30 July 1998 As claimed, no strong homologies: wu-blast finds nothing (top 1ncs 0.47) double-blast finds nothing t75.t98_6 has plenty of homologs, finding no strong ones in PDB: 1vhh -3.42 1vhh sonic hedgehog 1ikl=1ikm=[12]il8[AB] -2.82 1tvxA interleukin template models 1edt -3.32 6hir -3.24 1edg -3.04 4thcI -2.63 1vhh -2.31 summed 1vhh -5.73 t75.remote_4 still finds 1vhh top, and does not have a huge number of sequences, so I'll use it as the search and alignment model. Note: t54, which we gave up on, also had 1vhh on top---maybe we should align t75 with t54? They are clearly NOT the same sequence. The beginning of 1vhh has a fairly good match to the END of T0075---this overlap may not be enough to make a good prediction. Is 1vhh part of a multi-domain protein? The 1edt alignment is similarly unpromising---just 13 residues in the middle of the chain. It can be edited into a marginal match (t75-1edt-hand1): T0075 .....MECADVPLLTPSSKEMMSQALKATFSGFTKEQQRLGIPKDPRQWTETHVRDWVMWAVNE V Q Q G P Q AV 1edt k57fnENVQRVLDNAVTQIRPLQQQGIKVLLSVLGNHQGAGFANFPSQQAASAFAKQLSDAVAK T0075 FSLKGVDFQKFCMSGAALCALGKECFLELAPDFVGDILWEHLEILQKEDVK...... L GVDF A G FV K 1edt YGLDGVDFDD---EYAEYGNNGTA--QPNDSSFVHLVTALRANMPDKIISLy103t. With two gaps and only 18 resdiue identities, this is not a very convincing alignment. The 1tvxA alignment is in three pieces, but can hand aligned to do OK (2 1-residue gaps and 14 identities). T0075 -MECADVPLLTPSSKEMMSQALKATFSGFTKEQQRLGIPKDPRQWTETHVRDWVMWAVNEFSLK GI Q E V E 1tvxA --------------------------LRCLCIKTTSGIHPKNIQSLEVIGKGTHCNQV-EVIAT T0075 GVDFQKFCMSGAALCALGKECFLELAPDFVGDILWEHLEILQKEDVK D K C A K LA D 1tvxA LKDGRKICLDPDA-PRIKKIVQKKLAGD------------------- NONE of these predictions agrees with the secondary structure prediction, which is mainly helical. The helix TETHVRDWVMWAV gets up to phat=0.98 in the center, but matches a beta turn in 1tvxA. 5 August 1998 Christian When the load on alpha/beta isn't so high, build constrained alignments for 1oya and 1edt. Thu Aug 20 11:23:55 PDT 1998 Kevin Karplus Moved the old stuff to subdirectory "old" and re-running with newest Makefile. wu-blast still nothing (top 1ncs 0.47) double-blast still nothing. Top hits with t75.t98_6 are all pretty weak: 1vhh -4.580 1vhh 1ctm -3.610 1hcz 2pcfB -3.610 1hcz 1hcz -3.600 1hcz 1azd[ABCD] -3.590 1nwpA 2sak -3.510 2sak 1ik[lm] -3.410 1tvxA [12]il8[AB] -3.410 1tvxA 3il8 -3.410 1tvxA The t75-1vhh-vit alignment still looks terrible I tried editing 1oya/t75-1oya-vit, and got a half-decent t75-1oya-hand1 alignment. Fri Aug 21 09:55:00 PDT 1998 Top hits with target98 library: 1edt -3.920 1hstA -3.750 1vhh -3.540 1edg -3.090 1fc2C -2.700 1gpmA_3 -2.570 Summing both ways: 1vhh -8.12 1oya -4.88 2bbkL -4.49 1oyc -4.25 1edt -3.920 1hstA -3.750 1ctm -3.610 Top hits with fssp viterbi: 256bA -5.400 1azh -5.270 1fct -4.690 1fokA -4.370 1aozA -3.520 1nfn -2.660 1occK -2.360 1pcfA -2.160 Top hits with target98-mixed library: 1aa2 -3.200 1agg -3.100 1fipA -3.030 1pmi -2.930 1ah7 -2.660 1allA -2.390 2bbkL -2.270 1cpcL -2.260 1phnB -2.260 1ai6A -2.150 1ayl -2.140 Top hits with target98 viterbi scoring: 1hstA -5.900 1fc2C -5.850 1vhh -5.770 1edt -5.490 1gpmA_3 -5.300 1edi -4.720 1edg -3.890 Top hits with t75.remote_4: 1vhh -4.460 1ik[lm] -3.770 [12]il8[AB] -3.770 3il8 -3.770 1oy[abc] -3.260 2sak -3.100 Fri Aug 21 10:24:38 PDT 1998 Ones worth considering: 1vhh -8.12 1vhh 1hstA -5.90 1hstA 1fc2C -5.85 1fc2C 1edt -5.49 2ebn (use 1edt) 256bA -5.40 256bA 1gpmA_3 -5.30 1gpmA 1azh -5.27 1azh 1oya -4.88 1oyc (use 1oyc) 1edi -4.72 1fc2C 70% (use 1fc2C) 1fct -4.69 1fct 2bbkL -4.49 2bbkL 1fokA -4.37 1fokA 1edg -3.89 1edg 1ikl -3.77 1tvxA (use 1ikl---the most recent average NMR structure for interleukin). 1ctm -3.61 1hcz (100%) 1aa2 -3.20 1aa2 1agg -3.10 1agg The top-scoring non-self alignments are 1vhh/t75-1vhh-vit 1vhh 157 -6.41 -7.57 2bbkL/t75-2bbkL-global 2bbkL 125 -2.25 -7.31 2bbkL/2bbkL-t75-global T0075 110 -1.56 -7.07 2bbkL/2bbkL-t75-fssp-global T0075 110 -1.04 -6.50 1oya/t75-1oya-vit 1oya 399 -5.31 -6.07 1oyc/t75-1oyc-vit 1oyc 399 -5.31 -6.07 1aa2/1aa2-t75-global T0075 110 -2.74 -5.98 1vhh/t75-1vhh-global 1vhh 157 -3.82 -5.92 1ikl/t75-1ikl-vit 1ikl 69 -6.32 -5.75 2sak/t75-2sak-vit 2sak 121 -3.84 -5.61 1hcz/t75-1hcz-vit 1hcz 250 -6.06 -5.60 1fc2C/1fc2C-t75-vit T0075 110 -3.67 -5.39 1hstA/1hstA-t75-global T0075 110 -3.23 -5.38 1fct/1fct-t75-vit T0075 110 -4.75 -5.05 1ikl/t75-1ikl-global 1ikl 69 -2.56 -5.01 2bbkL/t75-2bbkL-vit 2bbkL 125 -4.06 -4.92 1vhh/1vhh-t75-vit T0075 110 -4.52 -4.81 1fokA/t75-1fokA-vit 1fokA 568 -4.52 -4.77 1edt/1edt-t75-vit.pw.dist:T0075 110 -4.65 -4.66 1hcz/t75-1hcz-global 1hcz 250 -3.88 -4.57 1hstA/1hstA-t75-vit.pw.dist:T0075 110 -4.58 -4.38 1fokA/t75-1fokA-global 1fokA 568 -6.88 -4.11 2sak/t75-2sak-global 2sak 121 -3.75 -4.09 1azh/1azh-t75-fssp-global T0075 110 -6.79 -3.86 2sak/2sak-t75-global T0075 110 -0.02 -3.72 1oyc/1oyc-t75-vit T0075 110 -3.08 -3.70 2bbkL/2bbkL-t75-vit T0075 110 -3.95 -3.68 1fokA/1fokA-t75-vit T0075 110 -4.10 -3.62 1fc2C/1fc2C-t75-global T0075 110 -2.86 -3.59 1agg/1agg-t75-global T0075 110 -3.89 -3.15 1aa2/1aa2-t75-vit T0075 110 -2.33 -3.05 1oya/t75-1oya-global 1oya 399 -3.44 -2.96 1oyc/t75-1oyc-global 1oyc 399 -3.44 -2.96 2sak/2sak-t75-fssp-global T0075 110 0.68 -2.79 1agg/1agg-t75-vit T0075 110 -2.26 -2.76 1edt/t75-1edt-vit 1edt 265 -2.67 -2.15 1hstA/1hstA-t75-fssp-global T0075 110 -0.82 -2.12 1azh/t75-1azh-vit 1azh 36 -0.65 -1.77 1gpmA/1gpmA-t75-vit T0075 110 -1.91 -1.77 1azh/1azh-t75-vit T0075 110 -3.81 -1.69 1fc2C/1fc2C-t75-fssp-global T0075 110 -2.39 -1.64 1fct/1fct-t75-global T0075 110 -3.62 -1.41 1aa2/t75-1aa2-vit 1aa2 108 -0.08 -1.38 1fct/1fct-t75-fssp-global T0075 110 -4.36 -1.33 1edg/1edg-t75-vit T0075 110 -2.74 -1.19 1vhh/t75-1vhh-vit 7 residues conserved out of 19 aligned. 2bbkL/t75-2bbkL-global 9 residues conserved out of 33 aligned, same region as t75-1vhh-vit. missing middle of beta sheet. 2bbkL/2bbkL-t75-global 11 residues conserved out of 23+27=50 aligned. unbridgeable gaps. 2bbkL/2bbkL-t75-fssp-global very similar to 2bbkL-t75-global (both 1vhh and 2bbkL had poor match to 2ry structure prediction) 1oya/t75-1oya-vit 4 residues out of 13 aligned, perfect 2ry match 1oyc/t75-1oyc-vit identical alignment to 1oya 1aa2/1aa2-t75-global 12 residues conserved out of 68 aligned 1aa2/1aa2/t75-hand1 12 residues conserved out of 65 aligned (smaller gaps, good 2ry match) 1vhh/t75-1vhh-global 8 residues conserved out of 30 1ikl/t75-1ikl-vit 5 residues conserved out of 18 2sak/t75-2sak-vit 7 residues conserved out of 13+9 good 2ry match, but not extendible rest of template beta, rest of target helix 1hcz/t75-1hcz-vit 5 conserved out of 37 good 2ry match, but not extendible. 1fc2C/1fc2C-t75-vit 5 conserved out of 7 aligned. 1hstA/1hstA-t75-global 7 conserved out of 16+9=25 1fct/1fct-t75-vit 4 conserved out of 9 aligned 1ikl/t75-1ikl-global 6 conserved out of 19+9=28 2bbkL/t75-2bbkL-vit 9 conserved out of 32 aligned, poor 2ry match 1vhh/1vhh-t75-vit 8 conserved out of 23 1fokA/t75-1fokA-vit 9 conserved out of 29--long surface beta hairpin, hard to extend to compact structure. 1edt/1edt-t75-vit 7 conserved out of 15 extendible to cover half of 1edt barrel ? maybe dimer that forms beta sadwich? None of these look very promising. There are 4 hand alignments to consider: 1aa2/1aa2-t75-hand1.a2m 1hstA/1hstA-t75-hand1.a2m 1edt/1edt-t75-hand1.a2m 1oya/t75-1oya-hand1.a2m I think the 1aa2 alignment may be a little more promising than the rest, unless the 1edt is a dimerization prediction. I looked at the PRODOM description for ETS1_MOUSE (which t75 is taken from), and found that there was a domain split predicted just before KEQQRLG. Although ETS1_MOUSE is DNA-binding, the binding domain is already solved (2stwA), and comes at the end of ETS1_MOUSE, while t75 is near the beginning. 22 August 1998 Christian Of the 4 alignment alternatives, only 1hstA looks promising judging solely by functional similarity of related proteins. The 1hstA FSSP family is all repressors and activators. I haven't looked at the alignments though. When the DECs come back online, I want to investigate these 1hstA FSSP family members that are not in the model library: 1smtA 1ghc 2hfh 2fokA 23 August 1998 Christian Even though 1smtA-t75.cbarrett1.a2m is suspect, I think it has at least a little hope. A couple of the conserved residues as outlined in PMID:9009269 (which discusses the region of ets1 that is t75) are aligned by introducing five inserts. These conserved residues mostly occur in turns where they would have a stabilizing effect. Furthermore, the predicted and 2ary structures match decently. 1smtA also comes from a family that contains repressors and activators. While t75-2hfh.cbarrett1.a2m sorta/kinda/maybe preserves the key residues, I think the 1smtA alignment is better. 1ghc-t75.cbarrett1.a2m is a fairly dubious alignment that shows no sign of conserving residues as outlined in the above literature. 2fokA does not provide anything remotely resembling a compact structure. 1hstA doesn't fit the conserved residue profile at all. I think the 1oya alignment is interesting because of the (mainly) proline conservation pattern in the loopy region and because 3/4 of the residues from the paper are conserved without having to introduce any gaps. The fourth residue could be if 1-residue helix deletion was allowed. See t75-1oya.cbarrett1.a2m. I think our best bet is either with 1oya or 1smtA. Although I wouldn't bet much on either, I would place more on 1smtA.