questions about ncs copy in autobuild
Dear All, I am using phenix.autobuild .eff file to build a structure (version 1.6.1). For the input I have a pdb model file with 6 chains; a sequence file which contains residues of a single chain (the sequence file has a few more residues at N-terminal than the pdb model file); an mtz amplitude file; I also specified ncs_copies=6 (I know there are 6 copies in the au). What confuses me is the output pdb file only has one chain (huge Rwork&Rfree, >0.5). Should there be 6 chains? The other issue is that the output chain has fewer residues than my sequence file (both N&C-terminal). It also has fewer residues compared with the pdb model file. Is this because I do not have enough resolution to resolve those missing residues? Thanks in advance. Jason U Pitt
Hi Jason, I am guessing that your starting model is an MR solution and you have data that is not very high-resolution or the MR solution is not that close to the structure that you are trying to solve. In this case these results would indicate that autobuild has failed to create a useful map of your structure and then it is unable to build a useful model, unable to identify sequence register, resulting in a single set of chains not assigned to sequence (and probably mostly incorrect). So a key question is: if you take your starting model and simply calculate an R factor (with or without refinement), is this R much less than 0.5? If not, this will be very difficult to rebuild, particularly if the resolution is not high. An alternative question: the starting map in autobuild (cycle_best_2.mtz)...does it look like your starting model? Does it look like a protein? If not....this will be difficult. Let me know if that doesn't help! All the best, Tom T
Dear All,
I am using phenix.autobuild .eff file to build a structure (version 1.6.1). For the input I have a pdb model file with 6 chains; a sequence file which contains residues of a single chain (the sequence file has a few more residues at N-terminal than the pdb model file); an mtz amplitude file; I also specified ncs_copies=6 (I know there are 6 copies in the au).
What confuses me is the output pdb file only has one chain (huge Rwork&Rfree, >0.5). Should there be 6 chains?
The other issue is that the output chain has fewer residues than my sequence file (both N&C-terminal). It also has fewer residues compared with the pdb model file. Is this because I do not have enough resolution to resolve those missing residues? Thanks in advance.
Jason
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i have one 3.27 A data of one of my protein. it is a multidomain protein . i used molecular repacement (phaser ) to find individual domains i got density for 3 domains. with last 4th domain i am getting solution with 5 clashes and density is nort clear for that . so i used phenix find helix and strands module i got 43 fragments model . now i dont know how to proceed further how i can start fitting. suggest me for further step .
vandana wrote:
i have one 3.27 A data of one of my protein. it is a multidomain protein . i used molecular repacement (phaser ) to find individual domains i got density for 3 domains. with last 4th domain i am getting solution with 5 clashes and density is nort clear for that . so i used phenix find helix and strands module i got 43 fragments model . now i dont know how to proceed further how i can start fitting. suggest me for further step . ------------------------------------------------------------------------
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Hi, What happens when you carry the molecular replacement search using the entire protein (4 domains) as the search model? This is only applicable, of course, if the 4 domains have the same relative orientation in the model and in your structure. Sometimes, one has to resort to real space molecular replacement in order to position domains, an example is given in Cosenza et al. (2002), J. Mol. Biol. 318, 1417-1432 (one domain had to be placed "in real space" after suitable density modification, i.e. "protein flattening" - don't know if it is possible to do that in Phenix at the moment). Further, you may have one domain that is disordered (or much less ordered) than the other 3. An example is in Xia et al. (1987), PNAS 84, 2629. Fred.
Something we've been using recently in our own work is to average the density, cut it out, and then use the density as a model to look for additional copies. This works pretty well, so we will be making it easier to do within Phenix, but at the moment it's not trivial. I'm just en route to a holiday, so I won't be able to help for a couple of weeks, but if you'd like some help trying that option, get in touch with me (preferably off-list) in the second week of August. All the best, Randy On Jul 22 2010, vandana wrote:
i have one 3.27 A data of one of my protein. it is a multidomain protein . i used molecular repacement (phaser ) to find individual domains i got density for 3 domains. with last 4th domain i am getting solution with 5 clashes and density is nort clear for that . so i used phenix find helix and strands module i got 43 fragments model . now i dont know how to proceed further how i can start fitting. suggest me for further step .
Hi Tom, The ncs copy problem is fixed by setting rebuild_in_place=False. The outcome does have 6 chains in the au. Facts: -input: a sequence file, a pdb model file, an mtz file; the sequence file is the same as the pdb model except that the sequence file has 6 more residues at N-terminal; the pdb model is almost the same as my protein structure. -problem: the output chain sequence is not the same as the sequence file (not the same as the input model file either). It misses some residues at the N-terminal and it adds some residues at the C-terminal (but disconnected from the C-terminal). Is it normal in autobuild? How to correct it? Thanks. Jason U Pitt
participants (5)
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J J -
Randy J. Read -
Thomas C. Terwilliger -
vandana -
Vellieux Frederic