Yes, I agree that MR is worth a shot, though depending on the resolution your life may be vastly easier with experimental phases! We've had several cases where the following kind of procedure works: find related structures with a very sensitive homology search (we like HHPRED, though other options probably work), use the corresponding sequence alignment to prune the models (with sculptor or chainsaw or other tools) and make a trimmed ensemble with ensembler. The last step can be very important, in trimming off any bits of the collection of models that do not constitute a conserved core. Then provide the ensemble to Phaser as a molecular replacement model. The trimmed ensemble is usually the best model, in our experience, but one of the individual models may be better in some cases, so that's also worth testing.
Also, the MR-Rosetta pipeline has had a substantial number of successes when the highest sequence identity was in the range of 15-25%. Best wishes, Randy Read > On 21 Jun 2017, at 17:08, Mark J van Raaij <mjvanra...@cnb.csic.es> wrote: > > If your data is good enough, your SeMets alone might well be enough. > Soaking native crystals in Hg compounds may also work, avoiding SeMet > altogether. We have had a lot of success with methylmercury chloride binding > to free Cys. You may have to experiment with different soaking times and > protocols. > Finally, don't give up on MR too early, what matters is the structural > similarity, not directly the sequence identity. We've had success once with > 19% identity. > Native protein may be much easier to produce than the SeMet and SeMet/SeCys > versions (and may differ a lot between proteins). > > Mark J van Raaij > Dpto de Estructura de Macromoleculas > Centro Nacional de Biotecnologia - CSIC > calle Darwin 3 > E-28049 Madrid, Spain > tel. (+34) 91 585 4616 > http://wwwuser.cnb.csic.es/~mjvanraaij > <http://wwwuser.cnb.csic.es/~mjvanraaij> > >> On 21 Jun 2017, at 17:46, Vito Calderone <calder...@cerm.unifi.it >> <mailto:calder...@cerm.unifi.it>> wrote: >> >> I am working on a protein having 360 residues. In its sequence there are 3 >> Met and 5 free Cys. >> I will need MAD to solve the structure since based on the sequence the >> closest homologue has 20% identity匢 suppose MR would be very unlikely to >> work卻o I would like to express a selenium derivative to exploit MAD. >> Looking in the literature 1 Se-Met every 120 residues seems not to comply >> the threshold to get a good anomalous signal. For this reason I would like >> to exploit both Met and Cys so I would have 8 seleniums per 360 residues. >> Could somenone suggest a reference to a protocol to express the double >> mutant protein in NON auxotrophic strains of E. coli which you have >> experienced working efficiently? >> Thanks > ------ Randy J. Read Department of Haematology, University of Cambridge Cambridge Institute for Medical Research Tel: + 44 1223 336500 Wellcome Trust/MRC Building Fax: + 44 1223 336827 Hills Road E-mail: rj...@cam.ac.uk Cambridge CB2 0XY, U.K. www-structmed.cimr.cam.ac.uk
