Sat May 13 13:57:43 PDT 2006 Kevin Karplus There should not be much in this README file, as notes about dimers should be kept in the README file for the monomer. I'll put a few notes here about methods for building dimers, and later transfer that information to pce/casp7/README. This method assumes that you have a pretty good monomer that you want to dimerize and then optimize. It is not intended for creating dimers from scratch. 1) create a subdirectory dimer/ (or 3mer/, 4mer/ ...) 2) in dimer create a target fasta file with the lengthened target sequence. For example, T0284.a2m would de >T0284 PA4872, Pseudomonas aeruginosa PAO1, 287 res MHRASHHELRAMFRALLDSSRCYHTASVFDPMSARIAADLGFECGILGGSVASLQVLAAP DFALITLSEFVEQATRIGRVARLPVIADADHGYGNALNVMRTVVELERAGIAALTIEDTL LPAQFGRKSTDLICVEEGVGKIRAALEARVDPALTIIARTNAELIDVDAVIQRTLAYQEA GADGICLVGVRDFAHLEAIAEHLHIPLMLVTYGNPQLRDDARLARLGVRVVVNGHAAYFA AIKATYDCLREERGAVASDLTASELSKKYTFPEEYQAWARDYMEVKE MHRASHHELRAMFRALLDSSRCYHTASVFDPMSARIAADLGFECGILGGSVASLQVLAAP DFALITLSEFVEQATRIGRVARLPVIADADHGYGNALNVMRTVVELERAGIAALTIEDTL LPAQFGRKSTDLICVEEGVGKIRAALEARVDPALTIIARTNAELIDVDAVIQRTLAYQEA GADGICLVGVRDFAHLEAIAEHLHIPLMLVTYGNPQLRDDARLARLGVRVVVNGHAAYFA AIKATYDCLREERGAVASDLTASELSKKYTFPEEYQAWARDYMEVKE 3) Copy the Makefile to the dimer/ subdirectory, and add a macro (before the include) MONOMER_LENGTH := 287 4) Make a dimer/decoys/ directory 5) Create a script make-dimer.under in the main directory This script needs to have a properly dimerized template to copy the positioning from and a monomer to dimerize. 6) Create an alignment file that has the target and copies of the best alignment. For example, for T0284, we have T0284/1mumA/1mumA.dimer-a2m modified from T0284-1mumA-t04-local-str2+CB_burial_14_7-1.0+0.4+0.4-adpstyle5.a2m : >T0284 PA4872, Pseudomonas aeruginosa PAO1, 287 res MHRASHHELRAMFRALLDSSRCYHTASVFDPMSARIAADLGFECGILGGS VASLQVLAAPDFALITLSEFVEQATRIGRVARLPVIADADHGYGNALNVM RTVVELERAGIAALTIEDTLLPAQFGRKSTDLICVEEGVGKIRAALEARV DPALTIIARTNAELIDVDAVIQRTLAYQEAGADGICLVGVRDFAHLEAIA EHLHIPLMLVTYGNPQLRDDARLARLGVRVVVNGHAAYFAAIKATYDCLR EERGAVASDLTASELSKKYTFPEEYQAWARDYMEVKE >1mumA sl------HSPGKAFRAALTKENPLQIVGTINANHALLAQRAGYQAIYLS GGGVAAGSLGLPDLGISTLDDVLTDIRRITDVCSLPLLVDADIGFGsSAF NVARTVKSMIKAGAAGLHIEDQVGAKRCGHrPNKAIVSKEEMVDRIRAAV DAKTDPDFVIMARTDALAvEGLDAAIERAQAYVEAGAEMLFPEAITELAM YRQFADAVQVPIlaNITEFGATPLFTTDELRSAHVAMALYPLSAFRAMNR AAEHVYNVLRQegtqksVIDTMQTRNELYESINYYQYEEKLDNL------ farsqvk >1mumB sl------HSPGKAFRAALTKENPLQIVGTINANHALLAQRAGYQAIYLS GGGVAAGSLGLPDLGISTLDDVLTDIRRITDVCSLPLLVDADIGFGsSAF NVARTVKSMIKAGAAGLHIEDQVGAKRCGHrPNKAIVSKEEMVDRIRAAV DAKTDPDFVIMARTDALAvEGLDAAIERAQAYVEAGAEMLFPEAITELAM YRQFADAVQVPIlaNITEFGATPLFTTDELRSAHVAMALYPLSAFRAMNR AAEHVYNVLRQegtqksVIDTMQTRNELYESINYYQYEEKLDNL------ farsqvk 7) in dimer make try1.costfcn, then edit it to have a KnownBreak between the chains: KnownBreak M288 If you want any constraints on the optimization, it is necessary to make multiple copies in the cost function, renumbering the constraints in the later chains (a real pain). Alternatively, you can compute the constraints only on the first monomer. If the monomers are identical, this should not cause any problems. Getting the scoring for predicted alpha may be harder, as generating multiple alignments and predictions for the polyprotein chain will be harder. (We could write scripts to take the monomeric predictions and concatenate them with renumbering, but haven't yet done this.) It may be easiest just to comment out the CreatePredAlphaCost commands of the costfcn, and remove the pred_alpha components. Once you have an acceptable dimer, you want to optimize it, keeping it dimerized in roughly the same orientations. If you read in a dimer with ReadConformPDB, be sure to mark it as a dimer by following the read command with Multimer 2 You can do the optimization as usual, but use "multimer 2" in the OptConform arguments. Any alignments (for fragments and the like) can be gotten from the original monomeric runs. You probably want to reduce the duration of the run (by reducing num_gen, gen_size, super_iter, and/or super_num_gen), because multimeric runs take longer than monomeric ones. You can also read the Template.atoms file from the monomeric directory, avoiding duplicating that file. You might want to turn off TweakMultimer at first if you are trying to pack a tight interface, as it will tend to move monomers apart to reduce clashes. Sat May 13 19:27:41 PDT 2006 Kevin Karplus There is something very wrong in the try1 run. The best score in the superpool was *LARGER* than several of the scores at the ends of the iterations. What happened to the good models? Were they not symmetrized during the optimization? They were supposed to be. Were they not added to the superpool? Sat May 13 20:08:29 PDT 2006 Kevin Karplus Worse and worse--the try1 run crashes just as the second OptConform is about to start. There seems to be a bug in the symmtrizing operation. Mon May 15 07:50:29 PDT 2006 Kevin Karplus I *think* I've fixed the problems---at least, everything runs fine in the debug version of undertaker. There may still be occasional crashes though. Mon May 15 08:01:45 PDT 2006 Kevin Karplus It may be necessary to add some inter-chain constraints to hold the dimer together. Even without TweakMultimer on, undertaker may find a way to alleviate clashes by moving parts of the dimer away from each other as it did in try1 (of T0284/dimer). Mon May 15 11:00:55 PDT 2006 Kevin Karplus There *still* seems to be a bug in undertaker, as the cost for the input model is not the same in the ConformationPool as it was on input: dimer-1mumA-from-chimera-try3-1zlpA.pdb marked as a 2-mer, with transformation 6.12303e-17 0 0 1 226.747 32.9105 0 CostConform dimer-1mumA-from-chimera-try3-1zlpA.pdb costs 211.404 But # best score in initial pool out of 20: dimer-1mumA-from-chimera-try3-1zlpA.pdb 370.77 cost/residue, 494 clashes 0.463728 breaks It takes 17 generations before the cost gets back down to the initial level I suspect that the symmetrizing operation is doing some damage. Mon May 15 11:47:08 PDT 2006 Kevin Karplus AND undertaker crashed again trying to read in a SCWRL result!