Cryo-EM: problem with RosettaCM using multiple template models as input

[Anonymous]

 Hi,

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.

Any suggestions? 

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.

 

thank you

Karl

 

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