Sun Aug 27 13:40:00 PDT 2000 Kevin Karplus >T0125 Sp18 protein, Haliotis fulgens No real blast hits. No double-blast hits. Only 6 sequences in multiple alignment. Secondary structure predicted all helical. Target model finds TARGET SCORE EVALUE 3lyn[AB] -17.70 9.5e-04 2lyn[ABCD] -10.98 1.0e+00 1.19.1.1.1 1lyn[AB] -10.98 1.0e+00 1.19.1.1.1 1lis -10.98 1.0e+00 1.19.1.1.1 2lisA -10.98 1.0e+00 1.19.1.1.1 Top hits combined template/target: Structure AverageReverse AverageEValue TargetReverse TargetEValue TemplateReverse TemplateEValue SeqLabel 6S 12N 12N 12N 12N 12N 12N 12S 1lis -21.07 6.171344059217e-06 Infinity Infinity -21.07 6.6e-06 3lynB -17.67 0.000184918767030627 -17.67 3.6e-04 Infinity Infinity 2lisA -15.96 0.0010224086413087 -10.94 4.0e-01 -20.98 1.8e-05 1jkw -6.85 9.23820127845384 -6.85 2.2e+01 Infinity Infinity 1kxu -6.85 9.23820127845384 -6.85 2.2e+01 Infinity Infinity 1ral -6.42 14.1934240648707 -6.42 2.2e+01 Infinity Infinity 1afsA -6.38 14.7716884601475 -6.38 2.2e+01 Infinity Infinity 1lwiA -6.38 14.7716884601475 -6.38 2.2e+01 Infinity Infinity 2tbvC -6.31 15.8408130204592 -6.31 2.2e+01 Infinity Infinity 1dr8B -5.93 23.1443604147246 Infinity Infinity -5.93 5.8e+01 Note 2lisA is a 2-way hit. Top three hits all 1.19.1.1.1 Top 2-track hits: % Sequence ID Length Simple Reverse E-value SCOP 2lisA 136 -39.02 -17.07 1.4e-04 1.19.1.1.1 1lis 136 -39.02 -17.07 1.4e-04 1.19.1.1.1 1qq8A 214 -31.18 -9.47 4.2e-01 1.123.1.1.1 1pty 321 -22.58 -8.44 1.1e+00 3.40.1.2.1 1zac 90 -19.47 -7.38 3.1e+00 1.41.1.5.1 Tue Sep 5 10:35:26 PDT 2000 remaking 2track Top now: % Sequence ID Length Simple Reverse E-value X count 1lis 136 -39.02 -17.07 1.5e-04 2lisA 136 -39.02 -17.07 1.5e-04 1qq8A 214 -31.18 -9.47 4.3e-01 1pty 321 -22.58 -8.44 1.2e+00 1zac 90 -19.47 -7.38 3.2e+00 Sun Sep 10 19:01:20 PDT 2000 Kevin Karplus Mon Sep 11 11:05:38 PDT 2000 (note: 1lis and 2lisA are identical sequences, so only way to choose between them is on the info in the PDB files.) 2lisA is much newer and better resolution. Wait a second---we have further information in the doc file: "Crystals were grown at both pH 3 and pH 9.5, both forms produced monomeric crystals with one disulfide bridge. Protein has similar predicted structure to lysin (Genbank M59972, PDB 3LYN)" Also, the top-scoring sequence with the target model is 3lyn[AB], which scores much better than 2lisA=2lynA and 1lis. I'll have to make 3lynA a possible model! Top alignments now: 3lynA/T0125-3lynA-2track-global 3lynA 122 -36.23 -31.62 1.0e-13 One 5-residue insertion dvfrDVQGFrgpkmt, 24 identical residues. Unaligned unresolved residues at ends of chain to get 3lynA/T0125-3lynA-karplus1.a2m 3lynA/T0125-3lynA-global 3lynA 122 -18.05 -29.60 7.6e-13 Has a different alignment for first helix. I played around with the first helix alignment, choosing my favorite alignment as 3lynA/T0125-3lynA-karplus2.a2m 2lisA/2lisA-T0125-global T0125 141 -25.64 -28.14 2.1e-12 1lis/1lis-T0125-global T0125 141 -25.22 -27.27 5.6e-12 Looks pretty good. can improve residue id and put gaps in better places: 2lisA/2lisA-T0125-karplus1.a2m This alignment is somewhat longer than the 3lynA ones, but not as close a homolog. 1lis/T0125-1lis-2track-global 1lis 131 -30.65 -23.60 3.1e-10 2lisA/T0125-2lisA-2track-global 2lisA 131 -30.65 -23.60 3.1e-10 looks pretty good. 3lynA/T0125-3lynA-2track-local 3lynA 136 -44.63 -22.08 8.4e-10 just unaligns first residue of 3lynA/T0125-3lynA-2track-global ... 3lynA/1lis-fssp-3lynA-T0125-local T0125 141 -10.67 -2.01 3.6e-01 The fssp alignment splits the insertion on either side of the helix before the insertion in 3lynA/T0125-3lynA-2track-global. Moving this helix around does allow the charged and polar residues to come to the surface. 3lynA/T0125-3lynA-karplus3.a2m Note: all the structures have the disulphide bridge as adjacent residues. Trying undertaker to resolve the different alignments and fix up the inserts. Start with the long 2lisA/2lisA-T0125-karplus1.a2m, and force the 3lynA/T0125-3lynA-karplus3.a2m on top of it, but provide the other two 3lynA alignments as entries in the alignment library also. The undertaker run seems to do ok, though it leaves some steric clashes: t125-opt at pool[3] 22.5968 bits/residue, 15 clashes 3.58433 breaks t125-opt-rot at pool[4] 22.1796 bits/residue, 32 clashes 3.58433 breaks t125-opt-scwrl at pool[5] 22.3782 bits/residue, 21 clashes 3.58433 breaks t125-opt-scwrl-rot at pool[6] 22.2964 bits/residue, 21 clashes 3.58433 breaks Still, these are much better than the initial alignments, which had bad clashes at the inserts, and the helices still look good. We should probably try another run, starting from the best so far, as the SSbond is not quite close enough yet. Tue Sep 12 09:38:56 PDT 2000 try2/t125-opt.pdb gets a good cys bond and has only 5 clashes. Attempting to optimize rotamers did not improve scores, and SCWRL made the score worse, increasing clashes. OptimizeRotatmers after SCWRL did not recover. The score problem is mainly due to SCWRL losing the disulphide bridge, though the increased clashes don't help. We could try another round of optimization, trying to get slightly tighter packing, starting from try2/t125-opt.pdb, or we could declare t125 done at this point. Tue Sep 12 12:18:08 PDT 2000 try3 packed a little tigher, but increased clashes. Again SCWRL made things score worse in an unrecoverable way. try2/t125-opt.pdb at pool[1] 28.5728 bits/residue, 5 clashes t125-opt at pool[2] 28.3912 bits/residue, 7 clashes t125-opt-rot at pool[3] 28.3912 bits/residue, 7 clashes t125-opt-scwrl at pool[4] 30.4758 bits/residue, 11 clashes t125-opt-scwrl-rot at pool[5] 29.0783 bits/residue, 37 clashes Let's submit try3/t125-opt.pdb