Reply to Sleutel and Remaut, ^“ Structural insights into <i>Escherichia coli</i> CsgA amyloid fibril assembly revisited”

T he discovery of functional amyloids in bacteria dates back several decades, and our understanding of the Escherichia coli curli biogenesis system has steadily evolved over time.Since Chapman's seminal work in 2002, which involved isolation, characterization, and depolymerization of the major curli subunit CsgA, a variety of structural tools have been employed to elucidate curli architecture.Notably, later on, both solid-state nuclear magnetic resonance spectroscopy (NMR) and X-ray diffraction offered critical insights into the structure of E. coli CsgA fibrils, revealing their hallmark cross-β architecture (1, 2).Despite these advances, curli fibers have remained challeng ing to characterize structurally, primarily due to the high aggregation propensity and intrinsically disordered nature of monomeric CsgA. Structural insights into E. coli CsgA and P. korlensis R15.5 amyloid fibril assemblyMotivated by recent progress in cryo-electron microscopy (cryo-EM), we initiated efforts in 2022 to determine the high-resolution structure of E. coli CsgA amyloid fibrils.However, the disordered nature of wild-type CsgA posed significant obstacles for downstream cryo-EM analysis.In the course of our work, we encountered an innovative study by Chapman's group (3), which described the engineering of a double-cysteine mutant of CsgA, with mutations placed in non-amyloidogenic regions.They demonstra ted that " CsgA CC +TCEP formed fibril structures of identical morphology to wild-type fibers, " as confirmed by transmission electron microscopy.We found this mutant particularly advantageous for our study, as it enabled more controlled polymerization of CsgA.In addition, the engineered cysteines introduced two extra negative charges at pH levels above 8, potentially enhancing electrostatic repulsion and promoting fibril dispersion.To optimize structural analysis, we examined CsgA fibril morphology across a broad spectrum of buffer conditions.These efforts culminated in the acquisition of a cryo-EM density map that we were able to analyze.The resulting map revealed discernible secondary structure features of the CsgA fibrils and, for the first time, allowed us to measure the β-helical dimensions of CsgA (4), which we compared with previous computational predictions.Notably, these measurements aligned closely with the model proposed by Tian et al. ( 5), providing strong experimental validation.Additionally, our lower-resolution maps revealed how two fibrils make contact or bundle together, offering further insight into their supramolecular organization.However, our study also highlighted persistent challenges: "the unambiguous determi nation of monomer connectivity was not feasible at the current resolution" and "the detailed interactions between molecules within a fibril remain to be elucidated through future higher-resolution structural studies."During the submission of our manuscript, we became aware that Sleutel et al. ( 6) were also pursuing cryo-EM-based structural studies of CsgA amyloids.They subsequently published a study on fibrils of Pontibacter korlensis R15.5, a CsgA homolog.We were excited to see that both groups independently addressed the structural question of CsgA

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