Orientation Maps of Subjective Contours in Visual Cortex (original) (raw)

6. Escherichia coli ENR is a homotetramer (M r ϳ 28,000 per subunit) that was prepared from an overexpressing E. coli strain (2, 5). Crystals of the ENR-NAD ϩ complex (crystal form A) belong to space group P2 1 and have unit cell dimensions of a ϭ 74.0 Å, b ϭ 81.2 Å, c ϭ 79.0 Å, and ␤ ϭ 92.9°with a tetramer in the asymmetric unit (16). Data were collected to 2.5 Å ( , 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 , 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 the model was refined with the program TNT (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-NAD ϩ (form A) crystal for 1 hour in 0.1 mM ethylmercuriphosphate, 10 mM NAD ϩ , 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 , 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 ␤6 and ␣6; 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 ϭ ⌺(FobsϪF calc )/⌺(F obs ), where F obs  and F calc  are the observed and calculated structure factor amplitudes, respectively]. The average B factor for the tetramer is 30 Å 2 (24 Å 2 for main-chain atoms), where B ϭ 8 2 ( 2 ) and is the mean square displacement of the atomic vibration. 7. Crystals of the ENR-NAD ϩ -diazaborine complex (crystal form B) belong to space group P6 1 22 and have unit cell dimensions of a ϭ b ϭ 80.9 Å, c ϭ 328.3 Å, ␣ ϭ ␤ ϭ 90°, and ␥ ϭ 120°for the thienodiazaborine complex, and a ϭ b ϭ 80.6 Å, c ϭ 325.3 Å, ␣ ϭ ␤ ϭ 90°, and ␥ ϭ 120°for the benzodiazaborine complex with a dimer in the asymmetric unit (16). Data sets were collected on the ENR-NAD ϩthienodiazaborine complex to 2.2 Å and on the ENR-NAD ϩ -benzodiazaborine complex to 2.5 Å , data sets Thieno and Benzo) at the CLRC Daresbury Synchrotron and were processed as above. The structures of both ENR-NAD ϩ -diazaborine complexes were solved independently by molecular replacement with the use of an appropriate dimer from the E. coli ENR-NAD ϩ 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 Å 2 (22 Å 2 for main-chain atoms, 20 Å 2 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°f or angles. The average B factor for the dimer is 24 Å 2 (20 Å 2 for main-chain atoms, 20 Å 2 for diazaborine atoms). For the ENR-NAD ϩ complex and the ENR-NAD ϩ -thienodiazaborine complex, 244 C␣ atoms superimpose with an rmsd of 0.3 Å, whereas for the two ENR-NAD ϩ -diazaborine complexes, 256 C␣ atoms superimpose with an rmsd of 0.2 Å.