Wed Jun 28 15:50:18 PDT 2000 >T0103 Pepstatin insensitive carboxyl proteinase, Pseudomonas sp. Double-blast gets some fairly strong hits (all via gi|323087|pir||A44869_34:246). All have FSSP rep 1gci (SCOP 1.3.35.1). chain COST 1bh6A -14.29 1c3lA -13.72 1af4 -12.86 1av7 -12.86 1avt -12.86 1be[68] -12.86 1bf[ku] -12.86 1cseE -12.86 1sbc -12.86 1sc[abd]-12.86 1scnE -12.86 1sel[AB]-12.86 1vsb -12.86 2secE -12.86 3vsb -12.86 The T2K alignment gets 28 PDB files in the 113 sequences, which started coming in on iteration t2k_3, with 1dbiA (again FSSP rep 1gci). In the evolutionary tree, many of the PDB files cluster together in subfamilies 19 and 20, but the target is in subfamily 4. The closest PDB files in the tree are 1thm and 1dbiA, both in subfamily 6. We may want to create a trimmed alignment that removes subfamilies 19 and 20 for doing detailed alignment, though all the PDB sequences are quite similar (1dbiA is 43% identical to 1gci, 1thm is 47% identical, the others are mostly around 61% identical). Several templates get good scores: 1thm, 1s01, 1sup, 1a1yE, 1gci, ... (all at least 40% identical to 1gci). These are all proteases, so are quite reasonable hits for this target. I'm building a t99 alignment for 1dbiA (the closest hit in building the alignment), so it can be used as a template. I trimmed out subfamilies 19 and 20 from the t2k alignment, to get T0103.t2k_trimmed.a2m, and used the trimmed alignment to create the alignments. The best-scoring alignments are 1sup/T0103-1sup-vit 9.5e-84 1s01/T0103-1s01-vit 2.5e-83 1sup/T0103-1sup-local 4.1e-81 1s01/T0103-1s01-local 6.9e-81 1a1yE/T0103-1a1yE-vit 5.5e-78 1gci/T0103-1gci-vit 6.0e-78 ... The only fssp file (1gci) has a weak scoring alignment 8.9e-03. The best of the template models tried is 1thm/1thm-T0103-global 1.1e-28 with the top-scoring for the target close behind: 1s01/1s01-T0103-global 2.3e-28 1sup/1sup-T0103-global 1.2e-27 Thu Jun 29 11:17:20 PDT 2000 Kevin Karplus Almost all the alignments look pretty good, with large conserved parts. The hard job will be choosing which alignment is the best. Right now I favor the template alignments over the target alignments, because they do not omit the beta strands of the first sheet. Wed Jul 5 12:12:05 PDT 2000 Kevin Karplus CAFASP summaries not available yet, but SAM-T99 gets only target-model hits, with 1sb[hi] and 1yj[abc] as the top hits. These are again subtilisin hits (1gci as fssp rep). Sat Aug 26 00:20:58 PDT 2000 Remade 2track predictions Sat Aug 26 14:20:43 PDT 2000 Kevin Karplus Remade 2track predictions Top hits: % Sequence ID Length Simple Reverse E-value X count 1s01 275 -155.20 -133.33 5.9e-55 1sup 275 -153.78 -131.76 4.4e-54 1a1yE 274 -145.01 -126.05 6.5e-52 1cseE 274 -143.17 -124.06 4.8e-51 1gci 269 -141.55 -120.28 2.6e-49 1dbiA 280 -133.79 -112.44 7.8e-46 1thm 279 -130.48 -110.68 5.8e-45 2prk 279 -116.54 -95.34 1.9e-38 Remade 2track joint alignments. Need to look at 1s01 2track alignments and 1thm template alignments to make final decision. Tue Sep 5 10:35:22 PDT 2000 remaking 2track to get new FSSP reps included No significant change. Best alignments: 1sup/T0103-1sup-vit 1sup 275 -210.99 -203.57 1.2e-88 1sup/T0103-1sup-local 1sup 275 -214.65 -196.99 8.4e-86 1sup/T0103-1sup-global 1sup 275 -302.03 -187.68 9.3e-82 high residue identity, but several insertions and misses an interior beta strand. 1s01/T0103-1s01-local 1s01 275 -213.57 -196.18 1.9e-85 high residue identity, but several insertions and misses an interior beta strand. 1a1yE/T0103-1a1yE-vit 1a1yE 274 -199.06 -186.90 2.0e-81 high residue identity, but several insertions and misses an interior beta strand. 1s01/T0103-1s01-global 1s01 275 -300.93 -185.58 7.6e-81 1a1yE/T0103-1a1yE-local 1a1yE 274 -203.29 -183.40 6.7e-80 1gci/T0103-1gci-vit 1gci 269 -192.38 -183.16 8.6e-80 1a1yE/T0103-1a1yE-global 1a1yE 274 -290.92 -176.66 5.7e-77 1gci/T0103-1gci-local 1gci 269 -196.06 -175.89 1.2e-76 1dbiA/T0103-1dbiA-vit 1dbiA 280 -180.00 -173.10 2.0e-75 1thm/T0103-1thm-vit 1thm 279 -178.57 -166.94 9.5e-73 1dbiA/T0103-1dbiA-local 1dbiA 280 -184.49 -166.14 2.1e-72 1gci/T0103-1gci-global 1gci 269 -281.59 -165.41 4.4e-72 1thm/T0103-1thm-local 1thm 279 -182.17 -163.17 4.1e-71 1dbiA/T0103-1dbiA-vit 1dbiA 271 -167.64 -160.02 9.6e-70 1dbiA/T0103-1dbiA-local 1dbiA 271 -172.68 -156.42 3.5e-68 1dbiA/T0103-1dbiA-global 1dbiA 280 -273.39 -153.96 4.1e-67 1thm/T0103-1thm-global 1thm 279 -270.67 -153.92 4.3e-67 1dbiA/T0103-1dbiA-global 1dbiA 271 -257.86 -144.46 5.5e-63 1s01/T0103-1s01-2track-local 1s01 275 -155.20 -133.33 5.2e-58 only 1 residue different from 1s01/T0103-1s01-local (aligns initial alanine) 1sup/T0103-1sup-2track-local 1sup 275 -153.78 -131.76 3.8e-57 extends 1sup/T0103-1sup-local by one residue at each end 1s01/T0103-1s01-2track-global 1s01 275 -129.16 -127.96 2.1e-55 1a1yE/T0103-1a1yE-2track-local 1a1yE 274 -145.01 -126.05 5.7e-55 1sup/T0103-1sup-2track-global 1sup 275 -127.71 -126.21 5.7e-55 1gci/T0103-1gci-2track-local 1gci 269 -141.55 -120.28 2.3e-52 1a1yE/T0103-1a1yE-2track-global 1a1yE 274 -120.93 -119.87 6.3e-52 1dbiA/T0103-1dbiA-2track-local 1dbiA 280 -133.79 -112.44 6.9e-49 1thm/T0103-1thm-2track-local 1thm 279 -130.48 -110.68 5.1e-48 1dbiA/T0103-1dbiA-2track-global 1dbiA 280 -110.44 -109.06 1.4e-47 1gci/T0103-1gci-2track-global 1gci 269 -116.07 -108.34 3.7e-47 1thm/T0103-1thm-2track-global 1thm 279 -106.36 -107.01 1.0e-46 1dbiA/T0103-1dbiA-2track-local 1dbiA 271 -126.52 -103.47 5.6e-45 1dbiA/T0103-1dbiA-2track-global 1dbiA 271 -103.12 -100.45 1.1e-43 1thm/1thm-T0103-global T0103 372 -66.73 -65.48 1.1e-28 has full beta sheet, though with some 2ry mismatches. Looks like a better prediction than the target-model predictions. Missing an interior helix and has gaps in initial helix. 1s01/1s01-T0103-global T0103 372 -63.32 -64.74 2.3e-28 Looks a bit better than 1thm/1thm-T0103-global Some light editing to get insertions in slightly better spots yields 1s01/1s01-T0103-karplus.a2m THIS IS CURRENTLY MY FAVORITE ALIGNMENT. 1sup/1sup-T0103-global T0103 372 -61.41 -63.09 1.2e-27 Edited to match 1s01 editing 1sup/1sup-T0103-karplus.a2m (1s01 turned out to have SEQRES and ATOM discrepancies in PDB file) 1thm/1thm-T0103-local T0103 372 -79.46 -61.68 4.9e-27 1s01/1s01-T0103-vit T0103 372 -67.06 -60.27 2.0e-26 1s01/1s01-T0103-local T0103 372 -75.63 -57.99 2.0e-25 1sup/1sup-T0103-vit T0103 372 -63.20 -56.83 6.2e-25 1thm/1thm-T0103-vit T0103 372 -63.15 -56.78 6.6e-25 1sup/1sup-T0103-local T0103 372 -73.60 -56.49 8.8e-25 1a1yE/1a1yE-T0103-global T0103 372 -51.47 -52.61 4.2e-23 1gci/1gci-T0103-global T0103 372 -48.84 -49.04 1.5e-21 1dbiA/1dbiA-T0103-global T0103 372 -48.99 -48.31 3.1e-21 1a1yE/1a1yE-T0103-local T0103 372 -63.99 -45.94 3.4e-20 1dbiA/1dbiA-T0103-local T0103 372 -64.39 -45.33 6.2e-20 1gci/1gci-T0103-local T0103 372 -62.48 -44.66 1.2e-19 1dbiA/1dbiA-T0103-vit T0103 372 -54.17 -44.39 1.6e-19 1a1yE/1a1yE-T0103-vit T0103 372 -50.95 -43.85 2.7e-19 1gci/1gci-T0103-vit T0103 372 -49.74 -43.04 6.1e-19 1a1yE/1gci-fssp-1a1yE-T0103-local T0103 372 -20.24 -6.07 7.4e-03 1dbiA/1gci-fssp-1dbiA-T0103-local T0103 372 -20.24 -6.07 7.4e-03 1gci/1gci-fssp-1gci-T0103-local T0103 372 -20.24 -6.07 7.4e-03 1s01/1gci-fssp-1s01-T0103-local T0103 372 -20.24 -6.07 7.4e-03 1sup/1gci-fssp-1sup-T0103-local T0103 372 -20.24 -6.07 7.4e-03 1thm/1gci-fssp-1thm-T0103-local T0103 372 -20.24 -6.07 7.4e-03 1gci/1gci-T0103-fssp-global T0103 372 -152.59 -5.81 8.9e-03 1a1yE/1gci-fssp-1a1yE-T0103-global T0103 372 -8.35 -5.81 2.0e-02 1dbiA/1gci-fssp-1dbiA-T0103-global T0103 372 -8.35 -5.81 2.0e-02 1gci/1gci-fssp-1gci-T0103-global T0103 372 -8.35 -5.81 2.0e-02 1s01/1gci-fssp-1s01-T0103-global T0103 372 -8.35 -5.81 2.0e-02 1sup/1gci-fssp-1sup-T0103-global T0103 372 -8.35 -5.81 2.0e-02 1thm/1gci-fssp-1thm-T0103-global T0103 372 -8.35 -5.81 2.0e-02 The 1s01 alignments based of the 1gci FSSP file look much worse than the previous ones. Let's go with 1s01/1s01-T0103-karplus.a2m