DNA double helix with many molprobity bad clashes
Hi, I am refining a protein-DNA complex at around 3 A with a standard b-form dsDNA. R-factors are around 24/20 (kinda low for the resolution). Originally, I placed the DNA using coot and now, after several rounds of refinement in phenix, I get a long list of bad clashes from molprobity validation (in phenix) that covers the whole length of the DNA and almost exclusively lists hydrogens from the ribose moiety (whether or not in contact with protein). My question is, - why is phenix.refine not taking care of these clashes (i.e. optimizes the structure such that they go away)?. Am I giving experimental contribution to high a weight (I have "optimize x-ray/stereochemistry" checked)? - if so, can I (and how) restrain geometry for the DNA a little more but leave the protein alone (and does that make sense)? thanks for any help Christian
On Wed, Nov 21, 2012 at 5:12 AM, Benda, Christian
- why is phenix.refine not taking care of these clashes (i.e. optimizes the structure such that they go away)?. Am I giving experimental contribution to high a weight (I have "optimize x-ray/stereochemistry" checked)?
Each restraint term needs to be balanced against the rest of the restraints and the X-ray target, and there is no guarantee that all parameters will refine to reasonable values. In fact it is remarkably easy to come up with strained conformations with bad clashes which can't be fixed without deleting parts of the model and rebuilding from scratch. (This is partly because of the way our minimizer works - much more aggressive sampling like Rosetta uses often does a better job getting rid of clashes, at the expense of significantly longer runtimes.) In your case I'm not sure what would be the explanation - two other possibilities include: - something pathological about where the hydrogens are placed, e.g. inverted chirality which causes them to clash (this can usually be fixed by removing and re-adding them) - something specific to nucleic acids, where the geometry is traditionally not as well restrained in refinement Do you mean that the ribose hydrogens are clashing with each other, or are there other atoms involved in the contacts? I'd be interested in seeing the model and maps if you're willing to share them (off-list, of course!).
- if so, can I (and how) restrain geometry for the DNA a little more but leave the protein alone (and does that make sense)?
Currently there is an option to apply a constant scale to covalent restraint terms for an arbitrary atom selection, but it doesn't work for non bonded interactions yet (this may be problematic anyway). It is possible to set the weight on the nonbondeds much higher overall though - the nonbonded_weight parameter controls this. You could set this maybe as high as 200 before the refinement really starts to choke, but we haven't tested this very much other than to make sure that the default is somewhat sensible. I do have an unrelated suggestion however: the nightly builds of Phenix have completely new hydrogen parameters for Reduce, Probe, and the geometry restraints, and their handling in refinement has changed significantly. So I would recommend trying this: 1) download and install the latest nightly build (currently dev-1218) 2) remove and replace existing hydrogens in the model 3) re-run the refinement with weight optimization (definitely a good option to use at this stage) If you still see the same clashes there is something else wrong. -Nat
Hi Benda,
I am refining a protein-DNA complex at around 3 A with a standard b-form dsDNA. R-factors are around 24/20 (kinda low for the resolution). Originally, I placed the DNA using coot and now, after several rounds of refinement in phenix, I get a long list of bad clashes from molprobity validation (in phenix) that covers the whole length of the DNA and almost exclusively lists hydrogens from the ribose moiety (whether or not in contact with protein). My question is,
- why is phenix.refine not taking care of these clashes (i.e. optimizes the structure such that they go away)?. Am I giving experimental contribution to high a weight (I have "optimize x-ray/stereochemistry" checked)?
because if the model is in a local minimum minimization will not kick it out of it. Simulated annealing refinement (available in phenix.refine) is the tool for this. This is illustrated on pages 67-71 here: http://www.phenix-online.org/presentations/latest/pavel_refinement_general.p... Simply make sure you are using recent Phenix version (so you get consistent clash-scores with Molprobity), add H atoms and run SA refinement. Pavel
Hi Christian,
Check that your OP1 and OP2 aren't swapped. Phenix doesn't check for this
and is unable to resolve it automatically but your geometry gets distorted
if they are swapped.
Get back to us if you resolve the problem.
Cheers,
Morten
On 21 November 2012 17:17, Pavel Afonine
Hi Benda,
I am refining a protein-DNA complex at around 3 A with a standard b-form
dsDNA. R-factors are around 24/20 (kinda low for the resolution). Originally, I placed the DNA using coot and now, after several rounds of refinement in phenix, I get a long list of bad clashes from molprobity validation (in phenix) that covers the whole length of the DNA and almost exclusively lists hydrogens from the ribose moiety (whether or not in contact with protein). My question is,
- why is phenix.refine not taking care of these clashes (i.e. optimizes the structure such that they go away)?. Am I giving experimental contribution to high a weight (I have "optimize x-ray/stereochemistry" checked)?
because if the model is in a local minimum minimization will not kick it out of it. Simulated annealing refinement (available in phenix.refine) is the tool for this.
This is illustrated on pages 67-71 here: http://www.phenix-online.org/**presentations/latest/pavel_** refinement_general.pdfhttp://www.phenix-online.org/presentations/latest/pavel_refinement_general.p...
Simply make sure you are using recent Phenix version (so you get consistent clash-scores with Molprobity), add H atoms and run SA refinement.
Pavel
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-- Morten K Grøftehauge, PhD Pohl Group Durham University
On Fri, Nov 30, 2012 at 3:14 AM, Morten Groftehauge
Check that your OP1 and OP2 aren't swapped. Phenix doesn't check for this and is unable to resolve it automatically but your geometry gets distorted if they are swapped.
Following up on this (we had considerable discussion off-list), apparently the problem was an issue of nomenclature - some of the hydrogen atoms were not being recognized by Probe as covalently bonded to the DNA backbone, so it thought there were clashes there. (phenix.refine is more flexible about atom names because Ralf added a bunch of special cases to the PDB interpretation - but Probe has an entirely monolithic codebase.) What we haven't been able to do yet is reproduce the problem using only tools in Phenix, so if anyone else has come across the same thing, we'd be very interested in seeing the model with a description of how it was obtained. -Nat
participants (4)
-
Benda, Christian -
Morten Groftehauge -
Nathaniel Echols -
Pavel Afonine