Andy A. Freer,$ Gerry McDermott,*$ Neil GuthrieJ Neil Isaacs,$ J. Gordon Lindsayt and Miroslav Papizs .... Evans, M. B., Cogdell, R. J. and Britton, G. (1988).
Photosynthesis and Photosynthetic Membrane Systems
3. Rees, D. C., Komiya, H., Yeates, T. O., Allen, J. P. and Feher, G. (1989) Annu. Rev. Biochem. 58,607-633 4. Komiya, H., Yeates, T. O., Rees, D. C., Allen, J. P. and Feher, G. (1988) Proc. Natl. Acad. Sci. USA. 85, 9012-90 16 5. Smith, E. L. (1968) Harvey Lect. 62,231-256 6. Chothia, C. and Lesk, A. M. (1986) EMBO J. 5, 823-826 7. Eisenberg, D., Weiss, R. M. and Tenvilliger, T. C. (1984) Proc. Natl. Acad. Sci. USA. 81, 140-144 8. Cornette, J. L., Cease, K. B., Margalit, H., Spouge, J. L., Berzofsky, J. A. and DeLisi, C. (1987) J. Mol. Biol. 195,659-685 9. Overington,J. P., Johnson, M. S., Sali, A. and Blundell, T. L. (1990) Proc. R. SOC.London B. 241, 132-145 10. Overington, - J., Donnelly, D., Johnson, M. J., $ah, A. and Blundell,T. L. (1992) Protein Sci. 1,216-226 11. Donnelly, D., Overington, J. P., Ruffle, S. V., Nugent,
J. H. A. and Blundell, T. L. (1992) Protein Sci., in the
press 12. Deisenhofer, J., Epp, O., Miki, K., Huber, R. and Michel, H. (1985) Nature (London) 318,618-624 13. Sali, A. and Blundell, T. L. (1990) J. Mol. Biol. 212, 403-428 14. Bleasby. A. J. and Wootton, J. C. (1990) Protein Eng. 3, 153-159 15. Lee, €3. and Richards, F. M. (1971) J. Mol. Biol. 55, 379-400 16. Hubbard. T. J. P. and Blundell, T. I,. (1987) Protein Eng. 1, 159-171 17. Henderson, R., Baldwin, J. M., Ceska, T. A., Zemlin, F., Beckmann, E. and Downing, K. H. (1990) J. Mol. Biol. 213,899-929
Received 25 September 1992
A progress report on the crystallographic studies on the B800-850 antenna complex from Rhodopseudornonas acidophila strain I 0050 Richard 1. Cogdell,* Evelyn Halloren,* Anna M. Hawthornthwaite,$ Marjolein M. G . M. Thunnissen,S Andy A. Freer,$ Gerry McDermott,*$ Neil GuthrieJ Neil Isaacs,$ J. Gordon Lindsayt and Miroslav Papizs Departments of Botany," Biochemistryt and Chemistry,$ University of Glasgow, Glasgow G 12 8QQ and SERC Daresbury Laboratory,§Warri ngton, Cheshire
Summary Large single crystals (up to 1 mm in each dimension) of the H800-850 antenna complex from Rhodopseudomonas acidophila strain 10050 have been grown in the presence of p-octyl-glucoside. These crystals have the space group R32 and unit cell dimensions of a = b = 119.9 A and c = 297.0 A. Recently we have improved our crystallization procedures so that all crystals now diffract reliably to beyond 3.5 A, with some diffracting to below 3 8. A range of isomorphous heavy atom derivatives have been prepared and we are now engaged in locating the heavy atom sites within the unit cell.
Introduction The B800-850 antenna complex from Rps. acidophila strain 10050 is an oligomer of two lowmolecular-weight apoproteins, called a and p [ 1,2]. These apoproteins have been sequenced and the /3-apoprotein consists of 53 amino acids (5.8 kDa) while the p-apoproteins contains 41 amino acids (4.6 kDa) [2].The intact pigmented complex is an a&, oligomer, which contains 18 molecules of bacterio chlorophyll a and nine molecules of the carotenoid rhodopin glycoside [3, 41. All of these pigments are non-covalently bound to the apoproteins. W e have reported previously the crystallization of this complex [4]. However, at that time, the crystals were very unreliable, with only one in
every 20-40 crystals diffracting X-rays to high resolution. This made a realistic heavy atom search almost impossible. In this report we describe how we have overcome this problem and where we are with our heavy atom search.
Methods The €3800-850 antenna complex was prepared as described previously [4], with the following modifications: Analar grade lauryldimethylamine-N-oxide (LDAO) from Fluka was used throughout the purification and only the very centre of the column fractions were used. The crystals were grown by vapour diffusion, as described previously, once the complex had been exchanged into p-octyl-glucoside [4]. In our previous crystallization procedures we had p H d the solution containing the complex just prior to setting up the crystal trials. W e have now ceased this practice and this seems to be the most important reason for our improved crystals. We have also found that great care should be taken in preparing an artificial mother liquor for the heavy atom soaking. A solution of 1% (w/v) poctyl-glucoside in 20 mM Tris/HCl pH 8.0 was made to 2.5% (w/v) with benzamidine hydrochloride and then 4 M potassium phosphate pH 9.5 was added to give a final concentration of 1 M. The required heavy atom compound was made up to
I993
39
Biochemical Society Transactions
Table I
Crystallographic data from the heavy atom soaks with the Rps. acidophila 6800-850 crystals
40 Data Native ( I ) Native (2) PCMBA AuCI, TMPA K2Hgl4 ( 1 ) K M 4
(2)
UO(CH,CO), (2)* UO(CH,CO), (2)t
Conc.
Soak time
o=b
c
Total observa-
Indep.
Rmerge
+ R,,,
Resoln
Completeness
(mM)
(h)
(A)
(A)
tions
ref].
(%)
(%)
(A)
(%>
-
-
5 5 5 2 2
4 2 4 2 24 2 2
19.9 19.5 20.6 20.5 19.5 20.4 20.8 121.2 120.3
297.5 296.5 295.2 296.1 296.4 297.6 296.1 291.3 296.2
23983 18204 22394 44447 28409 13922 7862 4521 16698
5931 6113 3779 7408 3918 1693 3955 1039 6777
5.1 9.3 7.5 7.5 9.9 5.0 4.7 5.5 4.0
4.2 3.8 4.75 3.8 4.7 6.5 4.5 6.2 3.8
94.0 75.0 85.7 87.8 87.3 87.8 68.5 5 I .o 80.4
? ?
-
8.4 15.2 16.1 10.6 I 1.3 14.6 -
11.4
*Final concentration unknown since only sparingly soluble. +Using Native I. Abbreviations used: PCMBA, p-chloromercuribenzoic acid, TMPA. trimethylplumbyl acetate,
twice the required soaking molarity in 1.5 M potassium phosphate pH 9.5. The two solutions were then mixed together in equal volumes. Subsequently, 50-100 pl aliquots of this mixture were set up as ‘sitting drops’ in a vapour diffusion vessel, where the outer well contained 8 c m3 of 2.3M ammonium sulphate pH 9.5. This was allowed to come to equilibrium for 2 days. Crystals could now be put into this artificial mother liquor system and soaked for as long as required. The crystals were then mounted in sealed X-ray capillary tubes. Diffraction data were collected on a Siemens area detector mounted on a rotating anode source, operating at 50 KV and 80 mA.
The results so far are presented in Table 1. During our analysis of this data we discovered that we were getting small but significant changes in the unit-cell dimensions of the native crystals from batch to batch (see Table 1). W e have therefore now had to go back and recollect native data sets for each batch and only compare the putative heavy atom derivatives with native crystals from the same batch. This process is not yet complete, but clearly we now have five isomorphous derivatives and we hope when this process is complete to be able to locate their heavy atom-binding sites. This, however, is quite a complicated process with our orthorhombic space group because of the high degree of symmetry.
Results
This work was supported by the SERC and the EEC contract number SCI/0004.
Up until about December 1991 the crystals of the B800-850 complex were of variable quality, with only a very small fraction diffracting to beyond 5 A. We have therefore put a great deal of effort into trying to improve both their quality and reliability. We began by keeping a full ‘life history’ of each batch of the complex we prepared. Everything done to the complex was carefully recorded. This then enabled us to correlate which changes in the procedure led to improved crystals. W e have outlined in the Methods section which alterations proved to be important. Now, all our crystals diffract reliably to beyond 3.5 A and a detailed heavy atom search has begun.
Volume 21
1. Cogdell, R. J., Durant, I., Valentine, J., Lindsay, J. G. and Schmidt, K. (1983) Biochim. Biophys. Acta 72, 427-455 2. Bissig, I., Brunisholz, R. A., Suter, F., Cogdell, R. J. and Zuber, H. (1988) Z.Naturforsch. 43,77-83 3. Evans, M. B., Cogdell, R. J. and Britton, G. (1988) Biochim. Biophys. Acta 935,292-298 4. Papiz, M. Z.,Hawthornthwaite, A. M., Cogdell, H. J.. Woolley, K. J., Wightman, P. A,, Ferguson, I,. A. and Lindsay,J. G. (1989) J. Mol. Biol. 209,833-835
Received 28 September 1992
