The SAM-T06 hand predictions use methods similar to SAM_T04 in CASP6 and the SAM-T02 method in CASP5. We start with a fully automated method (implemented as the SAM_T06 server): Use the SAM-T2K and SAM-T04 methods for finding homologs of the target and aligning them. The hand method also uses the experimental new SAM-T06 alignment method, which we hope is both more sensitive and lass prone to contamination by unrelated sequences. Make local structure predictions using neural nets and the multiple alignments. We currently use 10 local-structure alphabets: DSSP STRIDE STR2 an extended version of DSSP that splits the beta strands into multiple classes (parallel/antiparallel/mixed, edge/center) ALPHA an discretization of the alpha torsion angle: CA(i-i), CA(i), CA(i+1), CA(i+2) BYS a discretization of Ramachandran plots, due to Bystroff CB_burial_14_7 a 7-state discretization of the number of C_beta atoms in a 14 Angstrom radius sphere around the C_beta. near-backbone-11 an 11-state discretization of the number of residues (represented by near-backbone points) in a 9.65 Angstrom radius sphere around the sidechain proxy spot for the residue. DSSP_EHL2 CASP's collapse of the DSSP alphabet DSSP_EHL2 is not predicted directly by a neural net, but is computed as a weighted average of the other backbone alphabet predictions. O_NOTOR2 an alphabet for predicting characteristics of hydrogen bonds from the carbonyl oxygen N_NOTOR2 an alphabet for predicting characteristics of hydrogen bonds from the amide nitrogen We hope to add more networks for other alphabets over the summer. We make 2-track HMMs with each alphabet (1.0 amino acid + 0.3 local structure) and use them to score a template library of about 8000 (t06), 10000 (t04), or 15000 (t2k) templates. The template libraries are expanded weekly, but old template HMMs are not rebuilt. We also used a single-track HMM to score not just the template library, but a non-redundant copy of the entire PDB. One-track HMMs built from the template library multiple alignments were used to score the target sequence. All the logs of e-values were combined in a weighted average (with rather arbitrary weights, since we still have not taken the time to optimize them), and the best templates ranked. Alignments of the target to the top templates were made using several different alignment methods (mainly using the SAM hmmscore program, but a few alignments were made with Bob Edgar's MUSCLE profile-profile aligner). Generate fragments (short 9-residue alignments for each position) using SAM's "fragfinder" program and the 3-track HMM which tested best for alignment. Residue-residue contact predictions are made using mutual information, pairwise contact potentials, joint entropy, and other signals combined by a neural net. The contact prediction method is expected to evolve over the summer, as new features are selected and new networks trained. Then the "undertaker" program (named because it optimizes burial) is used to try to combine the alignments and the fragments into a consistent 3D model. No single alignment or parent template was used as a frozen core, though in many cases one had much more influence than the others. The alignment scores were not passed to undertaker, but were used only to pick the set of alignments and fragments that undertaker would see. Helix and strand constraints generated from the secondary-structure predictions are passed to undertaker to use in the cost function, as are the residue-residue contact prediction. One important change in this server over previous methods is that sheet constraints are extracted from the top few alignments and passed to undertaker. After the automatic prediction is done, we examine it by hand and try to fix any flaws that we see. This generally involves rerunning undertaker with new cost functions, increasing the weights for features we want to see and decreasing the weights where we think the optimization has gone overboard. Sometimes we will add new templates or remove ones that we think are misleading the optimization process. New this year, we are also occasionally using ProteinShop to manipulate proteins by hand, to produce starting points for undertaker optimization. We expect this to be most useful in new-fold all-alpha proteins, where undertaker often gets trapped in poor local minima by extending helices too far. Another new trick is to optimize models with gromacs to knock them out of a local minimum. The gromacs optimization does terrible things to the model (messing up sidechains and peptide planes), but is good at removing clashes. The resulting models are only a small distance from the pre-optimization models, but score much worse with the undertaker cost functions, so undertaker can move them more freely than models it has optimized itself. We experienced some challenges with these tetramer models. Undertaker currently is able to handle cyclic multimers much better than it can handle linear ones such as the one for this target. Undertaker will try to optimize linear multimers into a cyclic structure. One possible way to overcome this tendency might be to specify some very heavily-weighted long range distance constraints to keep a linear model stretched out. Unfortunately, we did not have time to experiment with this method. None of the tetramers submitted for this target have been optimized as tetramers. Instead, they are tetramers that have been build from optimized monomers. Model 1 is T0375.try13-opt2.4mer-1rk2.pdb. It is a tetramer built from the monomer model, T0375.try13-opt2.pdb. The template used to build the tetramer was 1rk2. The monomer is not the top scoring model with breaks or even the overall unconstrained cost function, but we believe that it is the best model that we have. Out of all the models that weren't based on the c.72.1.5 family, it does the best in terms of breaks. It has the second best unconstrained cost. This model started started from the automatic alignments, and went through several rounds of optimizing and polishing. The third and fifth rounds of optimization started with the gromacs models to remove breaks. Also, try5 added some helix constraints that joined some short, neighboring sections of predicted helices. Not only did try13 start with a gromacs model, but also the probabilities for the undertaker methods were increased for minimizing gaps and breaks. This monomer makes a nice-looking tetramer using 1rk2 for a template. Model 2 is T0375.try7-opt2.4mer-1rk2.pdb. It is a tetramer built from the monomer model, T0375.try7-opt2.pdb. The template used to build the tetramer was 1rk2. The monomer scores decently with breaks as well as overall. It is based on the automatic alignments, with several rounds of optimizing and polishing. It also makes a decent-looking tetramer using the 1rk2 template, with nice interfaces between the chains (especially in the sheets). Model 3 is T0375.try12-opt2.4mer-1rk2.pdb. It is a tetramer built from the monomer model, T0375.try12-opt2.pdb. The template used to build the tetramer was 1rk2. The monomer is based on the same automatic alignments that T0375.try13-opt2.pdb started from. However, the optimization runs were slightly different. This model never used a gromacs starting model. Model 4 is T0375.try8-opt2.4mer-1rk2.pdb. It is a tetramer built from the monomer model, T0375.try8-opt2.pdb. The template used to build the tetramer was 1rk2. The monomer originated from the top three alignments in the automatic run (T0375.best-scores.html). The IDs of the chains were 1v1aA, 2fv7A, and 1rkd. This model underwent one round of polishing. Model 5 is T0375.try9-opt2.4mer-1vi9.pdb. It is a tetramer built from the monomer model, T0375.try9-opt2.pdb. The template used to build the tetramer was 1vi9. This monomer is different from most of the other ones we've been working on. It was made from alignments to 1vi9A, 1td2A, and 1lhpA. These are from the SCOP family called c.72.1.5. This model underwent one round of polishing from agromacs starting point.