question for a (putative?) disulfide bridge
Dear Modeller caretaker, using mutate_model I replaced a residue in a protein with Cys. Looking at the mutant protein, I saw that this new Cys residue is near to another Cys residue that was previously in the protein. The two sulphur moieties are not facing each others as in a disulfide bridge, but are rotated away. However, manually rotating the side chains, they can approach their sulphur moieties until less than 3A of distance, so in my opinion a disulfide bridge could be formed. At present I have not experimental data to confirm if a disulfide bridge is forming or not in this mutant. How can I check with MODELLER if this newly introduced mutation is really able to form a new disulfide bridge into my protein? How can I model this new disulfide bridge? Many thanks and best regards Anna Marabotti
______________________________________________ Anna Marabotti, Ph.D. Laboratorio di Bioinformatica e Biologia Computazionale Istituto di Scienze dell'Alimentazione, CNR Via Roma 52 A/C 83100 Avellino (Italy) Tel: +39 0825 299651 Fax: +39 0825 781585 Skype: annam1972 E-mail: amarabotti@isa.cnr.it Web page: http://bioinformatica.isa.cnr.it/anna/anna.htm Coming soon: BBCC2008: http://bioinformatica.isa.cnr.it/BBCC/BBCC2008 ____________________________________________________ "If you think you are too small to make a difference, try sleeping with a mosquito"
Anna Marabotti wrote: > using mutate_model I replaced a residue in a protein with Cys. > Looking at the mutant protein, I saw that this new Cys residue is > near to another Cys residue that was previously in the protein. The > two sulphur moieties are not facing each others as in a disulfide > bridge, but are rotated away. However, manually rotating the side > chains, they can approach their sulphur moieties until less than 3A > of distance, so in my opinion a disulfide bridge could be formed. At > present I have not experimental data to confirm if a disulfide bridge > is forming or not in this mutant. How can I check with MODELLER if > this newly introduced mutation is really able to form a new disulfide > bridge into my protein? How can I model this new disulfide bridge?
You can use model.patch() to add a disulfide bridge (see http://salilab.org/modeller/9v5/manual/node24.html) and build a model in the usual fashion (you could use your original model prior to mutate_model as your template, and use the mutated sequence as your target, or you could use the output from mutate_model as your template and use a 1:1 alignment). The standard assessment scores will give you an idea whether the resulting model is native-like, but you can't be sure that means a disulfide bridge exists.
Ben Webb, Modeller Caretaker
Dear Ben, thank you for your message, and sorry to bother you again. I tried to use model.patch() to add the disulfide bridge to my model, but I have a supplementary problem. In fact, my protein is a homodimer and the disulfide bridge should link two Cys residues in the two different subunits. I tried to indicate "A" and "B" in the script in this way: # Create the disulfide bond: def patches(mdl): mdl.patch(residue_type='DISU', residues=(mdl.residues['55:A.'], mdl.residues['180:B']))
but it doesn't work. What is the correct way to include this supplementary information about the chains? Many thanks again and best regards Anna
-----Messaggio originale----- Da: Modeller Caretaker [mailto:modeller-care@salilab.org] Inviato: giovedì 20 novembre 2008 18.58 A: Anna Marabotti Cc: modeller_usage@salilab.org Oggetto: Re: [modeller_usage] question for a (putative?) disulfide bridge
Anna Marabotti wrote: > using mutate_model I replaced a residue in a protein with Cys. > Looking at the mutant protein, I saw that this new Cys residue is > near to another Cys residue that was previously in the protein. The > two sulphur moieties are not facing each others as in a disulfide > bridge, but are rotated away. However, manually rotating the side > chains, they can approach their sulphur moieties until less than 3A > of distance, so in my opinion a disulfide bridge could be formed. At > present I have not experimental data to confirm if a disulfide bridge > is forming or not in this mutant. How can I check with MODELLER if > this newly introduced mutation is really able to form a new disulfide > bridge into my protein? How can I model this new disulfide bridge?
You can use model.patch() to add a disulfide bridge (see http://salilab.org/modeller/9v5/manual/node24.html) and build a model in the usual fashion (you could use your original model prior to mutate_model as your template, and use the mutated sequence as your target, or you could use the output from mutate_model as your template and use a 1:1 alignment). The standard assessment scores will give you an idea whether the resulting model is native-like, but you can't be sure that means a disulfide bridge exists.
Ben Webb, Modeller Caretaker
Anna Marabotti wrote: > thank you for your message, and sorry to bother you again. I tried to > use model.patch() to add the disulfide > bridge to my model, but I have a supplementary problem. In fact, my > protein is a homodimer and the disulfide > bridge should link two Cys residues in the two different subunits. I > tried to indicate "A" and "B" in the > script in this way: > > # Create the disulfide bond: > def patches(mdl): > mdl.patch(residue_type='DISU', residues=(mdl.residues['55:A.'], > mdl.residues['180:B'])) > > but it doesn't work.
What do you mean by "it doesn't work"? You get an error message (what is it?) or everything runs but the disulfide isn't created? In the latter case, I'd need to see the full script. In the former case, you probably have the wrong residue numbers and/or chains for your two Cys residues. The first residue should probably be '55:A' rather than '55:A.' but one way to check the numbers is to run modeling first without the patch, and look at the numbers in the generated model.
Ben Webb, Modeller Caretaker
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