A structure-guided classification framework reveals the diversity and catalytic architecture of BECR ribonuclease

Microorganisms across all domains of life engage in molecular conflict, deploying toxins to inhibit competitors or respond to biological threats. Among these, ribonuclease toxins are particularly widespread and diverse. A substantial fraction is associated with the BECR fold, a compact α/β architecture that supports RNase activity despite extensive divergence. Although several canonical members are well characterized, many BECR-fold proteins remain difficult to identify because of low sequence similarity, variation in catalytic residues, and structural elaborations that obscure evolutionary relationships. The growing availability of high-confidence protein structure predictions provides an opportunity to reassess this deeply divergent protein landscape. Here, we integrate iterative profile-HMM searches, profile-similarity networks, structural analyses, active-site mapping, and genomic context to examine BECR proteins across the tree of life. Our analysis resolves an expanded BECR-fold landscape comprising canonical BECR and BECR-like superfamilies, refines the organization of canonical BECR proteins and identifies previously unrecognized families. We further validate BECR-Tox2 as a toxin neutralized by a cognate immunity protein and show that its homologs occur in both Menshen-like anti-phage systems and polymorphic toxin loci. Together, these findings expand and clarify the BECR-fold landscape and provide a framework for identifying and interpreting highly divergent proteins of this fold.