Orientation Maps of Subjective Contours in Visual Cortex

1. A. Banerjee et al., Science 263, 227 (1994); A. Dessen, A. Quémard, J. S. Blanchard, W. R. Jacobs Jr., J. C. Sacchettini, ibid. 267, 1638 (1995). 2. H. Bergler et al., J. Biol. Chem. 269, 5493 (1994). 3. S. T. Cole, Trends Microbiol. 2, 411 (1994). 4. G. Högenauer and M. Woisetschläger, Nature 293, 662 (1981). 5. M. M. Kater, G. M. Koningstein, H. J. J. Nijkamp, A. R. Stuitje, Plant Mol. Biol. 25, 771 (1994). 6. Escherichia coli ENR is a homotetramer (Mr ; 28,000 per subunit) that was prepared from an overexpressing E. coli strain (2, 5). Crystals of the ENR-NAD1 complex (crystal form A) belong to space group P21 and have unit cell dimensions of a5 74.0 Å, b5 81.2 Å, c 5 79.0 Å, and b 5 92.9° with a tetramer in the asymmetric unit (16). Data were collected to 2.5 Å ( Table 1, data set Native-1) on a twin San Diego Multiwire Systems (SDMS) area detector with a Rigaku RU-200 rotating anode source, and the data set was processed with SDMS software (17 ). Data were also collected to 2.1 Å ( Table 1, data set Native-2) at the CLRC Daresbury Synchrotron and processed with the MOSFLM package (18), and the 2.1 and 2.5 Å data sets were then scaled and merged with CCP4 software (19). Initially, a model of B. napus ENR (10) was used as a basis for a molecular replacement solution of the structure, but the map, calculated after themodel was refinedwith the programTNT (20), was not of sufficient quality to confidently assign residues in regions of structural differences between the B. napus and E. coli enzymes. Therefore, to solve the structure, we obtained a heavy-atom derivative by soaking an ENR-NAD1 (form A) crystal for 1 hour in 0.1 mM ethylmercuriphosphate, 10 mM NAD1, 20% (w/v) polyethylene glycol (molecular weight 400), and 100 mM acetate (pH 5.0). Derivative data were collected at the CLRC Daresbury Synchrotron to a resolution of 3 Å ( Table 1, data set Hg) and were processed as above. The positions of the heavy atoms in this derivative were revealed by difference Fourier methods with the use of the approximate phases provided by the molecular replacement solution. The heavy-atom parameters were refined with the program MLPHARE (21) and resulted in a phase set with an overall mean figure of merit of 0.34 to 3 Å resolution. Using a map derived from these phases, we generated molecular masks for the molecule with the program MAMA (22) and performed 50 cycles of solvent flattening and fourfold molecular averaging with the program DM (19, 23). In the resultant electron density map, calculated from the averaged phases, we were able to find clear density for all but the first residue, the last four residues, and 10 residues from the loop joining b6 and a6; using the graphics program FRODO (24), we were able to build with confidence a model comprising 247 of the 262 amino acids of E. coli ENR. Several cycles of rebuilding and refinement gave a final R factor for the model of 0.157 (52,346 reflections in the range 10 to 2.1 Å, 7836 atoms including 324 water molecules), with an rmsd of 0.017 Å for bonds and 2.92° for angles [R 5 S(Fobs2Fcalc)/S(Fobs), where Fobs and Fcalc are the observed and calculated structure factor amplitudes, respectively]. The average B factor for the tetramer is 30 Å2 (24 Å2 for main-chain atoms), where B 5 8p2(m# 2) and m# is the mean square displacement of the atomic vibration. 7. Crystals of the ENR-NAD1-diazaborine complex (crystal form B) belong to space group P6122 and have unit cell dimensions of a 5 b 5 80.9 Å, c 5 328.3 Å, a 5 b 5 90°, and g 5 120° for the thienodiazaborine complex, and a5 b5 80.6 Å, c5 325.3 Å, a 5 b 5 90°, and g 5 120° for the benzodiazaborine complex with a dimer in the asymmetric unit (16). Data sets were collected on the ENR-NAD1thienodiazaborine complex to 2.2 Å and on the ENRNAD1-benzodiazaborine complex to 2.5 Å ( Table 1, data sets Thieno and Benzo) at the CLRC Daresbury Synchrotron and were processed as above. The structures of both ENR-NAD1-diazaborine complexes were solved independently by molecular replacement with the use of an appropriate dimer from the E. coli ENR-NAD1 structure and were refined against their respective data sets with the program TNT (20). The initial electron density maps were readily interpretable and unambiguous density could be observed for the location of the diazaborine compounds, which were then incorporated into the refinement. Clear density could be found for all but the first residue and the last four residues. Refinement of the thienodiazaborine complex gave a final R factor of 0.191 (30,825 reflections in the range 10 to 2.2 Å, 3936 atoms), with an rmsd of 0.012 Å for bonds and 2.9° for angles. The average B factor for the dimer is 27 Å2 (22 Å2 for main-chain atoms, 20 Å2 for diazaborine atoms). Refinement of the benzodiazaborine complex gave a final R factor of 0.169 (20,204 reflections in the range 10 to 2.5 Å, 3930 atoms), with an rmsd of 0.013 Å for bonds and 2.7° for angles. The average B factor for the dimer is 24 Å2 (20 Å2 for main-chain atoms, 20 Å2 for diazaborine atoms). For the ENR-NAD1 complex and the ENR-NAD1-thienodiazaborine complex, 244 Ca atoms superimpose with an rmsd of 0.3 Å, whereas for the two ENR-NAD1-diazaborine complexes, 256 Ca atoms superimpose with an rmsd of 0.2 Å. 8. M. A. Grassberger, F. Turnowsky, J. Hildebrandt, J. Med. Chem. 27, 947 (1984). 9. S. Zhong, F. Jordan, C. Kettner, L. Polgar, J. Am. Chem. Soc. 113, 9429 (1991). 10. J. B. Rafferty et al., Structure 3, 927 (1995). 11. H. Bergler, G. Högenauer, F. Turnowsky, J. Gen. Microbiol. 138, 2093 (1992). 12. C. Taillefumier, D. de Fornel, Y. Chapleur, Bioorg. Med. Chem. Lett. 6, 615 (1996). 13. J. T. Bolin, D. J. Filman, D. A. Matthews, R. C. Hamlin, J. Kraut, J. Biol. Chem. 257, 13650 (1982); C. Bystroff, S. J. Oatley, J. Kraut, Biochemistry 29, 3263 (1990). 14. H. G. Bull et al., J. Am. Chem. Soc. 118, 2359 (1996). 15. M. D. Sintchak et al., Cell 85, 921 (1996). 16. C. Baldock et al., Acta Crystallogr., in press. 17. R. Hamlin,Methods Enzymol. 114, 416 (1985); A. J. Howard, C. Nielson, N. H. Xuong, ibid., p. 452; N. H. Xuong, C. Nielson, R. Hamlin, D. Anderson, J. Appl. Crystallogr. 18, 342 (1985). 18. A. G. W. Leslie, Joint CCP4 and ESF-EACBMNewsletter on Protein Crystallography No. 26 (SERC Daresbury Laboratory, Warrington, UK, 1992). 19. Collaborative Computational Project No. 4, Acta Crystallogr. D50, 760 (1994). 20. D. E. Tronrud, L. F. Ten Eyck, B. W. Matthews, ibid. A43, 489 (1987). 21. Z. Otwinowski, in Proceedings of the CCP4 Study Weekend,W.Wolf, P. R. Evans, A. G.W. Leslie, Eds. (SERC Daresbury Laboratory, Warrington, UK, 1991), p. 80. 22. G. J. Kleywegt and T. A. Jones, ESF/CCP4 Newsletter No. 28 (1993), p. 56. 23. K. Cowtan, ESF/CCP4 Newsletter No. 31 (1994), p. 34. 24. T. A. Jones, J. Appl. Crystallogr. 11, 268 (1978). 25. T. E. Ferrin, C. C. Huang, L. E. Jarvis, R. Langridge, J. Mol. Graphics 6, 13 (1988). 26. R. Esnouf, personal communication. 27. P. J. Kraulis, J. Appl. Crystallogr. 24, 946 (1991). 28. A. C. Wallace, R. A. Laskowski, J. M. Thornton, Protein Eng. 8, 127 (1995). 29. We thank the support staff at the Synchrotron Radiation Source at Daresbury Laboratory for assistance with station alignment. Supported by grants from the UKBiotechnology and Biological Sciences Research Council (BBSRC) and Medical Research Council (D.W.R. and A.R.S.). C.B. is funded by a Zeneca Agrochemicals–supported CASE award. J.B.R. is a BBSRC David Phillips Research Fellow. The Krebs Institute is a designated BBSRC Biomolecular Science Centre.