[phenixbb] Re: inconsistency of Molprobity results between RealSpaceRefine and ValidationCryoEM

Hi Kevin, additionally, check to see if this behavior persists in the latest Phenix, say in 2.0-5824. You are using an 'ancient' version and I had an impression that I was fixing something relevant to this some time ago. Also, Nigel's CDL vs no CDL would reasoning can explain differences for bond/angle deviations but still does not explain discrepancies in other metrics (Ramachandran, clashes, rotamers). Pavel On 9/25/25 11:37, Nigel Moriarty wrote:

Kevin

All of this arises from a simple miscommunication. The default restraints for refinement in Phenix is the Conformation Dependent Library. It basically changes the ideal values for the protein backbone based on the phi/psi.angles. While Moprobity in Phenix uses the same libraries, it appears the communication has broken so it's not using the CDL but rather the single value restraints. As a result, .geo file (which is the gold standard) is correct and the Molprobity is slightly wrong. As a power user (you look in the .geo file), I suggest you check the .geo file until I correct this.

Cheers

Nigel

--- Nigel W. Moriarty Building 91, Molecular Biophysics and Integrated Bioimaging Lawrence Berkeley National Laboratory Berkeley, CA 94720-8235 Email : [email protected] Web : CCI.LBL.gov ORCID : orcid.org/0000-0001-8857-9464

On Tue, Sep 23, 2025 at 12:20 PM Kevin M Jude wrote:

...

I notice inconsistencies in the Molprobity results reported for the same structure from RealSpaceRefine and from ValidationCryoEM. As an example, for bond angle restraints, RSR reports that there are four >4sigma outliers (but does not list the outliers), while ValidationCryoEM reports 0.

RSR CryoEMValidation Number of restraints 13228 13228 RMS(deviation) 0.509 0.51 Max. deviation 9.997 9.998 Min. deviation 0 0 Number of outliers > 4sigma 4 0

Browsing through the .geo file, I don’t see any >4 sigma deviations among the highest residual angles, so I think this is an error.

I also see small discrepancies in Ramachandran favored, rotamer outliers, and clash score which seem to come down to minuscule differences in measurements at the margins.

This is for Phenix 1.21.2-5419. Happy to share files offline.

--

Kevin Jude, PhD

Structural Biology Research Specialist, Garcia Lab

Howard Hughes Medical Institute

Stanford University School of Medicine

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