Bottromycin is produced naturally as a series of products differing in methylation patterns. All products contain valine and phenylalanine methylation. Bottromycin A2 is singly methylated on proline, bottromycin B lacks methylation on proline, and bottromycin C contains a doubly methylated proline.
A partial structure of bottromycin was reported shortly after the initial discovery of bottromycin. The first structural studies relied on traditional methods of analysis. Its peptide-like structure, including the presence of glycine and valine, was first suggested by a combination of acidic hydrolysis, acetylation, ninhydrin staining, and paper chromatography, among other experiments. The presence of a thiazole ring, along with an adjacent β-methylated phenylalanine, was established by ninhydrin staining, potassium permanganate oxidation, and comparison to synthetic standards. A methyl ester substituent was reported in 1958. The same study also reported that the Kunz hydrolysis product lacking a methyl ester was biologically inactive. Nakamura and colleagues later reported that bottromycin contained tert-leucine and cis-3-methylproline. They also proposed a linear iminohexapeptide structure.
These early structural studies were not followed up until recent years with the renewed interest in bottromycin. The structure was confirmed in the 1980s and 1990s to be a cyclic iminopeptide based on NMR studies, with a linear side chain connected to the macrocycle via an amidine linkage.
Its absolute stereochemistry, however, was not characterized until 2009. Stereochemistry at carbon 18 and 25 was proposed by comparing predicted conformers obtained using  molecular dynamics to experimental constraints obtained through NMR experiments. Stereochemistry at carbon 43 was confirmed by comparing H NMR of authentic hydrolysis product to a chemically synthesized sample of the same fragment. Finally, optical rotation, H NMR, and HRMS experiments of chemically synthesized bottromycin matched that of biologically produced bottromycin.
The three-dimensional solution structure of bottromycin A2 was solved by NMR in 2012. The overall structure was obtained with good resolution (RMSD 0.74±0.59 Å), with a RMSD of 0.09±0.06 Å for the macrocycle. In this study, it was proposed that the methylated proline residue contributed to the restricted conformation of the macrocycle. The methylated proline and β-OMe alanine residues were found to be on the same face of bottroymycin A2 and it was suggested that this characteristic contributed to binding of bottromycin to the ribosomal A site.