Hi Claudia, To carry on from where you are, I would recommend fixing by hand any things that you can see are incorrect. If you know that the loops have been built connecting the wrong chains then I would definitely fix that. If the sequence has been incorrectly assigned I would fix that too. Your hand-fixed model can then be the starting point for another round of AutoBuild, either with "rebuilding_in_place=False" to try and add more backbone model, or with "rebuild_in_place=True" to just try to make a better fit of the model that you have built. You can then use "phenix.fit_loops" to try and fill in loops that have not yet been built. You might also try another approach from the beginning of model-building by starting with the MR model without trimming to poly-Ala. You might alternatively try the new Sculptor tools in the current PHENIX to set up your starting model. I am guessing that you may have a relatively low-resolution map as you have chosen to use "helices_strands_only=True". You might want to follow a different path and just use "rebuild_in_place=True" from the beginning, then cut out all the places where the starting model had the wrong sequence, and then use fit_loops to fill in these places. The advantage of this approach is that rebuild_in_place=True keeps your overall model, only adjusting where the main-chain goes and rebuilding side-chains from the Richardson rotamer library. If AutoBuild is doing a poor job at building your model from scratch then this might be a useful approach. All the best, Tom T

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Dear List,

Sorry fo r the easy question, but I have a problem with Autobuild.

I'm trying to rebuild a scFv, composed of two domains linked by a linker.

I've managed to find an MR solution with a polyalanine model deprived of the loops, which is my starting model for rebuilding.

I've given Autobuild three input files: the mtz, the model coordinates and the sequence of the scFv, asking to model only the beta strands and helices.

Problem is that in the final model Autobuild seems to guess incorrectly the density of two beta-strands, inserting the linker there instead of the expected side chains.

Any suggestions on how to correct this? Shall I manually rebuild this part to avoid problems? Or is it crucial to prevent errors, for example, that the model and the sequence are numbered so that they are in register?

Thanks in advance,

Claudia

Claudia Scotti Dipartimento di Medicina Sperimentale Sezione di Patologia Generale Universita' di Pavia Piazza Botta, 10 27100 Pavia Italia Tel. 0039 0382 986335/8/1 Facs 0039 0382 303673

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