The multi-wire proportional chamber as an area detector for protein crystallography in comparison with photographic film; Guidelines for future development of area detectors

NUCLEAR

INSTRI.;MENTS

AND

METHODS

!52 (1978)

205-208

, ©

NORTH-HOLLAND

PUBLISHING

CO

THE MULTI-WIRE PROPORTIONAL CHAMBER AS AN AREA DETECTOR FOR PROTEIN CRYSTALLOGRAPHY IN COMPARISON WITH PHOTOGRAPHIC FILM; GUIDELINES FOR FUTURE DEVELOPMENT OF AREA DETECTORS G E O R G 1:. SCIa.ULZ

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29. 69 IIcMelherg. German).

G E R D P,O S E N B A UM

E,,ropean :$f(,le:'.,h," B,G/o.~;' Labo,ator~. .'~!~:;:er/:o/s/, . 6 ~) Ite~delbcrg. Germapc

l-he multlwlre proporlmm.[ c h a m b e r is c o m p a r e d to p h o t o g r a p h i c film for protein crystallography W i t h t h e electromc de. rector, t h e a c q m r e d mlormalJO.'- per p h o t o n mcbdem on t h e s p e c : ) r e n is h~gher and a m u c h better tm~e resolution can be achieved F u t u r ~ dc'~elopmen,.~ of s u c h detectors s h o u l d plov~ctc a better spatial r e s o l u n o n and m u c h higher c o u n t i n g rates /

1. Introduction Protein crystallography has now b e c o m e a classical m e t h o d with not m u c h incentive for those w h o hke fascinating mett:odolog~cal d e v e l o p m e n t s But In contrast to m o s t fascinating new m e t h o d s it works and ~t c o n t i n u e s to put Noiogy on a m o r e and m o r e solid molecular basis Up to now structure analyses were pretty m u c h restr,cted to the m o s t stable protein crysta,s But s~.ablhty borders death, ~ e. the solved [3|OtelLS are ~lot the m o s t interesting ones for a life science, they were m o s t l y e n z y m e s working with small suostra~cs which do not h a v e to u n d e r g o large mtnns~c m o v e m e n t s In the future m o r e laNle proteins h a v e to be tackled M o r e o v e r , o n e ~s interested in g e m n g a reasonable t i m e resolution so that biological a c n v m e s can be followed in the crystalline state T h e r e f o r e , ~t is w o r t h w h d e to i m p r o v e the presently available tools Th~s d e v e l o p m e n t should go '.nto two d~rect,ons 1) i m p r o v e m e n t o f the acqmred r e f o r m a t i o n per photon incident on the s p e o m e n , 2) i m p r o v e m e n t o f the n i n e r e s o l u u o n For both purposes a multi-wire proportional c o u n t e r ( M W P C ) c o m p a r e s favorably with photographic film P h o t o g r a p h i c film is a c o n v e n i e n t , cheap and fairly efficient m e d i u m o f recording and s t o n n g Xray diffraction data In contrast an M W P C with associate electronics is a c o m p l e x and e x p e n s i v e m a c h m e Similar to m o s t electronic p h o t o n c o u n t ing s y s t e m s the a d v a n t a g e s o f the M W P C are - very low intrinsic noise b a c k g r o u n d ,

- i m m e d i a t e read-out o f intensities for any poslt~on In digitized f o r m a t , - very fast sw:tching from one f r a m e to the next, -- energy resolution T h e a d v a n t a g e s of p h o t o g r a p h i c film are - very high spatial r e s o l u n o n , - acceptance of ver.~, h~gi; p h o t o n fluxes For stat;c protein crystallography intrinsic backgro~.md, spat.al reso~ut;on and m a x i m u m tolerable p h o t o n flux are the p a r a m e t e r s that d e t e r m i n e the u s e f u l n e s s of the detector in practice For d y n a m ical protein crystallography (as for all d y n a m ' c a l Xray e x p e r i m e n t s ) fast access to the m t e n s a y data and fast switching characteristics m a k e the M W P C an u n n v a l e d detector

2. Acquired information per photon hitting the specimen T h e acquired l n l o r m a t l o n ~s the accuracy of the intensity m e a s u r e m e n t s of all reflections at a given r c s o l u n o n W e use 3 A as the reference resolution To c o m p a r e the i n f o r m a t i o n acquired with film and M W P C we wdl first c o m p a r e the accuracy of a single reflection m e a s u r e m e n t and subs e q u e n t l y consider the m e a s u r e m e n t of all reflections within the reference resolution A typical accuracy is 3% P h o t o g r a p h i c film has a fairly high intrinsic b a c k g r o u n d ( - - c h e m i c a l fog) o f a b o u t 0 15 O D In order to obtain an accuracy of 3% with film d e n s l t o m e t r y the a v e r a g e optical density a b o v e b a c k g r o u n d o f all recorded reflecn u n s m u s t be a r o u n d 0 3 0 D , i n d e p e n d e n t o f spot size T o c o n v e r t t h e s e values to p h o t o n intensities VI

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incident on the film we used the average o f the conversion factors d e t e r m i n e d by M o n m o t o and Uyeda ~) and H a r m s e n et al. 2) o f 0 . 8 3 × 10 6 phot o n s / m m 2 for 1 . 0 0 D T h e results are g w e n m table 1 In an M W P C with ~ts low lntrms~c background ( = c l e c t r o m c noise and spurious s i g n a l s ) 3 % accuracy ts obtained w~th 1000 detected p h o t o n s (Polsson stat~sucs) Using x e n o n as c o u n t i n g gas the el'fic~ency o f the M W P C ~s about 5096 T h u s 3% accuracy reqmres 2000 photons m a d e n t on the detector, i n d e p e n d e n t o f spot stze T h e results in table 1 show that the comparison between film and M W P C depends cmttcally on the spot stze As a spot o f 0 3 × 0 3 m m 2 ~s about the smallest size that can be obtained eastly, the MWPC y~elds 11 times more mformauon per photon m a reflection than film However, thts superiority Js d~mm~shed when one constders the number of reflecuons whtch can be recorded s~multaneously Using CuK~ radiation the 3 A cone occupies 0.85 sr A film of 115× 115 mm 2 at a d~stance of 100 mm from the crystal accepts 1009,; of th~s cone and it spatmlly resolves all reflections of a normal protein crystal (all axes around or below 100 A) In contrast the MWPC designed by Xuong, which we take as worked example, could only record part of this cone for a crystal of hydrofolate reductase with axes 9 3 × 9 3 x 7 5 A 3 ~) W~th ~ts absolute spatial resolutton of 2 mm a crystal-detector distance of 500 m m was ncccssary to resolve the reflections At this d~stance the detector area of 290×290mm: corresponds to a sohd angle of 0 3 4 s r , ~e 40% o f the 3 A cone. T h e r e f o r e , the resulting a d v a n t a g e of a X u o n g - t y p e M W P C over film at 3 A resoluuon reduces to 1 1 x 4 0 % / 100% = 4 5 w~th respect to photons hlttmg the specimen, ) e to crystal hfe t)me. For 2 5 A resolution th~s factor reduces to 3.

3. Acquired information per real time As each gram o f a film Js a separate detector there ts practically no saturation effect as long as the beam s h u t t e r can be activated fast e n o u g h T h e u m e o f a m e a s u r e m e n t lS only h m n e d by the intensity of the X-ray sources T h e s e m t e n s m e s can be very high m the case o f electron synchrotrons or storage rings 2) In contrast an M W P C ~s a single c o u n t e r and the c o u n t i n g rate ts hm~ted by the electromcs that d e t e r m i n e s the p o s m o n s o f the recorded photons. For protein crystals the

G

ROSENBALIM

TABLE 1

Spot s,ze (nlnl 2)

Photons incident on film for a reflecnon of 0 3 OD

0 2 ×o 2 03×03 04xO4

10000 22 500 40 000

Photon ratio film/dctector for 3% accuracy

5 11 20

hm~t is not g w e n by the reflecuon lntens~ues but by the unavoidable background radlauon Background m t e n s m e s m expertmental set-ups adjusted for low background are stated m table 2 T h e s e b a c k g r o u n d mtens~Ues have to be compared with the reflection m t c n s m e s . In the g~ven example 4) about 80 reflecuons are s~multaneously recorded m the M W P C , l e m 40% of the 3 A cone Accordingly, about 200 reflections are s~multaneously recorded m the 3 A cone In the dfffract o m e t e r set up one would expect an average countmg rate of about 50 p h o t o n s / s e c for a reflecuon, ~e a total rate of 10 000 p h o t o n s / s e c m the reflecuons at any u m e , which ~s only 10% of the background rate This value agrees reasonably w~th the value of 1076 reported by U Arndt (private c o m m u m c a u o n ) a n d the value o f 20";, g w e n m ref 4 The X u o n g - t y p e M W P C accepts a rate of up to 30 kHz At this rate there is a r a n d o m electromc loss of 17% due to dead t~me m the electromcs

TABLE2 Experimental set-up

Total background of 3 A cone (photons/scc)

I)lffractometer source 0 4 x 8 nlm 2 , 40 kV, 25 mA, Nl-filter s o u r c e - c r y s w l dtstancc 350 m m beam d m m e t e r at cr.~stal 1 mm crystal s)zc (0 5 m m ) 3

100 (L~O

Oscfllat,on camera source 0 2 × 2 r a m 2.

40 kV, 40 nlA, m~rror-mtrror focussing crystal stze ( 0 4 ram) 3

250 (300

MUI.II-V¢IRE

PROPORTIONAL

CHAMBER

In those M W P C s where the posluon of the photons ~s derived from the delay t;me o f pulses travelhng on external delay hnes, h~gh c o u n t rates not only cause dead t~me loss but also g~ve rise to false position decoding, tf a second p h o t o n hits the c o u n t e r before the pulses from the first have reached the ends o f the delay line If the real pattern consisted of only two strong reflections the d,splay o f a pattern recorded at a high rate would show an a d d m o n a l streak between the reflections If the pattcrn consisted o f a uniform background the false positions would be randomly distributed and would not change the pattern T h e latter descr,bes the normal s~tuat~on m protein crystallography, whereas the f o r m e r may occur ,n specml d y n a m i c e x p e r i m e n t s where large parts of the background may be obscured by absorbers m order to record only an interesting small part of the pattern A rate of 30 kHz corresponds to 70 000 incident p h o t o n s / s e e when absorpuon efficiency ( = 5 0 % ) and electronic loss arc accounted for Therefore, the X u o n g M W P C could not possibly accept the full 3 ,~ cone ~n the a r r a n g e m e n t s g w e n m table 2 As it can a n y h o w accept only 40% o f the 3 cone lbr resoluuon reasons, the X u o n g - t y p e M W P C would be fast e n o u g h for the dd'frac~omcter beam (table 2) but a factor of 1 5 too slow for the oscdlat~on camera beam (t~ble 2) In conclusion we find that for protein crystallography m u c h higher c o u n t i n g frequencies are required to use even conventional X-ray sources efficiently The examples o f table 2 apply by far not to the strongest c o n v e n t i o n a l beams available 2) S y n c h r o t r o n radiation would be a waste If we do not consider the time elapsed during a m e a s u r e m e n t but the time r e s o l u u o n one can achieve, the M W P C ts quite superior to photographic film Even to record a pattern of less than 3 / k resolution, a dozen different films have to be exposed m an osc,llat~on camera m order to avo,d reflecuon overlap As a f, lm change requires about a second the m i n i m u m t~me scale a m o u n t s to about 10see In contrast the p~cture frame of an M W P C can be changed m m~lhseconds, leading to a m u c h smaller t , m e scale For a given accuracy r e q u , r e m e n t th~s time scale is only limited by the m a x i m u m c o u n t i n g rate of the M W P C Moreover, in d y n a m i c m e a s u r e m e n t s often only part of the pattern has to be recorded (e g ~f reflection pos~u o n s arc known), so that m u c h stronger X-ray sources can be applied

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DEI-ECTOR

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4. Guidelines for development 1)

Improvement o/ reso/utwn

T h e i m p r o v e m e n t o f the rat,o o f mformat~on per photon incident on the specimen, that means saving of crystal hfe t~me, can only be e n h a n c e d ,f the n u m b e r e f separable p,cture e l e m e n t s on the detector area ts increased Th,s can be. dor, e e~ther by increasing the size o f thc detector or by dccreastag the absolute resolut,on Xuong reports that he can resolve 150x 150 picture elements m practice In the given example -~) th~s suffices to resolve the reflection w~thln 40% of the 3/~ cone So one should a,m at 3 0 0 x 3 0 0 resolvable p,cture elements m order to record the full 3 A cone T h e information per photon ,nc~dent on the spec,men would then be aga,n about 11 t,mes better than with film Presumably the M W P C developed by Gabr,el will finally fulfill these requirements S) 2)

Impro~,ement o/" speed

Any , m p r o v e m e n t of detector speed does not ~ncrease the mlbrmat~on per photon me,dent on the spcc~men In other words, ,t does not saxae crystal hfe time but only crystallographers" tmae For normal prote~,l crystals this is not the major problem H o w e v e r , speed becomes most essentml tbr d y n a m i c m e a s u r e m e n t s as described above T h e slow part of an M W P C is the t~me to pulse he,ght to digital conversion procedure One way to c , r c u m v e n t this ,s the m e t h o d of Charpak Calculating the center of gravity o f the reduced charge l¥om the s~gnal o f a n u m b e r of individual cathode electrodes T h e other way ~s to use a fast clock and counters for d,rect t~me to d,g,tal conversion We have developed an electromc stop watch for 5 to 256 nsec using a c o u n t e r working at 700 MHz W e also have designed a check electromc to detect false decoding W~th these two m~provements counting rates o f 1 Mllz should be possible Th~s counting rate ~s approximately the background rate mid the 2 5/k cone us,ng a br,ght X-ray tube, e g an Elhott GX13 rotating anode S y n c h r o t r o n rad m u o n , which ~s one or two orders o f m a g m t u d e more intense2), would totally flood e v e n this M W P C in the case of an ordinary s~ze protein crystal

We thank K C H o l m e s and J Hendr,eks for d~scusslons and W. Kabsch for providing us w~th an oscillatten photograph to calculate the background value o f table 2 ~,1

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S C H U L Z AND G

References i) 11 Morm-loto and R Uyeda (1963), Acta Cryst

16 (1963)

1107 2) A Harmsen, R Leberman and G E Schulz, J Mol Blol 104 (1976) 311 3) D A Matthews, R A Alden. J T Bohn, S T Freer, R Harnhn, N Xuong, J Kraut. M Poe, M Wdhams and K

ROSENBAUM

Hoogsten Sctence 197 (1977) 452 4) R Hamhn, Ph D Thes~s, Umverslty of Cahforma, San Dtego, 1975, Xerox University M,crofilms No 75-29. Ann Arbor, MJch 48106. U S A , p 439 5) A Gabnel, F Dauvergne and G Rosenbaum, th,s tssue, I: 191