Hi, >> 9. The selenomethionine dataset was solved using MIRAS in SHARP/autoSHARP. >> The experimentally phased electron density yields contiguous tracts of >> density in the right place, unbiased density indicates a good solution. >> Model building was conducted in P212121, initially into the experimental >> maps and later with refinement in refmac using HL-coefficient-based >> restraints. In some regions, sequence can easily be deduced from clean >> electron density (for the resolution). In other regions, side chains are >> missing, and in others, density is completely inconsistent with the >> connectivity of the chains and highly conserved structural elements. As >> occurs sometimes with twinned data, many loops cannot be modelled at all, >> and the Rfree does not drop below 0.41 with an Rfactor of 0.34. The >> result >> is a model that is about 60-70% complete. Refinement was performed with >> and >> without B-factor refinement.
It is not uncommon to have some severe density truncations in density modification from difficult/poor data/phases. Since you have a partial model, I would recommend a procedure we've used over the last years quite a few times: feed-back of the model into density modification (see e.g. http://scripts.iucr.org/cgi-bin/paper?S090904950302394X). It is easily done in the SHARP/Sushi interface: - copy your latest/best model into sharpfiles/datafiles/model_xyz.pdb - start a new density modification run with this initial model as an additional phase information: our SOLOMON script will then start not just from the SHARP phases, but from the SHARP+model phases. There are some decisions you might need to do * how many cycles to use this model: if you're worried about bias, just use it for a single cycle. Usually, I would expect a model that is based on some initial experimental map to be fairly unbiased - unless you used some homologuous structure as an initial model. I usually use the model for say 10 cycles and doing 40 cycles of density modification. * you might want to redo the optimisation of the solvent content: since you now start with a better initial map (hopefully), this might give you a very different solvent content - initially poor regions that were flattened might pop up. - have a look at the resulting map (FBshasol/PHIBshasol column if you don't use the tools in Sushi) and try to correct your model and - maybe - complete it. - go back to the start with a now improved model. I tend to do this 3-4 times at least (sometimes not a lot seems to be happening at the first 1-2 cycles, but often it suddenly picks up). If you want to help your refinement (e.g. in REFMAC) with HLs from SHARP: you want to use FP/SIGFP and HLA-D from the eden.mtz file (or FPsha/SIGFPsha and HLA-D from a eden_flat_*.mtz file). These are in synch with each other. Also: remember to build MSE residues and adjust your f' value. If you want to improve the HA refinement in SHARP you could use phase information from a model or from a density modification run throughout the HA refinement and residual map calculation (but _not_ the phasing): if you're still missing a few Se or there are some alternate conformations for the Se, this might be a good way of picking them up. >> 11. phenix.autobuild is able to build a polyalanine model that covers >> about >> 25% of the molecule. I've had some very nice results recently with Kevin Cowtans BUCCANEER (latest binary from his WWW page): it seems especially good in building a lot of connected regions. If you use such an automatic built model in the iterative procedure mentioned above, you don't really have to worry about bias I guess. Cheers Clemens -- *************************************************************** * Clemens Vonrhein, Ph.D. vonrhein AT GlobalPhasing DOT com * * Global Phasing Ltd. * Sheraton House, Castle Park * Cambridge CB3 0AX, UK *-------------------------------------------------------------- * BUSTER Development Group (http://www.globalphasing.com) ***************************************************************
