Model validation: local diagnosis, correction and when to quit

Abstract

Traditionally, validation was considered to be a final gatekeeping function, but refinement is smoother and results are better if model validation actively guides corrections throughout structure solution. This shifts emphasis from global to local measures: primarily geometry, conformations and sterics. A fit into the wrong local minimum conformation usually produces outliers in multiple measures. Moving to the right local minimum should be prioritized, rather than small shifts across arbitrary borderlines. Steric criteria work best with all explicit H atoms. `Backrub' motions should be used for side chains and `P-perp' diagnostics to correct ribose puckers. A `water' may actually be an ion, a relic of misfitting or an unmodeled alternate. Beware of wishful thinking in modeling ligands. At high resolution, internally consistent alternate conformations should be modeled and geometry in poor density should not be downweighted. At low resolution,CaBLAMshould be used to diagnose protein secondary structure andERRASERto correct RNA backbone. All atoms should not be forced inside density, beware of sequence misalignment, and very rare conformations such ascis-non-Pro peptides should be avoided. Automation continues to improve, but the crystallographer still must look at each outlier, in the context of density, and correct most of them. For the valid few with unambiguous density and something that is holding them in place, a functional reason should be sought. The expectation is a few outliers, not zero.

MolProbity: More and better reference data for improved all‐atom structure validation

“…Except in a small structure at high resolution, zero outliers usually means overfitting the data. A recent overview from a CCP4 Study Weekend presentation gives our best current guidance on how to tackle rebuilding the different types of validation outliers, with examples, and advice on when to stop.…”

“…The change seems to be leveling off, as it must, since it cannot go below zero. In fact, this measure is sometimes interpreted too stringently: the goal is few clashes, as in the best reference data, not zero clashes …”

Nucleobase carbonyl groups are poor Mg2+inner-sphere binders but excellent monovalent ion binders—a critical PDB survey

“…Thus, crystallographic structures deposited to the PDB (Berman et al 2016) remain the main source of information regarding Mg 2+ binding modes. Nonetheless, correctly assigning an ion to an experimental electron density pattern is notoriously difficult and, unfortunately, the PDB embeds a significant number of well-and not so well-documented assignment errors (Williams 2005;Wlodawer et al 2008Wlodawer et al , 2013Wlodawer et al , 2018Kleywegt 2009;Cooper et al 2011;Joosten et al 2012;Pozharski et al 2013;Dauter et al 2014;Echols et al 2014;Jain et al 2015;Weichenberger et al 2015;Minor et al 2016;Raczynska et al 2016;Rupp 2016;van Beusekom et al 2016;Richardson et al 2018). Incorrectly identified ions considerably bias database analysis results.…”

Challenges and opportunities in cryo-EM single-particle analysis

“…6). There are numerous good reviews on validation topics that go into much more detail (178,179). Furthermore, with the recent "Frontiers in Cryo-EM Validation" meeting held in Hinxton, United Kingdom, in January 2019, we can soon expect a timely update to the established standards set several years back (180).…”