Thanks for that suggestion, Phil. After several weeks, I've finally found solid answers to all my questions, which I'll write here in case anyone reading this message is curious: Q1: Is there any way to apply different map weights to different pieces of the structure during refinement? A1: Pavel tells me the answer is no—this feature is not available in Phenix yet. The way I was able to accomplish something to a similar effect was by performing full-model refinements at different weights. Then, in a final refinement job, use torsion restraints from each of those output models (as reference models) across the desired stretches of residues. Clunky, but it solved a few problems for me. Q2: How is the generic "weight" parameter at the bottom of the Phenix GUI applied to the refinement? A2: The target function is T = Tmap * weight + Trestraints. At the time of writing this message, there is a typo in the Phenix Real-Space Refine documentation, which incorrectly reports the target function as "T = Tmap + weight * Trestraints" (Pavel confirmed). Q3: Why is the PDB validation telling me I have geometric issues that were not acknowledged by Phenix? A3: Phil is right. The ESD for several nucleic acid bond lengths and angles is wide as compared to the sigma tolerated by wwPDB. To fix this, I went into the CIF files within the geostd folder and made the following changes to the troublesome RNA and DNA nucleotide files: *data_AD.cif, data_TD.cif, data_CD.cif, data_GD.cif:* length: C1'-N1, ESD=0.020 C3'-O3', ESD=0.020 angles: O4'-C1'-N9/N1, ESD=0.30 *data_U.cif* angles: C5-C6-N1, ESD=0.5 C6-N1-C2, ESD=0.5 *data_C.cif* angles: C6-N1-C2, ESD=0.5 *data_A.cif* angles: N7-C8-N9, ESD=0.5 Best, Josh On Mon, Sep 13, 2021 at 7:23 AM Philip D. Jeffrey wrote:

:: most of the outliers are not acknowledged as such (by Phenix), and I only become :: aware of them when the wwPDB validation tells me they are there.

Check that the equilibrium values that PDB are using and the ones that Phenix are using are the same. If they're not (or if the sigmas are way different) you're not going to be able to change the outcome just by changing weights.

Phil Jeffrey Princeton

------------------------------ *From:* [email protected] < [email protected]> on behalf of Josh Cofsky < [email protected]> *Sent:* Sunday, September 12, 2021 9:42 PM *To:* [email protected] *Subject:* Re: [phenixbb] Restricting real-space refinement and weights to specific atom selections

I now realize that I am confused as to what the mentioned "weight" parameter refers to. The Phenix RSR documentation contains a line that reads "T = Tmap + weight * Trestraints"—if I'm understanding correctly (?), this means a larger "weight" value should yield greater conformity to geometric restraints at the expense of model/map fit. In reality, I observe the opposite trend in my refinements (which is why I assumed it was a "map weight"). Can someone clarify what "weight" (Refinement Settings > "Other Options") refers to?

The reason I am exploring these questions is that Phenix RSR is introducing bond angle outliers into a low-resolution area of a DNA molecule in my model—most of the outliers are not acknowledged as such (by Phenix), and I only become aware of them when the wwPDB validation tells me they are there. I assumed this was the result of overfitting to the map because setting "weight" to 0.0 prevented these outliers from appearing, but at the expense of map-model fit in the remainder of the structure (which is why I wanted to apply different weights to different segments of the model).

Thanks, Josh

On Sun, Sep 12, 2021 at 12:21 PM Josh Cofsky wrote:

Hi all,

I'm using phenix.real_space_refine to refine a model into a cryo-EM map. My local map resolution is highly variable across the molecule, and using a single map weight (the parameter shown in the Refinement Settings > "Other Options" in the GUI) on the whole model leads to poor map-to-model fit in some locations and overfitting in others. Potential solutions could be to: 1. assign different map weights to different segments of the model (based on a user-specified atom selection) OR 2. perform iterative refinements with different weight settings, holding different (user-specified) segments of the model fixed during each iteration (I suspect this isn't possible based on Pavel's message here: http://phenix-online.org/pipermail/phenixbb/2021-May/025037.html)

Is either of these approaches (or another one that accomplishes the same goal) possible? I am using the Phenix GUI, but if this is only possible in command line, and you're willing to share an example command that accomplishes either of these tasks, please let me know.

Thanks! -Josh

(also, apologies if this is a duplicate message...still trying to figure out the phenixbb listserv)