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However, insufficient binding affinity between BoNT\/A and its receptor SV2C could lead to mild to severe side effects. An open active conformation of BoNT\/A cryo\u2010EM structure at a resolution of \u223c2.85\u00a0\u00c5 was resolved. Guided by the BoNT\/A cryo\u2010EM structure, saturation mutagenesis libraries were constructed and screened by BACTH system to identify mutants that can boost toxin\u2010SV2C affinity. The engineered toxin binding domain achieved up to approximately six\u2010fold improved affinity in SPR analysis, and the engineered toxins exhibited significantly improved binding capacity, and enhanced SNAP25 cleavage efficacy in cultured neurons. Preclinical animal studies, including MLB, DAS, sweat test, and PET\/CT assays, demonstrated that the engineered BoNT\/A VLTS has higher potency, lower diffusion, and significantly better safety profiles than the BoNT\/A wt, which can reduce side effects and benefit future therapeutic applications.<\/jats:p>","DOI":"10.1002\/advs.202516713","type":"journal-article","created":{"date-parts":[[2026,4,7]],"date-time":"2026-04-07T11:02:19Z","timestamp":1775559739000},"update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Cryo\u2010EM Structure Guided Engineering of Botulinum Neurotoxin A With Advanced Receptor Binding Affinity and Therapeutical Benefits"],"prefix":"10.1002","volume":"13","author":[{"ORCID":"https:\/\/orcid.org\/0009-0008-1442-0459","authenticated-orcid":false,"given":"Wenrui","family":"Wang","sequence":"first","affiliation":[{"name":"School of Pharmacy Joint Drug Development and Innovation Centre For Neurological Disorders of Lanzhou University\u2010China National Biotec Group\u2010Lanzhou Biotechnology Development Co. 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