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.