First of all, I am not sure about the category of my post, but the "Phenix/MR rosetta" category seems to be the most relevant, considering the topic of this post.
I have been folllowing the cryo-EM tutorial from 2015 to produce a model, which would be derived from the fragments of multiple templates and nicely fit to cryo-EM map. However, all produced models are out of density. I understand that the purpose of this protocol is to perform a more exhaustive sampling, but still several top-scored models are simply out of density, not even close. The problem is with both ccp4 and mrc maps.
Templates are aligned to the density. I checked in both COOT and Chimera, so it is not a problem of headers...I think.
Adding -relax::constrain_relax_to_start_coords flag with different values of of cst improve, but do not solve the problem.
rosetta_scripts.linuxgccrelease \ -database database \ -in::file::fasta fasta.fasta \ -parser::protocol xml.xml \ -edensity::mapfile mrc.mrc \ -edensity::mapreso 4.3 \ -edensity::cryoem_scatterers \ -relax:minimize_bond_angles \ -relax:min_type lbfgs_armijo_nonmonotone \ -relax:jump_move true \ -relax:default_repeats 2 \ -default_max_cycles 200 \ -nstruct 50 \ -out::suffix _ref2_$1 \
<ROSETTASCRIPTS> <SCOREFXNS> <stage1 weights="score3"> <Reweight scoretype=atom_pair_constraint weight=0.25/> </stage1> <stage2 weights="score4_smooth_cart"> <Reweight scoretype=atom_pair_constraint weight=0.25/> </stage2> <fullatom weights="talaris2013_cart"> <Reweight scoretype=atom_pair_constraint weight=0.25/> </fullatom> </SCOREFXNS> <MOVERS>¨ <Hybridize name=hybridize stage1_scorefxn=stage1 stage2_scorefxn=stage2 fa_scorefxn=fullatom batch=1> <Template pdb="thr_1.pdb" weight=1.0 cst_file="AUTO"/> <Template pdb="thr_2.pdb" weight=1.0 cst_file="AUTO"/> <Template pdb="thr_3.pdb" weight=1.0 cst_file="AUTO"/> <Template pdb="thr_4.pdb" weight=1.0 cst_file="AUTO"/> </Hybridize> </MOVERS> <PROTOCOLS> <Add mover=hybridize/> </PROTOCOLS> </ROSETTASCRIPTS>
2.) Additionally, I am not sure whether the tutorial itself is up to date. According to the tutorial, the "AUTO" flag should be "auto", but actually it should not be.
When I change to "auto" I get error = [ERROR] Unable to open constraints file: auto
3.) Finally, which flags specify rescoring againts density? Shouldn't we use any density scoring function? I mean, does the hybridizer follows any density data when building structures? The main script file also does not contain any density function.