The acyl-CoA N-acyltransferase domain (ypeA) of putative acyltransferase from Escherichia coli str. K-12 substr. MG1655 was co-crystallized with acyl-CoA, and its structure was determined by molecular replacement and refined to 1.95 A resolution. The P21 asymmetric unit contains 8 copies of the domain. Each chain binds acyl-CoA at the solvent exposed groove between oligomerization and core subdomains (see Annotation for apo-structure; PDB entry 4qus). Acyl-CoA occupies only half of the groove’s length (about 40 A). While the nucleotide portion of the ligand is positioned between the helix from the oligomerization subdomain and two helices from the core subdomain (at one end of the groove; an area of positive surface charge distribution), the aliphatic tail of the ligand extends along the groove into a small pocket that accommodates the acetyl group. Another end of the groove ends with heavily positively charged surface area.