contact (G8oU12), the three symmetry-related 29-OH groups of G point towards a water molecule located on the threefold axis (Fig+ 3B)+ The relation of G8 to the threefold axis results in the location of U12 at a remote distance from the crystallographic axis+ As a consequence of the packing, two sulfate ions are positioned on the same crystallographic axis only in noncontiguous layers of RNA duplexes (Fig+ 4)+ Three types of RNARNA packing contacts are observed (Fig+ 5A)+ The 29-hydroxyl group of A5 forms one hydrogen bond with the O1P atom of G4 in a symmetry-related duplex+ The same type of contact is observed between the 29-hydroxyl residue G11 and the O1P atom of residue dC18 of another symmetry-related duplex+ A third type involves the 29-hydroxyl group of G17 with the N2 atom of G2+ Hydration of the duplex The duplex is hydrated by 84 clearly identified water molecules+ Among these, seven are located at special positions with an occupancy of one third+ Sixteen other

FIGURE 3. Side-by-side stereo drawings of symmetry-related GoU pairs that cluster in a propeller twist fashion in two different ways around the threefold axis+ A: The sulfate ion located on the axis is trapped by three GoU pairs that contact directly the ion with the N2 amino group of the G residues+ Three water molecules also interact directly with the ion+ Symmetry operations raise the number of hydrogen bonds from four in the asymmetric unit to 12+ It is worth noting that the riboses of the GoU pairs perform van der Waals contacts with riboses of the symmetry-related GoU pairs+ B: A water molecule located on the threefold axis interacts with the 29-OH groups of the three symmetry-related G residues participating in the GoU pair+ The U residues are thus located far away from the axis and face the shallow groove of another duplex+ Only the water molecules and the nucleotides from the asymmetric unit are labeled for clarity+ All hydrogen bonds drawn imply a distance between heavy atoms of between 2+3 and 3+5 +
solvent molecules are linked with intermolecular distances below 2+4 and occupancies of 0+50+ W174 interacts with U3, which is involved in one of the GoU pairs trapping the sulfate ion+ The close proximity of the very well-defined sites W103 and W105W174 indicates that the latter has a low occupancy (0+20) coupled to the occupancies of W103 (0+95) and W105 (0+85) (Fig+ 3A)+ Seventy-eight hydration sites belong to the first hydration shell and six to the second (W111, W154,
W162, W168, W176, and W182)+ An average of 10+5 water molecules per base-pair step is observed, in agreement with other high-resolution RNA structures (for reviews, see Egli et al+, 1996; Auffinger & Westhof, 1998; Masquida & Westhof, 1999)+ Four water molecules (W101, W104, W123, and W133), because of their location on the threefold axis, are bound to oxygen atoms of three symmetry-related duplexes simultaneously+ This occurs for the 29-OH of
in addition to the N2 atom of G17 (Fig+ 3A)+ Those three water molecules form a hydration cone centered around the S-O2 bond of the sulfate ion+ W103 bridges the N2 atom of G17 to the 29-OH group of a symmetry-related G17+ W105 is bound to the 29-OH of U3 and contacts the N3 atom of a symmetry-related G17+ W145 does not contact tightly any RNA atom of the pocket (the distance W145(O29(G4) is 3+21 ), but is close to the sulfate ion (the distance W145 + + + O4(SO4 ) is 2+47 )+ All three water molecules interact with some other water molecules and RNA groups in the shallow groove+ Some differences in the resulting hydration patterns of the shallow grooves of GoU pairs are, however, worth noting+ In the sulfate trap, the water site linking the amino group of the G to the O2 and O29 atoms of the U (W174) has an occupancy of 0+20+ This site, described as characteristic of GoU pairs, is thus underpopulated in the GoU pair surrounding the sulfate ion+ Although the hydration patterns of the GoU of the sulfate trap diverge from the hydration pattern canonically observed for GoU pairs, the network of water molecules in the region around the second GoU pair (G8oU12) is in agreement with previously reported structures (Auffinger & Westhof, 1998; Mueller et al+, 1999)+

In this work, we describe the structure of a nonameric RNA obtained at 0+97 of resolution and containing six WatsonCrick and two GoU wobble pairs+ The latter are formed in two distinct structural contexts+ Whereas one of them is hydrated as previously observed (for review, see Auffinger & Westhof, 1998), the second one constitutes the first reported RNA-binding site for a sulfate ion in a helical context+ Protein structures in which a sulfate ion is bound show that a protein pocket can hold a sulfate ion solely by a set of hydrogen bonds (often with side chains of Asn residues), as in the sulfatebinding protein of Salmonella typhimurium (Pflugrath & Quiocho, 1985) or in the satellite of the tobacco mosaic virus crystal structure (Larson et al+, 1998)+ The hydrogen-bonding network is sometimes reinforced by several salt bridges, as in the human theta class glutathione transferase (Rossjohn et al+, 1998)+ The network of solvent molecules in contact with the sulfate ion could be water molecules or ammonium ions+ However, in the present case, the observed number of 12 hydrogen bonds is consistent with a coordination of the ion purely formed by hydrogen-bond donors such as amino groups or water molecules+ The basis for the biological role of GoU pairs has been emphasized in the crystal structure of the P4P6 domain of the group I intron of Tetrahymena (Cate et al+, 1996)+ The deep groove of tandem GoU pairs binds magnesium ions that can be replaced under special conditions by a variety of metal ions like osmiumor cobalt-hexamine (Cate & Doudna, 1996)+ Here, we present a crystal structure in which a sulfate dianion interacts in the shallow groove of three GoU pairs, clustered around the crystallographic axis+ Although the presence of the sulfate ion is obviously due to the crystallization conditions, its ability to bind to GoU pairs is somehow puzzling+ An octameric RNA (Portmann et al+, 1995) has been recently crystallized in conditions containing ammonium sulfate+ The space group (R32) together with the lattice parameters make the
FIGURE 6. Side-by-side stereo representation of the hydration of the deep groove of the duplex+ Three to five water molecules interact with the deep groove sites of each pair+
TABLE 2+ Distances between non-hydrogen RNA atoms (A) or sulfate ion (B) and water molecules+ The O1P and O2P atoms correspond to OS and OR oxygens of the phosphate groups, respectively+ The symmetry operation is not indicated when both heavy atoms belong to the same asymmetric unit+
A+ Distances between non-hydrogen RNA atoms and water molecules Strand 1 (Residue) atom type Water number Distance () (Residue) atom type Strand 2 Water number Distance ()

Symmetry operation

(G2) O2P 2(G2) O5T

W181 W163

2+96 2+35
(G11) O5T (G11) N2 (G11) N3 (G11) O6

(G2) N3 (G2) O6

1 y, x y, z
W106 W120 W130 W124 W137 W166 W155 W174 W149 W124 W120 W114 W105 W174 W131 W173 W171 W146 W155 W108 W114 W149 W134 W145 W101 W108 W145 W148 W133 W118 W119 W131 W173 W161 W156 W119 W173 W148 W184 W147 W140 W116 W157 W129 W167 W147 W184 W135 W142 W113
2+91 3+08 2+96 2+88 2+84 2+81 3+33 2+95 3+32 3+47 3+03 2+69 2+74 2+88 3+00 2+89 2+80 2+73 2+69 2+84 2+80 2+72 2+87 3+21 2+86 2+69 3+21 3+14 2+59 2+64 2+85 3+24 2+55 3+24 2+91 2+92 3+29 2+84 2+72 2+82 2+66 2+80 2+84 2+71 2+73 2+92 3+40 2+85 3+20 2+54
y x 2/3, x 1/3, z 1/3 x 1/3, y 1/3, z 1/3 x 1/3, y 1/3, z 1/3 y x 2/3, x 1/3, z 1/3 x 1/3, y 1/3, z 1/3

W110 W106 W106 W130

2+68 2+91 3+49 3+01
(G2) N7 (G2) O29 (U3) O1P (U3) O2P (U3) O2 (U3) O4

(G11) N7 (G11) O29

W175 W110

2+84 2+84

(U12) O2 (U12) O4

(U3) O29

(U12) O29

W109 W160 W183 W144 W109

3+29 3+33 2+68 3+25 2+76

(G4) O1P

(G4) O2P (G4) (G4) (G4) (G4) (G4) N3 N2 O6 N7 O29
(G13) O2P (G13) N3 (G13) O6 (G13) N7 (G13) O29
W122 W180 W126 W144 W122 W159
3+02 2+57 2+93 2+74 2+85 2+71

1 y x, 1 x, z

(A5) O1P (A5) O2P (A5) N3 (A5) N6 (A5) N7 (A5) O29 (U6) O1P (U6) O2P (U6) O2 (U6) O4 (U6) O29 (C7) O2P
1 y x, 1 x, z 1 y, x y, z
(A14) O2P (A14) N3 (A14) N6 (A14) N7 (A14) O29 (U15) O1P (U15) O2P (U15) O2 (U15) O4
W180 W115 W172 W138 W115 W102 W107 W127 W112 W136 W116 W152
3+09 2+97 2+95 2+74 2+82 2+79 2+80 2+89 2+87 2+77 3+37 2+41
1 y x, 1 x, z 1 y x, 1 x, z
(C16) O2P (C16) O2 (C16) N4 (C16) O29 (G17) O1P

(C7) N4 (G8) O1P

W139 W169 W121 W164 W121 W125
2+53 2+75 3+10 3+46 2+73 2+77

continued

TABLE 2+ Continued
A+ Distances between non-hydrogen RNA atoms and water molecules (continued) Strand 1 (Residue) atom type Water number Distance () (Residue) atom type Strand 2 Water number Distance ()

(G8) O2P (G8) N3 (G8) N2

(G8) O6 (G8) N7 (G8) O29 (C9) O1P
W178 W141 W170 W109 W126 W170 W160 W143 W123 W158 W153
3+13 2+74 3+06 2+87 3+15 2+67 2+62 2+50 2+78 2+53 2+70
(G17) O2P (G17) N3 (G17) Ny x, 1 x, z 1 y, x y, z (G17) O6 (G17) N7 (G17) O29 (C18) O1P (C18) O2P 1 y x, 1 x, z
W128 W105 W174 W103 W120 W117 W179 W174 W103 W106 W150 W128 W125 W150 W103 W117 W132 W104 W110
2+95 2+92 2+60 3+10 3+07 2+77 3+41 3+48 2+68 2+82 2+87 2+92 2+81 3+39 2+80 3+49 2+86 2+66 2+73

(C9) O2P

y x 2/3, x 1/3, z 1/3

W181 W165

3+39 2+71 2+99 (C18) O2 (C18) N4 (C18) Oy, x y, z

(C9) N4

B+ Distances between sulfate ion and water molecules
(SO419) O1 (SO419) Oy, x y, z 1 y x, 1 x, z (SO419) O3 (SO419) Oy, x y, z 1 y x, 1 x, z 1 y, x y, z 1 y x, 1 x, z
W103 W105 W145 W103 W174 W103 W105 W145 W145 W145
2+84 3+20 3+05 2+71 3+40 2+97 3+30 3+39 2+47 2+83
packing in both structures very similar+ The region corresponding to the cluster of GoU pairs trapping the sulfate ion in the present structure is replaced in the related structure by a cluster of three GC pairs with the accompanying network of water molecules (Fig+ 7)+ The comparison between these structures clearly indicates that the N2 groups of the G residues are closer to the threefold axis when belonging to a GoU pair (3+6 ) than when belonging to a GC pair (4+9 )+ The resulting narrowing of the hydration channel could be invoked to enable the trapping of the ion+ Since sulfate and phosphate are structural analogs, one can expect that guanine amino groups could intervene in the folding of complex RNA molecules through binding of a phosphodiester in the shallow groove of helices+ Such an interaction has been observed in a recent crystal structure of an RNA pseudoknot (Su et al+, 1999)+ The O2P atom of a phosphate group from a single-strand junction interacts with the G amino group of a GC
pair in exactly the same fashion as the sulfate ion does with the GoU pair in the present structure (Fig+ 8)+ The topology of the pseudoknot forces a single strand to pass in the shallow groove of a helix and leads to a contact between a phosphate with the N2 group of a G+ Thus, the guanine N2 positions seem to constitute favorable and specific binding sites for negatively charged groups+ More generally, it seems reasonable to suggest that negatively charged amino acid side chains such as Asp or Glu could interact with the amino group of guanines to mimic the described interaction+ GoU pairs isolated in a WatsonCrick helix adopt characteristic twist values with the preceding and following base pairs in such a way that the step 59 of the U is overtwisted (compared to the usual 338 value) and the step 39 of the U undertwisted+ We have therefore compared the values in our structure to values in two other high-resolution crystal structures of RNA duplexes containing GoU pairs (Lietzke et al+, 1996; Shi

FIGURE 7. Comparison between the crystal structures of (A) r(CCCCGGGG)2 (Portmann et al+, 1995) and (B) the one of the present work+ The regions of the packing where ribose rings of symmetry-related duplexes are interacting through van der Waals contacts are depicted+ The narrowing of the hydration channel leading to the binding of the sulfate ion to the amino groups of the guanines is clearly seen+ Distances between C19 atoms and between amino groups of symmetry-related residues as well as the distance of the amino group to the crystallographic axis are indicated in angstroms+
et al+, 1999)+ We have also refined ab initio models of the duplexes depicted in the crystal structures with NUCLIN/NUCLSQ (Westhof et al+, 1985) to check if the observed trend was purely geometric or due to stacking (and possibly to crystal packing)+ The values presented in Figure 9 show that the effect of the packing is negligible, as average values of crystal and modeled structures are very similar+ The same sets of values and trends are obtained upon least-squares geometrical and stereochemical refinement of standard RNA helices (Arnott et al+, 1973)+ Thus, the 59 high-U-low 39 twist alternation has essentially a geometric origin stemming from the nonisosteric nature of GoU pairs+ On the basis of a crystal structure containing one GoU pair, Mueller et al+ (1999) concluded that there is no recognizable structural distortion of the helical fragment away from the wobble pair+ In Figure 10, we have superimposed the C19 atoms of a GoU and a UoG pair at the base of two helical fragments to illustrate the propagation of the high versus low twist angles+ Deviations up to 2 between the two helices can be observed 5 bp away from the two nonisosteric wobble pairs+ In Escherichia coli tRNAAla , there is a large difference in aminoacylation efficiency between a G3oU70 and a U3oG70 pair (McClain et al+, 1988; Musier-Forsyth et al+, 1991; Frugier & Schimmel, 1997), leading to the suggestion that the protruding orientation of the 2-amino
group is important+ As illustrated in Figure 10, if a synthetase locks in the GoU pair via the guanine amino group, a reversal of the GoU pair into a UoG could reorient the -CCA 39 end in a sufficiently off-track direction to hamper aminoacylation+ Finally, the present crystal structure illustrates the context dependence of small RNA motif consisting of noncanonical base pairs+ The NMR determinations of two fragments of the crystallized P4P6 domain (Cate et al+, 1996) present two examples of local structural rearrangements; one in the tetraloop receptor, the internal J6a/b loop (Butcher et al+, 1997), and one in loop L5c (Wu & Tinoco, 1998)+ Interestingly, in both cases, phylogenetic analyses, which reflect the structurally related functional constraints on sequences, identified the correct junctions linking the secondary structure elements (Michel et al+, 1982; Cech et al+, 1994)+ MATERIALS AND METHODS Synthesis and purification

The nonamer was produced at a 5 mmol scale using phosphoramidite chemical synthesis on an automated DNA/RNA synthesizer (Applied Biosystems, model 392) with a specially dedicated program (Oubridge et al+, 1994; Kyoshi Nagai, pers+ comm+)+ A dC residue was added at the 39 end to promote
FIGURE 8. A: View of the interaction between the sulfate ion and the N2 group of the G residue involved in the GoU pairing in the present crystal structure+ B: The possibility for amino groups of guanines to interact with negatively charged groups is exemplified by the crystal structure of a frameshifting RNA pseudoknot (Su et al+, 1999) in which an N2 group of a guanine of a GC pair interacts with the phosphate group of the backbone of the single strand topologically forced to pass in the shallow groove of the helix+
butylammonium fluoride (TBAF) 1+0 M in THF, which reacted during 24 h to complete the deprotection of the 29-hydroxyls+ At the end of the reaction, 40 mL of butanol and 0+3 M sodium acetate were added to the solution and stored at 20 8C+ The pellet was separated from the liquid phase and dissolved in 20 mL of Millipore water+ The quality of the synthesis was assessed by anion-exchange HPLC (Nucleopac-PA-100; DIONEX) using a salt gradient with solutions A (1 mM NaClO4 , 20 mM MES, pH 6+2, 4 M urea) and B (400 mM NaClO 4 , 20 mM MES, pH 6+2, 4 M urea) with 1570% of solution A over 48 min+ Finally, 200 ODs of RNA were loaded on the column to perform a preparative scale purification+ The fractions containing the product of correct length were desalted by gel filtration (NAP 25 Pharmacia prepacked column) and evaporated to dryness+
Crystallization and data collection
FIGURE 9. Twist values (8, standard deviations indicated by parentheses) at different UoG steps in various WatsonCrick contexts, computed with the program CURVES (Lavery & Sklenar, 1989)+ The first set of values (left column) is calculated with crystal structures containing isolated UoG pairs: (1) Shi et al+, 1999, (2) the present work, (3) Lietzke et al+, 1996+ The values (second column) stay in the same ranges when computed from ab initio models with the corresponding sequences refined geometrically with NUCLIN/NUCLSQ (Westhof et al+, 1985)+ (4) The twist value is thus higher on the 59 side than on the 39 side of the U of the UoG+
intermolecular contacts between symmetry-related molecules as observed elsewhere (Cruse et al+, 1994)+ yet, the presence of the 39 dC promoted the slippage of one strand in the 59 direction, resulting in a significant stabilization of the canonical base-pair scheme away from the mismatched one+ The RNA was then cleaved from the support and deprotected directly in the column by flowing 8 mL of a mixture of NH4OH/ MeOH (3:1) at regular time intervals during 24 h at room temperature+ The solution was filtrated and evaporated to dryness in a speedvac concentrator+ Millipore water (250 mL) was added to the pellet to help it dissolve in 8 mL of tetra-

Prior to crystallization, the RNA was renatured at a concentration of 10 mg/mL in 5 mM MgCl2 and 10 mM sodium cacodylate, pH 6+0, by heating during 10 min at 85 8C followed by slow cooling to room temperature+ One volume of this solution was then mixed with one volume of the reservoir solution (2+22+6 M ammonium sulfate, 550 mM magnesium sulfate, 50 mM sodium cacodylate, pH 6+0, and 1 mM spermine) on a cover-slip and sealed with vacuum grease on the wells of a Linbro box+ Crystals, observed after several days, typically looked like extruded hexagons of regular or irregular geometry with size ranging from 100 to 400 mm+ Four data sets were subsequently collected under cryogenic conditions (110 K), using three different crystals+ The first two were collected on a Enraf-Nonius rotating copper anode source operating at 45 kV and 100 mA, coupled to a MacScience detector (dip 2,000), to 1+83 and 1+57 resolution, respectively+ Data sets were collected to 99+1% and 99+8% completeness, merging to Rsym 3+6% and 3+8%, respectively+ A majority of the reflections (.95%) presented I/s ratios greater than 10 even in the last resolution shells+ The third data set, collected on beamline DW32 at the Laboratoire pour lUtilisation du Rayonnement lectromagntique (LURE) synchrotron facility using a Mar Research detector (mar 345 cm), was collected to 98+9% completeness at 1+12 (Rsym 3+5%)+ The distance crystal detector was set to 120 mm with l 0+92 + The fourth data set was collected on beamline ID14-EH4 at the European Synchrotron Radiation Facility (ESRF) (l 0+934 )+ The decentering of the CCD camera towards the beam enabled the collection of data to 0+92 of resolution, but data were complete enough only to 0+97 + Data were processed with the HKL package (Otwinowski & Minor, 1996) in space group R3 for the four crystals+ To get a complete data set at high resolution, medium resolution passes were merged with the high resolution passes of the ESRF+ The resulting lattice parameters are a b 39+958 , c 67+445 , g 1208+ The data set consists of 23,838 unique reflections representing a completeness of 99+8% (last shell 99+9%) and the Rsym is 4+1% (last shell 44+6%)+
FIGURE 10. Side-by-side stereo representation of two regular WatsonCrick duplexes containing a GoU or a UoG pair (bottom)+ The C19 atoms of the riboses involved in the wobble pairs have been superimposed to emphasize the relative deviation of the backbone coordinates+
Structure solution and refinement
The structure was solved by the molecular replacement method using the program AMoRe (Navaza, 1994) with the
data set collected to 1+83 (a b 39+90 , c 67+21 , a b 908; g 1208)+ The refinement was completed with the highest resolution data set+ We started by searching both strands of a model of the four mismatches exhibited by the three-dimensional model of the SECIS element sandwiched between two CG or GC pairs on each side (Walczak et al+, 1996)+ Between 8+0 and 3+5 , several solutions were found with correlation factors around 37% and R-factors around 48%+ We thought that the backbone distortion in the mismatched region was impeding the molecular replacement process and decided to search two copies of the four 59 base pairs to give sufficient leeway at the step in the middle of the eight base-pair duplex+ This significantly raised the correlation to 52% and lowered the R-factor to 44%+ At this time, no modification of the search parameters could improve the solution+ The search for two WatsonCrick helices of four random base pairs generated with the program NAHELIX (Westhof, 1993) was then performed as a negative control+ Strikingly, the statistics indicated that it was the best solution, with correlation of 66+5% and R-factor of 38+3% in a packing showing no bad contacts+ A rigid-body refinement of a model with correct sequence was performed using CNS (Brnger et al+, 1998) and a 3Fo 2Fc map was calculated to inspect the positions of base pairs and phosphate groups at high contour level (3+0 sigma units)+ All phosphates could be superimposed to a given peak in the map, meaning that the structure of the backbone was basically solved+ We then carefully inspected the packing generated from the molecular replacement solution at the step between the two halves of the model+ This step adopted a very low twist value, indicating we were considering the junction between two symmetry-related duplexes instead of the step between the two halves of the model+ The duplex was then screwed 4 bp along the helical axis to place the apical base pair at the correct step and a new refinement performed, as described above+ The decrease observed both for R (33+1%) and R-free (38+6%) was in agreement with the hypothesis+ The subsequent inspection of a 2FoFc map indicated that the structure of the backbone was solved whereas the structure of the sequence was wrong+ Because omit maps clearly indicated that the 39 residue was of the deoxyribose type, we deduced that dC9 and dC18 were, unexpectedly, involved in base pairing+ The base-pair scheme could be rearranged by sliding 2 bp in the 59 direction and the formation of six WatsonCrick and two wobble pairs, resulting in the dangling of the 59-rC+ The model was annealed in CNS, and 41 water molecules were added, decreasing R to 26+6% and R-free to 26+2%+ The inspection of a 2Fo-Fc map showed the sequence was right but the dangling 59-rC could not be seen, meaning it was disordered or hydrolyzed+ This question was investigated by MALDI-TOF spectrometry experiments under conditions described elsewhere (Lecchi et al+, 1995)+ The spectra performed with the starting RNA solution exhibit peaks corresponding to the correct nonamer product+ A washed and subsequently dissolved crystal yielded peaks corresponding to the correct-length product as well as a 59-C-pruned product with a 59-OH end+ Because the correct product is synthesized, hydrolysis could intervene during the renaturation process, resulting in the loss of the phosphate group of the first G of the sequence+ yet, the removal of the phosphate results in the increase of both R and R-free while its presence in the model contributes correctly to the maps+

The structure was refined to 1+12 and then to 0+97 with SHELX (Sheldrick & Schneider, 1997) without any s cut-off for reflections+ R-free was composed of 5% of the reflections+ R values are communicated for 21,657 reflections with intensity over 4 s+ The refinement started with the naked RNA and yielded an R-factor of 22+51% and an R-free of 25+11%+ The identification of a disordered sulfate ion on the threefold axis decreased R and R free to 21+29 and 23+92%, respectively+ The addition of 44 water molecules decreased R to 17+57% and R-free to 21+05%+ At this stage, R and R-free could be reduced to 16+85% and 20+19% by an anisotropic description of the B factors+ The 40 remaining water molecules were added step by step to the model and minor adjustments, including occupancy check of water molecules, led to an R-factor of 14+66% and an R-free of 19+31%+ A final cycle of refinement with the 5% of reflections composing the R-free was performed+ The final R-factor is 14+81%+ The coordinates and the reflection data have been deposited in the Nucleic Acid Database (NDB AR0019)+

Computer programs

Model building and electron density map inspections were performed with the O graphical system (Jones et al+, 1991)+ Pictures were prepared with SETOR (Evans, 1993), and DRAWNA (Massire et al+, 1994)+

ACKNOWLEDGMENTS

B+M+ was supported by a Bourse-Docteur-Ingnieur CNRS/ Rhne-Poulenc-Rorer and a Human Frontier Science Program grant (to E+W+)+ C+S+ acknowledges a grant from the Ministre de lenseignement suprieur et de la recherche+ Special thanks are due to the Agence Nationale pour la Recherche sur le SIDA (ANRS) for financial help towards the cost of the in-house X-ray generator and detector (grant to Bernard Ehresmann)+ We are most grateful to P+ Dumas for the use of the diffractometer+ We acknowledge S+ McSweeney and colleagues at ESRF and R+ Fourme and the team at LURE for their help during data collection+
Received June 4, 1999; returned for revision July 15, 1999; revised manuscript received July 26, 1999

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