Structure of rabbit muscle aldolase at low resolution

THEJOURNAL OF BIOLOGICAL CHEMISTRY

Vol. 260, No. 28, Issue of December 5, pp. 15286-15290.1985 Printed in U.S.A.

0 1985 by The American Society of Biological Chemists, Inc.

Structure of Rabbit Muscle Aldolaseat Low Resolution* (Received for publication, December 24, 1984)

J. Sygusch, H. Boulet, and D. Beaudry From the Departement de Biochimie, Faculte de Medecine, Universite de Sherbrooke, Fleurimnt, Quebec, Canada J l H 5N4

X-ray diffractiop datawere measured by x-ray dif- been completely determined by amino acid sequencing (4, 13, fractometry to 5-A resolution for both the monoclinic 14) and confirmed by nucleotide sequencing (15) have been form of rabbit skeletal muscle aldolase (EC 4.1.2.13) carried out at various times (6-7). These structural studies, and a platinumderivative. The heavy atom difference however, did not result in a model for a three-dimensional patterson was solved at 6-A resolution yielding eight structure of the enzyme. distinct heavy atom sites. Choice was made of the enRecently it has been shown that themonoclinic form of the antiomorph and protein phases were calculated on the rabbit enzyme is kinetically active in thecrystalline state (16). basis of single isomorphous replacement differences. The catalytic cycle of the rabbit muscle enzyme, a type I The electron densitymap calculated from these phases aldolase, proceeds through two Schiff base intermediates was averagedaccordingtothenoncrystallographic molecular symmetry. Rotational symmetry analysis of which have been isolated (3). The structural study of rabbit native patterson andsite symmetry analysis of refined muscle aldolase offers a possibility of detailed investigation heavy atom positions are consistent with the aldolase at the molecular level of the catalytic mechanism of the tetramer possessing very a high degreeof 222 internal enzyme. In a preliminary step we wish to report on aspects of symmetry. The subunits in the tetramer are positioned the three-dimensional structure of rabbit muscle aldolase at inatetrahedralconfigurationdisplayinga slight low resolution. square planar deformation. Each subunit is roughly ellipsoidal in shape with the majoraxis nearly parallel EXPERIMENTAL PROCEDURES~ to a local2-fold axis. Prominent at the surface of each subunit were structural features resembling a helices. RESULTS Each subunit contributes to its boundary surface at A portion of the noncrystallographic syqmetry averaged least six helices which are arranged in a barrel-like manner and possessing a right handed twist with re- electron density map representing a 14-A thick section spect to each other. Density associated with binding of through the rabbit muscle aldolase tetramer is shown in Fig. substrate on the enzyme was located on the surface of 1. The electron density map was of sufficient quantity such that molecular and subunit boundaries were readily traced in eachsubunit.Cooperativeaspects of theconformational changes produced upon substrate binding are most parts on the map. The over911dimension of the tetramer discussed. is approximately 90 x 90 x 65 A along the local dyad directions with the shortest dimension roughly parallel to the crystallographic 2-fold screw axis. The subunits associate in Fructose-1,6-diphosphatealdolase (EC 4.1.2.13) is an ubiq- a tetrahedral-like configuration when viewed along the local uitous glycolytic enzyme which catalyzes the reversible aldol dyad nearly parallel to the crystallographic screw axis. The cleavage of fructose 1,6-diphosphate to glyceraldehyde3-phos- shape of each subunit is best charatterized by a spherical phateand dihydroxyacetone phosphate. The enzyme isa ellipsoid (dimensions 40 X 45 X 50 A) whose major axis is tetramer with a molecular weight of 158,000and in vertebrates slightly inclined with respect to the previous local dyad axis exists in three isozyme forms (1).In rabbit tissues, aldolase nearly parallel to the screw axis. Each subunit contacts preA has been isolated from muscle, aldolase B from liver, and dominantly only two of its nearest subunits. Contacts between aldolase C from brain. The three forms have been purified to subunits relatedby the local %fold axis nearly parallel to the homogeneity and extensively characterized (1-3). The iso- crystallographic screw axis appear to be fewer bycomparison. zymes can be readily distinguished in terms of their kinetic It is possible to trace within each subunit long stretches of and electrophoretic properties (1-3). Comparison of amino density which become difficult to follow only at the surface acid sequences of muscle and liver aldolases suggests an early of the tetramer. In particular, at theboundary surface of each subunitare discernable long thick rod shaped densities evolutionary divergence from a common ancestral gene (4). Aldolase A which is present in large amounts in muscle suggestive of a helices. Six of these a helices make up part of tissue, about 5% of the total soluble protein and which is a barrel-like structure at the subunit boundary with each a equal in amount to actin ( 5 ) , has been crystallized in many helix being twisted in a right handed manner with respect to different forms (6-12). Of the various aldolase A crystallized, Portions of this paper (including “Experimental Procedures,” an only human muscle aldolase has been studied by x-ray crystallography at low resolution (8). Crystallographic investiga- additional Fig. 1, and Tables 1-3) are presented in miniprint at the tions of the rabbit muscle enzyme whose protein sequence has end of this paper. The abbreviation used is: SIR, single isomorphous replacement. Miniprint is easily read with the aid of a standard

* This

investigation was supported by Grant MA-8088 from the Medical Research Council of Canada. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solelyto indicate this fact.

magnifying glass. Full size photocopies are available from the Journal of Biological Chemistry, 9650 Rockville Pike, Bethesda, MD 20814. Request Document No. 84M-3886, cite the authors, and include a check or money order for $4.40 per set of photocopies. Full size photocopies are also included in the microfilm edition of the Journal that is available from Waverly Press.

15286

Structure of Aldolase at Low Resolution

FIG. 1. Electron density map at 5-A resolution of rabbit

skeletal muscle aldolase averaged according to the noncrystallographic 222 symmetry of the aldolase tetramer. The orientation of the 14-A thick section corresponds to a view down the molecular 2-fold axis nearly parallel to the crystallographic 2-fold screw axis. The intersection of this local 2-fold with the plane of the section is indicated 0. The horizontal direction corresponds to the direction of the local dyad parallel to the crystallographic c* axis. A tracing of the boundary of one subunit is indicated by a thick continuous line. Prominent density features resembling CY helices forming a barrel-like structure are indicated by arrows. The tentative location of the active site near one of the assigned helices is shown

@. its neighbor. Contacts between subunits aremediated in part by this barrel-like structure. The electron density distribution is consistent with the binding of the heavy atom derivative which appears to bind to features on the surface of the subunits near their interface (Pt 1 sites) or in a crevice along the local dyad parallel to c* (Pt 2 site). The region of intersection of the local dyads is free of density and in only six instances are small regions of density shared between subunits contacting along the local 2folds. To identify the active site on the enzyme, the intensity data measured for the crystalline enzyme covalently inactivated by its cleavage product, ~-glyceraldehyde-3-P,was employed for analysis. A difference Fourier map averaged according to the noncrystallographic symmetry was computed for the same grid spacings as the native Fourier and is shown in Fig. 2. The coordinates of the strongest peak on the noncrystallographic symmetry averaged difference Fourier corresponded to a position on the surface of the tetramer distantfrom the subunit contacts and located close to a feature of density previously associated with an helix on the native electron density map (see Fig. 1). This site is readily accessible to solvent in the crystals. Although this site is not strongly discriminated from the remainder of the peaks found on the difference Fourier it is the only site among the firstsix peaks which does not map a t or in the vicinity of the subunit interface. The peaks which mapped near subunit interfaces were for the majority part of rod shaped density featuresthat

15287

FIG. 2. An electron densitydifference map at 8-Aresolution averaged according to the noncrystallographic 222 symmetry and comparing the glyceraldehyde-3-P inactivatedenzyme to the unmodified enzyme. The orientation and cross-section of the difference electron density map as well as features showing the boundary of a monomer and the intersection of the molecular dyad axis with the plane of the map section are the same as for the native electron density map shown in Fig. 1. The density feature tentatively associated with substrate binding to theactive site locus @ is shown in the outlined monomer. Positive density is contoured by a continuous line whereas negative density is contoured by a dotted line.

could be associated with an equivalent negative density features in the map and suggestive perhaps of movement of a helices upon reaction of ~-glyceraldehyde-3-Pwith the enzyme. DISCUSSION

The interpretation of the electron density map at 5-A resolution suggests a packing of the aldolase subunits corresponding to a compact tetrahedral or pseudotetrahedral spatial arrangement of subunits that has been proposed from observations of electron micrographs of aldolase (1).Each subunit in the electron density map is in close contact with only two neighboring subunits related through the local 2fold axes that are oriented nearly parallel to the crystallographic a and c* axis, respectively. The dimensivns for the aldolase tetramer of approximately 90 X 90 X 65 A along the local symmetry axes are consistant with the packing of the tetramer in the crystal lattice with the reported unit cell dimensions. The structure resembles that of the low resolution structure of aldolase from human muscle (8) also a tetramer but in this case the molecular symmetry coincided with crystallographic symmetry. Both forms of aldolase, rabbit, and human muscle, react with Pt (11)series compounds. Comparison of the heavy atom positions between the two structures suggest that they do not bind at the same sites with respect to the internalmolecular symmetry. Nevertheless the disposition of the Pt (11) sites with respect to the molecular symmetry is similar. The molecular symmetry averaged electron density map was of sufficient quality that many features of continuous

15288


density could easily be traced to the surface of the molecule before difficulty in connectivity would be encountered. Notable among the structural features were densities resembling barrel-like supersecondary structures of CY helices at the subunit boundaries reminiscent of similar right handed twist barrel-like structuresof CY helices in triose isomerase, pyruvate kinase, and 2-keto-deoxy-6-phosphogluconate aldolase (17). This barrel-like structure of six a! helices is not inconsistent with the prediction of a /3a/3a!/3/3a!Ba!/3structureinrabbit muscle aldolase (18). It is interesting to note that the great majority of barrel-like CY helices structures have a righthanded twist whereas if the incorrect enantiomorph had been chosen this structural motif in aldolase would have possessed a left handed twist. The correct assignment of the catalytic site at the surface of the tetramer issuggested by the fact that monomers of the aldolase tetramer retain up to 50% of their catalytic activity upon dissociation (19). Had the position of the active site corresponded to one of the sites which map at or in the vicinity of the subunit interfaces considerable a loss of activity could be predicted upon dissociation of the tetramer. Additionally, the Pt (11) sites which are located at the subunit interfaces do not influence catalytic activity of the aldolase crystals. The accessibility of the proposed active site to solvent is consistent both with the absence of apparent steric hindrances at theactive site locus from the electron density map and with the kinetics of aldolase crystals which are comparable with solution kinetics (16). Similarly, affinity elution of the enzyme from CM-52 ion exchanger with Fru-P2 substrate is not inconsistent with the location of the active site locus at the surface of the enzyme and accessible to solvent. The precise location of the active site locus on the native enzyme, however, cannot be assigned with certainty on the basis of the computed difference electron density map since covalent reaction of glyceraldehyde-3-P with the enzyme results in substantial conformational changes throughout the aldolase monomer (see Fig. 2). Aldolase from rabbit muscle when reacted with p-chloromercuribenzoate also crystallizes in monoclinic space group P21 (6), however, the latticeparametersare significantly different from crystallized native aldolase such that themercury-reacted aldolase crystals are not isomorphous. We have observed on occasion especially if the quality of the native crystals ispoor, i.e. large intensity profiles, upon reacting the native crystalswith Pt (11)series compounds produces lattice parameter changes resulting in unit cell dimensions identical with those of the p-chloromercuribenzoate-reacted aldolase crystals. This lattice parameter transition can be characterized by a rotation of the aldolase tetramer which brings a local molecular dyad axisintobetteralignment with the crystallographic screw axis. This interpretation follows from the magnitude of a pseudo-origin peak present on the Harker section of the Patterson map and resulting from interaction of a local molecular dyad axis with the crystallographic screw axis. The height of this peak which represents a measure of the degree of mutual parallelism of the two rotation axes approaches 70% of the theoretical value in p-chloromercuribenzoate aldolase (6), whereas in native aldolase this peak height corresponds to only 10% of the ideal value. In addition to the rotational mobility exhibited by the tetramer in the crystalline state, covalent reaction of aldolase with ~-glyceraldehyde-a-P at the active site locus induces substantial conformational changes in each of the aldolase subunits. Conformational mobility in the crystalline state is essential for catalytic activity and thus entirely expected in the vicinity of the active site. Structural changes associated

with binding at the active site but distant from it such as possible movement of helices at thesubunit interfacesreflects a conformational fluctuation that probably the entire subunit must undergo to accommodate D-glyceraldehyde-3-Pbinding at the active site. The fact that the crystals remain isomorphous and do not crack upon substrate binding to the active site suggests that the tetramer does not undergo a major volume change even though each subunit demonstrates substantial structural changes especially at the subunit interfaces. Structural changes at subunit interfaces usually suggests evidence of cooperativity between adjacent subunits. All aldolase kinetics including that of inactivation by D-glyceraldehyde-3-P, however, do not display any evidence of cooperativity between subunits. An explanation consistent with all of the above observations is that each subunit undergoes a conformational fluctuation such as possibly a rotation in a manner consistent with the internal 222 symmetry of the tetramer that is stabilized upon binding to a single subunit. Kinetics under these circumstances will not display cooperativity. It is not possible to rule out that each subunit reacts independently of the other subunits. This possibility, however, is less probable since aldolase is a practically nondissociable tetrameric enzyme (20) suggesting a very tight association of the aldolase subunits at their interfaces. Conformational changes consequently are expected to be cooperative between subunits when conformational changes occur at subunit interfaces. REFERENCES 1. Penhoet, E., Kochman, M., Valentine, R., and Rutter, W. J. (1967) Biochemistry 6, 2940-2949 2. Penhoet, E., Rajkumar, T., and Rutter, W. J. (1966) Proc. Natl. Acad. Sci. U. S. A . 56, 1275-1282 3. Horecker, B. L., Tsolas, O., and Lai, C. Y. (1972) The Enzymes (Boyer, P. D., ed) Vol.7, pp. 213-258, Academic Press, New York 4. Benfield, P. A., Forcina, B. G., Gibbons, I., and Perham, R.N: (1979) Biochem. J. 183,429-444 5. Penhoet, E., Kochman, M., and Rutter, W. J. (1969) Biochemistry 8,4396-4402 6. Eagles, P. A. M., Johnson, L. N., Joynson, M. A., McMurray, C. H.. and Gutfreund. H. (1969)J. Mol. Biol. 45,533-544 7. Heidner, E. G., Weber, B. H.,.and Eisenberg, D. (1971) Science 171,677-680 8. Miller, J. R., Shaw, P. J., Stammers, D. K., and Watson, W. C. Lond. Biol. Sci. 293, 209-214 (1981) Philos. Trans. R. SOC. 9. Sawyer, L. (1972) J. Mol. Biol. 71,503-505 1'0. Weismam, L. (1979) Ph.D. dissertation, University of California 11. Goryunov, A. I., Andreyeva, N. S., and Shpitsberg, V.L. (1969) Biofizika 14,1116-1117 12. Brenner-Holzach, O., and Smit, J. D.G. (1982) J. Biol. Chem. 257,11747-11749 13. Lai, C. Y., Nakai, N., and Chang, D. (1974) Science 183, 12041206 14. Sajgo, M., and Hajos, C. (1974) Acta Biochem. Bwphys. Acad. Sci. Hung. 9,239-241 15. Tolan, D.R., Amsden, A. B., Putney, S. D., Urdea, M. S., and Penhoet, E. E. (1984) J. Biol. Chem. 259, 1127-1131 16. Sygusch, J., and Beaudry, D. (1984) J . Biol. Chem. 259, 1022210227 17. Richardson, J. S. (1979) Biochem.Biophys.Res. Conmun. 90, 285-290 18. Stellwagen, E. (1976) J. Mol. Biol. 106,903-911 19. Rudolph, R., Westhof, E., and Jaenicke, R. (1977) FEBS Lett. 73,204-206 20. Ovadi, J., Salerno, C., Keleti, T., and Fasella, P. (1978) Eur. J. Biochem. 90, 499-503 21. Sygusch, J., Lehoux, J. C., and Beaudry, D. (1984) Bwchem. Biophys. Res. Commun. 123,1069-1075 22. Fletterick, R. J., Sygusch, J., Murray, N., Madsen, N.B., and Johnson, L. N. (1976) J. Mol. Biol. 103,l-13 23. Reeke, G. N. (1984) J. Appl. Cryst. 17, 125-130


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24. Crowthers, R. A. (1972) T h e MolecularReplacementMethod (Rossman, M. G., ed) pp. 174-178, Gordon and Breach, New York 25. Rossmann, M. G., and Blow, D. M. (1962) Acta Cryst. 15,24-31 26. van Schaik, E. J. M., Schutter, W. G., Gaykema, W. P. J., Schepman, A. M. H., and Hol, W. G. J. (1982) J. Mol. Biol. 158,457-485 27. Sygusch, J. (1985) Acta Cryst., in press 28. Bricogne, G. (1976) Acta Cryst. Sect. A Cryst. Phys. Dqfr. Theor. Gen. Crystallogr. 32, 794-802

29. Matthews, B. W. (1966) Acta Cryst. 20, 230-239 30. Lai, C. Y.,Martinez-de Dretz, G., Bacila, M., Marinello, E., and Horecker, B. L. (1968) Biochem. Biophys. Res. Commun. 30, 665-672 31. Sygusch, J., and Beaudry, D. (1985) Biophys.Biochem.Res. Commun. 128,417-423 32. Reeke, G. N. (1984) ROCKS Crystallographic Computing System, pp. 26, Rockefeller University, New York 33. Rollett, J. S. (ed) (1965) Computing Methods in Crystallography, pp. 35-37, Pergamon Press, Oxford

S u p p l e m e n t a r y Material t o S t r u c t u r e of R a b b i t M u s c l e A l d o l a s e a t Law Resolution

RottatJonfunction calculauon. Rotation functions were calculated wing the fast rotation function of Cmwther (24). intsgratron olume for the Patterson functron was taken a8 a sphere wrrh a r a d i s varyrngfrom 23.3AJo 55.Oi. Theresolutionlimitsutilized 1" the calculationr varied between 50.OA lower h i t and 5.OA upper h i t . The c u t off for weakreflecttons was also varred.Whenever posrlble the resolution limm and cut-off for weak reflections ,were chosen vrch that largest number of reflections contributed per calculation subject to the constrants imposed by memory sire hmrtations of therotatien program. Variation in theintensity cut-off lefttherotationfunction results virtuallymaltered. Eulerian cowdinater of the maxima of thefastrotatmnfunction were tmwfarmedforinterpretationinto the sphericalparameters of Rossman & Blow (25) as derived prev~ourly(26).

J . Sygusch, H . B o u l e t ,

and 0. B e a u d r y

Cr stalllratmn. Crystals of rabbrt muscle aldolase were grown by precrprtatlon in ammonlum sulfate mltmns a t Doc accordingto a modrflcatmn (16) of the crystall~zatlon COndltmnS arlglnally described (6). Aldolase Used forcryrtallrzatlon war extherpurchasedfromBoehrrnger Mannhelm Ltd. or It was p u r h e d by affmlty elution on a CM52 ion exchanger (21). Crystallmeplaterappeared wrrhm sevecal daysand grew to 0 . 3 ~ 1 . 5 ~ 2mm fmal dzmenmons w t h m 2 weeks. The crystals were mvarlablytwinnedaboutthecryrtallographrc a ax!% Untwrnned vngle cryrtalS Were obtaned by Cuttlng the twlnned crystals wLth a d of a polarlrlng mcrorope t o an average plate drmenrron of 0.6xO.b mm. Verlflcarron thatthe cut unglecrystal$ were mdeedentwinned was carriedout by vannrng characteriS1reflect onr an a Iffractometer. The q a c e group of thecrystalsappeared to be P2 wlfh a=Ib4.0# b=57.78r, c=SS.Lfand E = 102.7". Theasymmetric unn Contams one tetramer or f o u r subunits of approxlmately molecular werght 40000 daltons each. Equrpment; Ddfractedmtenrrlier were measured on an Enraf-Nonius CAD-4 d i f f r a c t m e t e r modifled The x-ray source was a Rtgak2-Denki Ru-200 rotating a n d e generator for piatern datacollectmn. operated at RKV and 53mA with a focal spot s z e of 900 p , take off msle 6', and CuK radiation. To acccomdate the physical constrarntr Imposed by x-ray generator, the CAD4 diffractoameter war mountedupside down inmde a rigrdmetal cage. TheCuradlation was moncchromatued by parrage through two focusvng 6cm Nlsoated glass mirror% % ' e reflected x-ray beam was f o c u s e dt o produce maximum crossfireatthecrystal position. Profileanalys~s of thereflectedinadent x-ray beam usmg a 200 p test crystal yielded a good ht when nngle gauruan fmctlons were employed to model the beam c r o r r f r e in each of the 2 6 and X planer at the crystal position. The halfwuldthr were comparable wlthm expermental error for the two drrectmns having valuer of t h e a d e r of 2OOp. Dlffractmeter Alignemem.A O . h m Ni wcre mounted on a gonmmeterhead was employed for allgment. The N i wire was carefully centered optically and then centered until t h e rhadow produced by the w r e in the x-ray beam was stationary a~ function of The dlffractometer was then d q l a c e d on tranrlatron sledges in the 2 e planeuntil the Ni wire, positionedvemcally, split the incident x-ray beam and its shadow corncidd with the positmn of the vertical slit in the recelvmg detector aperture at the 2 0 zero poution. To ensure that the Ni coated wire was properly centered m the x-ray beam the abave test had t o be satisfiedforboth a 0 . h m and hnm colltmator.The dlffractometer was then displaced by screws m the vertical direction prpendrcular to the 2 B plane until t h e N1 wire IX)W posrtioned horizontally rplrt the tnddent x-ray beam and the shadow of the Ni wlre cornclded with thehorirmtaldetector sht. Again the test had t o be %tisfredforboth collrmaton. Thr procedure oar iterated to stability. Routinely one cycle of adjustment was sufficient. The diffractometer cage was thenblocked by a series of rct x ~ e w sto dinimlze mvement about thecentered position during data cOlleCtmn. A final checkwar then carried out using the alignment procedure suggested by the diffractmeter manufacturer. The average

+.

discrepancies were less then .OH0 between equivalent reflections at + and -28, and less than 0.0150 on 0 forthe Jame equivalentreflectionsand less than 0.02' far x a t t h e + a = position. Durmg m a f u r e ~ n tt h ed i f f r a c t m e t e r moved less than 25p horuontally and less lop v?rtrcally aboutIts mean position.The average change m allgnmntbefore and afterdata collection, for fypxally 2500 d a t a measurements, was less than mu.

- 9 0 '

w s a n was rrred for intenuty measurement using I.4mm horizontal Datameanlrement.AContmuws and 2mm vertical detector apertures. The average peak scan width war 0.25O MU. The Y P ~profiles were symmetrrc and crystals exlbifing arymctric profiler and/or =an widths in excess of0.35O on w were rejectedfordata collecuon. Maximum a total of 12840 reflectronr were measured to 4.5A resolution from 5 differentnativecrystals. Atypicaldatasetconsisted of 2500 uniquereflections measured aver a 2 day period.

Heavyatom parameter and phrefinement. Initially, a difference Patterson at b x Rrolution of the platinum derivative MIcalculated in sectionsperpendiculartothe baxis. The twolargestpeaks 111 the Harker section, Figure I. The position of the Second largest peak coincidedwith the D O I L T L O ~of a larse oeak in the native Patterson. This a a k has been mteroreted as the result of the $esence of a l~al'molecular dyadparallel t o the cryhallograpic ~ r e wdyad axis (6). Both the shape and height of the strongest peak lying close to the oripn of the Harker section suggested that tll peak was probably due to result of mme fortituour interaction of the cross vectors of the heavy atompontronr.Otherthanrhese two peaks virtudly all of thestrongpeaks in the Patterson Were concentratedaboutthe X Y plane. Four strmg wellrounded p a k r were observed in the Harker sectLon lying more t o t h e X Y plane (Figure I). Assuming thatthestrongest peak 1" the Harker sites, m s t the sectionrepresented a fortrtuousinteractton among the four potentialheavyatom strong peaks in the Patterson CQUM be explained. Several of the remaining strong p e a k s were elongated in the Y direcnon suggesting perhapsthat at 6A resolution additionalheavyatomsites were mresolved.Evidence [or theresites could be found on the HarkeT sectionaboutthe strong peakresulting from the presence of a molecular dyadparallel tl) twofoldcrystallographic screw axis (Figure 1). Rdlnement of theoccupanciesandcoordinates of the heavyatom Sites as Well aS protein phases war carried out using ROCKS crystallographic camputing system (23). The temperature factors were held fired at l 5 f ' throughoutthe refinements.The 8 heavyatom sites were first The fact that agreement residualsvariedsharply reflned with data collected mly to 6Aresolution. Y a fmction of re lution aboutthetranstion from data measured in the 8 A data xt and data measured in the 6 data set, the two d a t a o t r a e r e refinedseparately. The Centric R factor plummeted by 0.10 for each of the data sets. The 5A hell derivative data ret was added to the data alm refined separately.AtthispointtheROCKScomputingsystem was modified to baseand constrainlikeheavyatomsites of eachderivativetothe Lame v a l u during t h e course of t h e refinement.Refrnement of all parameters using fuU normal equatlonf matnx was continued untll all heavy atom shifts were less then 0.5 standard deviatronr and the average cosine of the phase change from one cycle to the next was greaterthan 0.995. At theend of therefinementthe heavy atom sites could be divided into two groups m the basts of their accupancier. Occupancieswith m each group varied by less than 15%, e largestvariation berng in the group whoseheavyatom positions are mt totally resolved a t 5 2 resolutron. Contrary to current practice it was possible to discrimmare the absolute configumm of a protein on the basis of a angle isomorphousderivative (27).

were located

Site symmetry analyur. Utes was analyzed according to the symmetry The qatial relationship of the heavyatom was based upon least squares aperations defmed by point group 222. The site symmetryanalysis rmnimization of the followin(? sum 5.

h t a reduction. Accurate centering of the c ~ y s t din the direct beam is necessary when t h e d i r e t beam dimenuom and crystal &menman$ are comparable. Slight miventering can prodwe inequalittes among equrvalent reflections. Transmisum a w e analyrlrfurnisher only correction formircentering errors m Wrtical plane. Centering errws m the 2eplane are thus not corrected by the transmmston

curve. Transmission Curve measurement for a Conventtonal diffractometeraboutthe x ;OD posltion are not fcauble. The mly information thw available C I M S fromanalyrss of equivalentreflectnons. AMIYW w h c h depend upon differences among equivalent reflectmns in the case of space group pz are not rigorous since any even cosine and ane fmctron used for analysis of the transmission cur,! highly correlates w t h even trigonometricterms of the Same orderemployed in the misormntatron analysis. The following transmisscan factor was thusemployed m the care of a plate-hkealdolase crystal. ,

c&

=

Cl'0S

Txzs0 = &(an >d