Thu Jul 13 11:51:52 PDT 2000 Kevin Karplus >T0114 Antifungal protein AFP-1, Streptomyces tendae disulphide C7-C25 no known homologs. blast finds weak hits to 1qh[67][AB] very weak to 1xnd double blast finds only 1qh[67][AB] t2k alignment contains only the sequence itself, and the longer sequence it comes from in NR. Secondary structure prediction is all beta Target model search finds essentially the same as BLAST (no surprise there, with only one sequence in alignment). 1qh[67][AB] 5.4 1axkA ? 2.28.1 1xnd 22 1axkA 2.28.1.11.3 1xyo[AB] 36 1axkA 2.28.1.11.5 1enx[AB] 36 1axkA 2.28.1.11.5 1xyp[AB] 36 1axkA 2.28.1.11.5 1re[def][AB] 36 1axkA 2.28.1.11.5 The template hits are 1qtsA 10 1qtsA 2.1.9,4.88.1 1b6tA 53 1b6tA ? 3.19.1 (based on FSSP similarity to 1cozA,1gtrA) ? 3.23.1 (based on FSSP similarity to 1mjhA) ? 3.21.1 (based of FSSP similarity to 1qnf) Since we have an all-beta structure prediction, we probably want to go with one of the 2.*.* SCOP families. Trying 1qh7A, 1qtsA, 1axkA, top-scoring alignments: 1qtsA/1qtsA-T0114-vit T0114 87 -14.04 -10.07 1.3e-04 1qh7A/T0114-1qh7A-local 1qh7A 207 -16.64 -8.33 7.2e-04 1qtsA/1qtsA-T0114-local T0114 87 -17.30 -7.71 1.3e-03 1qh7A/T0114-1qh7A-vit 1qh7A 207 -10.12 -6.69 3.7e-03 1qh7A/1qh7A-T0114-local T0114 87 -12.80 -3.40 9.7e-02 1axkA/T0114-1axkA-vit 1axkA 394 -7.13 -3.38 9.9e-02 1axkA/1axkA-T0114-local T0114 87 -13.53 -3.10 1.3e-01 1qh7A/1qh7A-T0114-vit T0114 87 -6.44 -2.91 1.6e-01 1axkA/T0114-1axkA-local 1axkA 393 -12.32 -2.68 1.9e-01 1axkA/1axkA-T0114-vit T0114 87 -6.13 -0.86 8.9e-01 1qtsA/T0114-1qtsA-vit 1qtsA 247 -2.55 0.11 1.6e+00 1qtsA/T0114-1qtsA-local 1qtsA 247 -8.13 0.23 1.7e+00 1qtsA/1qtsA-T0114-vit gapless, picks out 4 strands of a beta sandwich/barrel, unfortunately missing a strand in the middle. 1qtsA/1qtsA-T0114-local same as 1qtsA/1qtsA-T0114-vit 1qh7A/T0114-1qh7A-local two small inserts, picks out 3 strands of beta sandwich. 1qh7A/T0114-1qh7A-vit one insert, again picks out 3 strands. 1qh7A/1qh7A-T0114-local not compact 1axkA/T0114-1axkA-vit only 13 residues long, but matches 8 residues. good beta-sandwich turn, but too tiny a fragment for anything but mini-threading. 1axkA/1axkA-T0114-local not compact None of these hits look promising, except as mini-threading fragments to assemble. Wed Jul 26 15:31:18 PDT 2000 Kevin Karplus CAFASP results fold level fold #server #predict #first #signif? 2.1 10 28 7 0 2.28 3 8 1 0 7.3 6 9 2 0 7.39 3 3 2 0 1.26 3 3 2 0 2.38 4 4 0 0 2.77 2 4 0 0 9.1 2 2 0 0 3.42 2 2 0 0 2.6 2 2 0 0 4.27 1 2 1 0 2.49 1 2 1 0 2.22 1 3 0 0 superfamily level 16 2.1.1 9 2.1.2 8 2.28.1 4 7.3.15 4 2.77.1 4 2.38.2 It looks like a moderately strong consensus for 2.1.1, with 2.1.2 and 2.28.1 as runners up. We have the 2.28.1 superfamily, but no hits for 2.1.1 or 2.1.2. Even trying looser thresholds for T0114 (make remote) doesn't seem to find anything more. 27 July 2000 Kevin Karplus Trying "make 2track" finds 1ap0 11. 1ap0 4.8.2.2.1 1epaA 26. 1epaA 2.56.1.1.9 1gsoA 29. 1gsoA 2.79.2.1.2, 3.25.1.1.2, 4.121.1.2.2 1drmA 38. 1drmA 4.91.2.2.1 Still no 2.1.1 or 2.1.2 matches. We should probably look at these (using the 2-track model for one of the alignments). 31 July 2000 Kevin Karplus Tried adding a 2.1.1 match (1qfhA, the FSSP rep for 1ksr found by bioinbgu). The alignments only got one-strand fragments. Trying to run undertaker with the knowledge of the SSbond. Getting some consistency in the beta strands, but this may be an artifact of the rather small set of alignments it has to work with. 31 July 2000 Kevin Karplus Our 2ry prediction has 6 strands Looking in SCOP for 6-strand structures. Try 2.4 (1hoe) 31 July 2000 Rachel Karchin The primary reference in ExPasy for AC Q9RC18 is "Characterization of a Novel, Antifungal, Chitin-Binding Protein from Streptomyces tendae". The web page of one of the authors, Ingmar Hoerr at url: http://www.uni-toebingen.de/jung/people/hoerr contains this summary of the low resolution structural analysis of AFP: "We could not discover a prevalent secondary structure except at 200nm a comparable spectrum refers to a beta-sheet. HYCON analysis confirmed these results showing no significant occurence of alpha-helical domains. Amino residues 30-40 and 60-70 showed similarity to beta-sheet areas. Disulfide bridges between Cys6 and Cys24 were conceivable." This supports T2k all-beta secondary structure prediction. GenThreader predictions for T114: Conf Prob Epair Esolv AlnSc Alen DLen Tlen PDB_ID ======================================================= GUESS 0.155 -5.3 1.0 34.0 39 44 87 1ryt02 GUESS 0.107 -14.0 5.3 42.0 40 76 87 1dgwX1 GUESS 0.107 -14.0 5.3 42.0 40 76 87 1dgwX0 GUESS 0.103 -3.7 1.0 28.0 22 31 87 1d4vA3 GUESS 0.062 -1.9 1.6 22.0 21 30 87 1rmd01 GUESS 0.037 -35.1 5.5 31.0 85 136 87 1bkb00 GUESS 0.035 -23.8 5.3 31.0 52 108 87 1cewI0 GUESS 0.034 -11.9 6.2 30.0 46 91 87 1cz4A1 GUESS 0.032 -21.3 5.0 31.0 77 133 87 1qtsA1 GUESS 0.029 -24.9 8.2 27.0 57 93 87 1an9A2 However, GUESS is <40% confidence level. 1qtsA is one of our template hits (2.1.9, 4.88.1) 1qtsA/1qtsA-T0114-vit is our top-scoring alignment with 1.3e-04 31 July 2000 Christian The 1hoe alignments, for instance 1hoe/1hoe-T0114-fssp-global.pw, look promising. The secondary structure prediction is in fairly decent agreement, the target and template are proteins of the same size, and the suspected or known cysteines are in proximity (although not ideal proximity). The only other protein in the FSSP neighborhood is 1wapA 31 July 2000 Kevin Karplus Redid "make 2track" with modified hmmscore. Now get three hits: % Sequence ID Length Simple Reverse E-value 1ap0 73 -13.37 -5.19 1.3e+01 1epaA 164 -12.69 -4.33 3.6e+01 1gsoA 431 -12.55 -4.21 3.6e+01 T0114-1ap0-2track-local has 9 conserved residues (in 41 residues aligned), but gets a helix where we predict a strand, and we don't expect any helices (based on the experimental data Rachel found). T0114-1epaA-2track-local has 14 conserved residues in a long gapless alignment, but it forms one sheet of a beta sandwich missing one interior strand, and has a helix. It seems like an unlikely structure, without the other half of the sandwich. T0114-1gsoA-2track-local gets two helices where we want strands and is not compact. I suspect that 2track hmm alignment is still not working right, as the alignments are all coming out gapless. 4 Aug 2000 Kevin Karplus Modified 2ry prediction to use 3 decimal points, and not allow 0 or 1 as probabilities (truncating at 0.001 and 0.999). Modified T0114.t2k-2d.mod to have a GENERIC node, and modified Make.main to use GENERIC node for FIMs and inserts in 2track.dist and 2track alignments. Top two-track distances (for fssp-in-scop) are now % Sequence ID Length Simple Reverse E-value 1aozA 552 -19.61 -5.77 1.3e+01 2.5.1 1avmA 201 -16.21 -5.44 1.3e+01 1.2.7, 4.38.1 1d4vA 117 -14.95 -4.84 3.6e+01 7.24.1 1qksA 567 -22.81 -4.74 3.6e+01 1.3.1, 2.65.2 1uok 558 -19.08 -4.70 3.6e+01 2.66.1, 3.1.7 1fuiA 591 -17.51 -4.68 3.6e+01 2.41.2, 3.79.1 1hoe 74 -16.67 -4.65 3.6e+01 2.4.1 1jmcA 246 -18.42 -4.39 3.6e+01 2.38.4 1clc 639 -17.75 -4.39 3.6e+01 1.97.1, 2.1.1 1msc 129 -19.37 -4.37 3.6e+01 4.70.1 1befA 181 -17.29 -4.25 3.6e+01 2.44.1 1bqsA 209 -19.32 -4.14 3.6e+01 2.44.1 1pmi 440 -16.87 -4.10 3.6e+01 2.77.2 Looking at the scores for 2-track alignments made by "make joints", the top ones are 1ap0/T0114-1ap0-2track-global.pw.dist:1ap0 73 -3.03 -12.45 1.8e-05 11 conserved residues, but two predicted strands unaligned and one strande matches a helix. 1hoe/T0114-1hoe-2track-global.pw.dist:1hoe 74 -8.10 -10.62 1.4e-04 9 conserved residues, one predicted strand missing. Possible template, but C7 not aligned, and location NOT near C25 1aozA/T0114-1aozA-2track-local.pw.dist:1aozA 552 -19.61 -5.77 2.0e-02 good 2ry match, but NO conserved residues. Template has larger beta sheets that this match is only part of. 1avmA/T0114-1avmA-2track-local.pw.dist:1avmA 201 -16.21 -5.44 2.0e-02 3-strand 2ry match, no conserved residues 1clc/T0114-1clc-2track-local.pw.dist:1clc 541 -17.88 -4.39 5.4e-02 9 conserved residues, gapless, good 2ry match. Can extend to cover last sheet as well. Possible template, but disulphide atoms NOT near each other. 1d4vA/T0114-1d4vA-2track-local.pw.dist:1d4vA 117 -14.95 -4.84 5.4e-02 lacks disulphides needed to stabilize structure, but does make 7-25 disulphide. 1fuiA/T0114-1fuiA-2track-local.pw.dist:1fuiA 591 -17.51 -4.68 5.4e-02 good 2ry match on 3 strands (gapless), 8 conserved residues. C7 not aligned, and extending alignment doesn't bring it close to C25. 1hoe/T0114-1hoe-2track-local.pw.dist:1hoe 74 -16.67 -4.65 5.4e-02 disagrees with global alignment. Doesn't preserve disulphide. 7 conserved residues in gapless alignment. 1jmcA/T0114-1jmcA-2track-local.pw.dist:1jmcA 238 -18.45 -4.39 5.4e-02 Good gapless alignment for first 4 strands, not easily extended. C7 not aligned, and extending alignment wouldn't bring it close to C25. 1qksA/T0114-1qksA-2track-local.pw.dist:1qksA 559 -22.82 -4.74 5.4e-02 Good gapless alignment for first 4 strands, can be extended to get all six strands, but template is beta propellor, and we have only 1.5 blades of propellor. C7 not aligned and C25 not near anything it could align with. 1uok/T0114-1uok-2track-local.pw.dist:1uok 558 -19.08 -4.70 5.4e-02 gapless alignment first 5 strands, 13 conserved residues. posible template, but C7-C25 NOT near each other. Fri Aug 4 14:42:51 PDT 2000 Kevin Karplus Running "make 2track" again, with a full set of t99 sequences, not just fssp-in-scop. Also, corrected a bug in the ../Make.main file (insert_method score mis-spelled and inset_method_score) Top scores are now: 1xnb 185 -24.92 -7.29 3.1e+00 1jmcA 246 -21.11 -6.49 8.5e+00 1obwA 175 -19.30 -5.53 2.3e+01 1aozA 552 -20.34 -5.42 2.3e+01 1lnh 857 -21.90 -5.19 2.3e+01 1avmA 201 -16.42 -5.05 2.3e+01 The best alignments are 1qtsA/1qtsA-T0114-vit.pw.dist:T0114 87 -14.04 -10.07 1.3e-04 : Antifungal protein AFP-1, Streptomyces tendae 1qh7A/T0114-1qh7A-local.pw.dist:1qh7A 207 -16.64 -8.33 7.2e-04 1hoe/T0114-1hoe-2track-global.pw.dist:1hoe 74 -9.69 -8.99 1.0e-03 Same as before, but maybe the missing disulphide bridge is not so bad---the N-terminus could fold back the other way to make it. 1qtsA/1qtsA-T0114-local.pw.dist:T0114 87 -17.30 -7.71 1.3e-03 : Antifungal protein AFP-1, Streptomyces tendae 1xnb/T0114-1xnb-2track-local.pw.dist:1xnb 185 -24.92 -7.29 2.7e-03 All six strands align, but there are middle strands of template missing. Not very convincing, despite 15 conserved residues. 1jmcA/T0114-1jmcA-2track-local.pw.dist:1jmcA 238 -21.14 -6.49 7.4e-03 All six strands align with 11 conserved residues (1 gap). Disulphide partners can't be made close. 1aozA/T0114-1aozA-2track-local.pw.dist:1aozA 552 -20.34 -5.42 2.0e-02 Last 5 sheets align gaplessly, but only 4 conserved residues. 1ap0/T0114-1ap0-2track-global.pw.dist:1ap0 73 -4.95 -5.74 2.0e-02 11 conserved residue. Helix where sheet predicted, disulphide not close. 1avmA/T0114-1avmA-2track-local.pw.dist:1avmA 201 -16.42 -5.05 2.0e-02 9 conserved residues aligning only 3 strands. 1lnh/T0114-1lnh-2track-local.pw.dist:1lnh 836 -21.92 -5.19 2.0e-02 14 conserved residues, aligning all residues gaplessly Missing middle strands of sheets. disulphide residues at opposite ends of protein. 1obwA/T0114-1obwA-2track-local.pw.dist:1obwA 175 -19.30 -5.53 2.0e-02 6 conserved residues, aligning all strands, but C7 not aligned and can't be brought close to C25.