3.2. How AlphaFold2 models helped improve the structure
The 2.6 Å resolution crystal structure of TSP4-N was determined by Molecular Replacement using a low-resolution structure (3.2 Å) that was initially built using the automated tracing program Autobuild42. Although the refinement progressed well, a strong residual difference electron density associated with Ile247 in the XD2 domain suggested that the experimental model required modification. A close examination of the AlphaFold2 model and the crystal structure revealed a polypeptide tracing error due to a wrong assignment of two neighboring proline residues (Pro236 and Pro239) carried over from the initial 3.2 Å structure. To better fit the electron density map, Pro236, located on a tight turn, was assigned a cis conformation, and Pro239 was assigned a trans conformation (Fig. 6A). Guided by the AlphaFold2 model, the two proline conformations were switched (now trans Pro236 and cis Pro239), and their positions shifted. The position of 20 amino acids were adjusted concomitantly so that Tyr249 was placed at the initial Ile247 position, which eliminated the residual difference electron density (Fig. 6B). This example demonstrates that in the future, the highly accurate models generated by AI methods will guide correct interpretations of low-resolution electron density maps generated by x-ray crystallography and cryo-EM, whenever difficulties exist in differentiating between cis and trans peptides.