Supplementary Information The structure of the neuraminidase from H5N1 avian influenza suggests new opportunities for drug design Rupert J Russell+, Lesley F Haire, David J Stevens, Patrick J Collins, Yi Pu Lin, G Michael Blackburn$, Alan J Hay, Steven J Gamblin & John J Skehel MRC National Institute for Medical Research, The Ridgeway, Mill Hill, London NW7 1AA $ + Department of Chemistry, University of Sheffield, Sheffield S3 7HF, UK Present address Centre for Biomolecular Sciences, University of St Andrews, St Andrews KY16 9ST, UK Supplementary Table 1: Crystallographic statistics N1 N1 + N1 + oselt oselt amivi N8 N8 + N8 + N8 + N8 + N8 + DANA Oselt Oselta Zana Pera amivi amivi mivir mivir mivir r (20 r r (3 day M) (0.5m (30mi soak) M) ns 2HT 2HT Q U I4 I4 a=b =90 41, c=10 9.30 a=b =89 78, c=9 3.2 N4 N4 + 2HT 2HTW P432 I432 soak) PDB 2HTY 2HU0 2HU4 2HT5 2HTR 2HT7 2HT8 ID Space C2221 C2221 C2221 I4 I4 I4 I4 a=20 1.74, b=20 1.51, c=21 2.43 a=202 82, b=203 07, c=216 79 a=200 62, b=200 70, c=210 48 a=b =90 67, c=10 9.4 8 1 1 1 2.5 3.0 2.5 2.4 2.5 2.6 2.4 2.2 2.2 2.8 3.5 Rsym 11.9 18.5 8.5 10.3 7.0 14.7 9.6 7.9 11.5 14.7( 23.9 (%) (39.3 (53.1 (27.1 (26.7 (21.2 (48.3 (24.3) (27.6 (31.4 44.9) (59.6 ) ) ) ) ) ) ) ) 7.3 4.6 11.2 10.7 13.2 12.6 7.6 9.9 6.3 8.0 6.0 (2.0) (1.5) (4.3) (3.0) (3.8) (3.8) (2.5) (2.5) (2.1) (2.8) (2.4) Compl 98.0 79.4 93.0 94.5 92.5 99.9 93.0 91.0 93.5 94.3 99.2 etene (92.6 (71.9 (65.8 (71.4 (60.0 (99.8 (63.7) (54.6 (74.8 (68.4 (99.1 ss (%) ) ) ) ) ) ) ) ) ) ) Uniqu 1433 7067 1367 16 14 13 16 20 17 29 52 94 195 343 732 124 267 595 345 998 3.1 2.5 3.7 3.2 3.2 7.3 3.3 2.4 2.8 3.9 4.4 group Unit cell dimen sions (Å) Z Resolu a=b =90 38, c=11 1.49 a=b= 90.58, c=110 78 a=b= 90.42, c=109 71 a=19 3.79 a=1 93.4 tion (Å) I/I e ) Reflec tions Redun dancy Rwork 23.2 21.8 23.7 21.7 21.7 22.9 25.2 27.1 23.4 22.2 21.7 26.2 29.5 27.3 26.1 26.7 28.6 27.3 33.4 28.6 27.3 29.5 2370 2370 2370 3002 3002 3002 3002 3002 3002 5992 2996 4 643 - - 155 - - 91 103 - 318 - 0.010 0.023 0.007 0.007 0.007 0.008 0.008 0.00 0.00 0.00 0.00 7 (%) Rfree (%) Protei n atoms Solven t atoms Rmsd bonds (Å) Rmsd 1.80 2.28 1.55 1.50 1.500 1.53 1.57 1.57 1.51 1.55 1.58 26.3 22.0 21.4 31.6 15.6 22.8 29.4 27.1 19.0 24.2 16.0 - 46.4 25.1 - 16.8 33.3 31.6 27.2 15.6 - 20.0 - - 36.3 - - 39.4 - - 26.8 - Angles (°) Bfactor protei n (Å2) Bfactor ligand (Å2) B- 27.4 factor solven t (Å2) Values in parentheses refer to the highest resolution shell Z is the number of monomers in the asymmetric unit Rsym = j| - Ij|/ where Ij is the intensity of the jth reflection and is the average intensity Rwork = | |Fo| - |Fc| |/ |Fo| Rfree = T | |Fo| - |Fc| |/T |Fo|, where T is a test data set of 5% of the total reflections randomly chosen and set aside before refinement Supplementary Figure Comparison of the superposed active sites of Influenza B neuraminidase with that of the Influenza N9 neuraminidase The active sites of the two enzymes are shown in Ribbons representation with the N9 NA coloured yellow and the Influenza B NA coloured blue The orientation of the view is very similar to that used in Figure 1d,e In the vicinity of the active site and the 150-loop the two structures are very similar Supplementary Figure The ‘open’ structure of N1 with bound oseltamivir The figure shows the active site of N1 in a similar orientation to that shown in Figure of the main manuscript with the N1 structure shown as ball-and-stick representation with the carbons coloured green Also shown in grey is the corresponding 2Fo-Fc electron density using the weighted coefficients from REFMAC5 The density map is contoured at 0.9 sigma The figure shows that oseltamivir is able to bind to N1, which crystallises under quite different conditions to N8, with the 150-loop in its open conformation The diffraction data used were recorded from a crystal of N1 that was soaked in 20µM oseltamivir for 150 minutes The data were recorded on a MAR345 image plate mounted on a Rigaku MicroMax 007 HF generator All other data handling and computational steps were carried out according to the main Methods section Supplementary Figure Binding of DANA, Zanamivir, and Peramivir neuraminidase inhibitors to the active sites of Group-1 and Group-2 NAs Superposition of the active sites of N8 (green) and N9 (yellow) NAs in complex with (A) DANA, (B) zanamivir and (C) peramivir show that the structures of the two NA groups are very similar after inhibitor binding