On Thu, Oct 9, 2014 at 10:30 AM, Tim Gruene wrote:

a CC of 0.49 is quite strong

No no no no no. This would be true in the general case, but it is absolutely *not* a good CC for comparing local density, especially when judging LigandFit results. A LigandFit CC > 0.8 is almost certainly correct, > 0.7 is probably correct but may have some misfit atoms or poor density, > 0.6 is maybe correct but frequently wrong, and < 0.6 is almost certainly wrong. For comparing the refined model with 2mFo-DFc density, the thresholds are higher - a ligand with refined CC < 0.8 should be treated with extreme suspicion. Obviously we need a better measure than CC!

Is there a better way to fit the peptide into density?

LigandFit isn't really the appropriate tool for this - but I don't think there's actually an easy, automatic way to do what you want in Phenix right now. You can run AutoBuild with the existing model defined as "ligands", but that's going to be very slow. Although I'm a little surprised that AutoBuild didn't build any peptide - are you sure it isn't already in your model somewhere? Or did it build something and you removed it? If you didn't include it in the sequence file, it may be a random set of residues (AutoBuild's guess for the actual sequence). -Nat

Yes Sneha.., "apo" structure and the tweaked-peptide should be merged (under the calculate pull-down menu in coot) before subsequent refinement cycle(s). Best Wishes, Partha On Thu, Oct 9, 2014 at 4:16 PM, Sneha Rangarajan wrote:

@Partha: My peptide is 13 residues long. I don’t see the density for all of it (just few of them). Pardon me for the naïve question but once I superimpose it in coot and adjust its position should I merge it with my “apo” structure (make it part of my model) and refine it to make it fit the density better or something like that?

@Nat: If I want to run autobuild to fit the ligand into the density, should I give it my “apo” model as ‘starting model’ and peptide.pdb as ‘ligands’ alongwith the mtz?

And yes, the peptide was never part of any model or sequence file right from the start since I wanted to avoid any bias. While I see positive density, autobuild has not built any peptide into it.

Thanks a lot,

Sneha

*From:* Parthasarathy Sampathkumar [mailto:[email protected]] *Sent:* Thursday, October 09, 2014 2:06 PM *To:* Sneha Rangarajan *Cc:* Nathaniel Echols; [email protected] *Subject:* Re: [phenixbb] ligand fitting

Hi Sneha,

Easier option would be to superpose homolog-protein+peptide coordinates onto your "apo" structure (using SSM for example within coot), which might get the peptide closer to the binding-site of your protein, and then tweak it for better fit.

OR if the peptide is not too long, and its electron density is clear one might be able to build it manually from scratch.

Hope this helps,

Partha

On Thu, Oct 9, 2014 at 12:56 PM, Sneha Rangarajan wrote:

Hello everyone,

I have a question about ligand fitting into density.

At this point my maps look quite good with decent density for the peptide (ligand)[Rfactprs 26/31].

I tried using ligandfit by giving it the pdb and mtz of the ligand free model along with peptide.pdb (peptide stripped from a pdb where it was complexed with a homologous protein).

However the output was a ligand.pdb file with a CC of 0.49. I am not sure how to interpret this. Does this mean it could not find the density for the ligand?

Is there a better way to fit the peptide into density?

Thanks,

S

*From:* [email protected] [mailto: [email protected]] *On Behalf Of *Sneha Rangarajan *Sent:* Wednesday, October 08, 2014 10:30 AM *To:* Nathaniel Echols *Cc:* [email protected] *Subject:* Re: [phenixbb] (no subject)

This was a great idea. My Rfactors after a second round of autobuild are now 25/32. I think it might be getting there afterall J

S

*From:* Nathaniel Echols [mailto:[email protected] ] *Sent:* Friday, October 03, 2014 3:08 PM *To:* Sneha Rangarajan *Cc:* Pavel Afonine; [email protected] *Subject:* Re: [phenixbb] (no subject)

On Fri, Oct 3, 2014 at 11:58 AM, Sneha Rangarajan wrote:

I did another round of refinement with default settings (XYZ,realsp, IndB and occ) with and without weight optimization.

Without weight opt, the Rfactors are 23/36 with RMSbonds-0.0108 and RMSangles-1.750

One idea would be to run AutoBuild again. I've seen cases before where it didn't converge using the default settings, and feeding a previous result back into the program for a second run produced significantly better models. It might help get rid of the overfitting.

-Nat

_______________________________________________ phenixbb mailing list [email protected] http://phenix-online.org/mailman/listinfo/phenixbb

Thanks everyone for your suggestions. I have managed to fit my peptide into the density. My Rfactors are now 25/30 and geometry looks good too. One more question- When does one use “add hydrogens” in refinement? Does it help in the final stages of refinement? Thanks a lot, Sneha From: Nathaniel Echols [mailto:[email protected]] Sent: Thursday, October 09, 2014 5:21 PM To: Sneha Rangarajan Cc: [email protected] Subject: Re: [phenixbb] ligand fitting On Thu, Oct 9, 2014 at 1:16 PM, Sneha Rangarajan mailto:[email protected]> wrote: @Nat: If I want to run autobuild to fit the ligand into the density, should I give it my “apo” model as ‘starting model’ and peptide.pdb as ‘ligands’ alongwith the mtz? No, in AutoBuild terminology, "ligands" means "atoms I want AutoBuild to leave alone (but include in refinement)", which would mean your apo model. You wouldn't supply peptide.pdb at all, because AutoBuild isn't going to move it into the correct place; ideally you want it to just build new residues into the appropriate density. To be honest I think I would be tempted to simply build the peptide by hand, which shouldn't be too difficult at this resolution. I'm normally a big fan of letting the program do all of the heavy lifting, but this is one of those corner cases where the manual approach might be more efficient. (This is a point for future improvement in Phenix, of course.) Your own opinion of how easy it will be may be different than mine, however. -Nat

On Mon, Oct 13, 2014 at 9:31 AM, Sneha Rangarajan wrote:

One more question- When does one use “add hydrogens” in refinement? Does it help in the final stages of refinement?

In theory, yes. Early in refinement hydrogen atoms tend to just make phenix.refine much slower. Later in refinement, they can help with geometry and R-factors. Personally I've never seen much benefit from using hydrogens at moderate resolution, but it's worth trying them and seeing what happens. It's more important to pick optimal restraint weights (use the automatic weight optimization for this) and a good refinement strategy (add TLS, turn off real-space), and deal with any notable difference density peaks. -Nat

Thank you all. Yes, it has improved the geometry and the score slightly without much effect on the Rfactors. Sneha -----Original Message----- From: [email protected] [mailto:[email protected]] On Behalf Of Tim Gruene Sent: Monday, October 13, 2014 2:23 PM To: [email protected] Subject: Re: [phenixbb] ligand fitting Dear Sneha, the impact of hydrogens to the scattering is not very great, as the link provided by Pavel also illustrates. Their meaning for the geometry is much more important and - if properly implemented - the use of hydrogen atoms in refinement should give you a better molprobity score than not using them. I recommend to always use hydrogen atoms at any stage of refinement, although you won't notice it much in R-factors and such. They don't cost data, nor do they add to the number of parameters, so you don't loose anything, but you most likely gain better geometry. In my experience the rise in run time of the refinement program is negligible. Best, Tim On 10/13/2014 06:31 PM, Sneha Rangarajan wrote:

Thanks everyone for your suggestions. I have managed to fit my peptide into the density. My Rfactors are now 25/30 and geometry looks good too.

One more question- When does one use "add hydrogens" in refinement? Does it help in the final stages of refinement?

Thanks a lot, Sneha

From: Nathaniel Echols [mailto:[email protected]] Sent: Thursday, October 09, 2014 5:21 PM To: Sneha Rangarajan Cc: [email protected] Subject: Re: [phenixbb] ligand fitting

On Thu, Oct 9, 2014 at 1:16 PM, Sneha Rangarajan mailto:[email protected]> wrote: @Nat: If I want to run autobuild to fit the ligand into the density, should I give it my "apo" model as 'starting model' and peptide.pdb as 'ligands' alongwith the mtz?

No, in AutoBuild terminology, "ligands" means "atoms I want AutoBuild to leave alone (but include in refinement)", which would mean your apo model. You wouldn't supply peptide.pdb at all, because AutoBuild isn't going to move it into the correct place; ideally you want it to just build new residues into the appropriate density.

To be honest I think I would be tempted to simply build the peptide by hand, which shouldn't be too difficult at this resolution. I'm normally a big fan of letting the program do all of the heavy lifting, but this is one of those corner cases where the manual approach might be more efficient. (This is a point for future improvement in Phenix, of course.) Your own opinion of how easy it will be may be different than mine, however.

-Nat

_______________________________________________ phenixbb mailing list [email protected] http://phenix-online.org/mailman/listinfo/phenixbb

-- Dr Tim Gruene Institut fuer anorganische Chemie Tammannstr. 4 D-37077 Goettingen GPG Key ID = A46BEE1A