Cryo‐ <scp>EM</scp> Analysis in <scp>CASP16</scp>

Since CASP13, experimentalists have been encouraged to provide their cryo-EM data along with the derived atomic models to the CASP organizers to aid assessment. In CASP16, 38 cryo-EM datasets were provided for assessment, which represented most cryo-EM targets. The corresponding targets typically comprised a single derived atomic structure; however, that model may be only one of several valid conformations. Flexibility often manifests as low-resolution regions in a cryo-EM reconstruction, particularly in RNA but often also in protein complexes. We show that local resolution in the reconstruction correlates well with the root-mean-square fluctuations (RMSF) of residues of accurate CASP predictions. The correlation between the local resolution and pLDDT was less clear, especially when mobile domains were present. When the resolution allowed, assessment of features such as sidechains, using our variant of SMOC with local fragment alignment, indicated that even high-quality predictions have room for improvement; on the other hand, some predictions fitted the density better in specific regions, indicating modeling discrepancies in the target. In one extreme case, a submitted target had regions of low-resolution that limited unambiguous model building. In such cases, part of the target structure is essentially a prediction itself, with implications for the assessment. Experimental data remain essential for model-free assessment of predictions and offer unique analyses such as comparisons to local resolution and thus flexibility.