{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,29]],"date-time":"2026-10-29T22:42:29Z","timestamp":1793313749831,"version":"4.5.0"},"reference-count":51,"publisher":"MDPI AG","issue":"1","license":[{"start":{"date-parts":[[2021,12,23]],"date-time":"2021-12-23T00:00:00Z","timestamp":1640217600000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100004359","name":"Swedish Research Council","doi-asserted-by":"publisher","award":["2018-03406"],"award-info":[{"award-number":["2018-03406"]}],"id":[{"id":"10.13039\/501100004359","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100002794","name":"Swedish Cancer Society","doi-asserted-by":"publisher","award":["20 1287 PjF"],"award-info":[{"award-number":["20 1287 PjF"]}],"id":[{"id":"10.13039\/501100002794","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Toxins"],"abstract":"<jats:p>Botulinum neurotoxins (BoNTs) are the causative agents of a potentially lethal paralytic disease targeting cholinergic nerve terminals. Multiple BoNT serotypes exist, with types A, B and E being the main cause of human botulism. Their extreme toxicity has been exploited for cosmetic and therapeutic uses to treat a wide range of neuromuscular disorders. Although naturally occurring BoNT types share a common end effect, their activity varies significantly based on the neuronal cell-surface receptors and intracellular SNARE substrates they target. These properties are the result of structural variations that have traditionally been studied using biophysical methods such as X-ray crystallography. Here, we determined the first structures of botulinum neurotoxins using single-particle cryogenic electron microscopy. The maps obtained at 3.6 and 3.7 \u00c5 for BoNT\/B and \/E, respectively, highlight the subtle structural dynamism between domains, and of the binding domain in particular. This study demonstrates how the recent advances made in the field of single-particle electron microscopy can be applied to bacterial toxins of clinical relevance and the botulinum neurotoxin family in particular.<\/jats:p>","DOI":"10.3390\/toxins14010014","type":"journal-article","created":{"date-parts":[[2021,12,23]],"date-time":"2021-12-23T21:40:21Z","timestamp":1640295621000},"page":"14","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":17,"title":["Structural Analysis of Botulinum Neurotoxins Type B and E by Cryo-EM"],"prefix":"10.3390","volume":"14","author":[{"given":"Sara","family":"Ko\u0161enina","sequence":"first","affiliation":[{"name":"Department of Biochemistry and Biophysics, Stockholm University, 10691 Stockholm, Sweden"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Markel","family":"Mart\u00ednez-Carranza","sequence":"additional","affiliation":[{"name":"Department of Biochemistry and Biophysics, Stockholm University, 10691 Stockholm, Sweden"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jonathan R.","family":"Davies","sequence":"additional","affiliation":[{"name":"Department of Biochemistry and Biophysics, Stockholm University, 10691 Stockholm, Sweden"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-9527-2310","authenticated-orcid":false,"given":"Geoffrey","family":"Masuyer","sequence":"additional","affiliation":[{"name":"Department of Biochemistry and Biophysics, Stockholm University, 10691 Stockholm, Sweden"},{"name":"Centre for Therapeutic Innovation, Department of Pharmacy and Pharmacology, University of Bath, Bath BA2 7AY, UK"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-4777-3417","authenticated-orcid":false,"given":"P\u00e5l","family":"Stenmark","sequence":"additional","affiliation":[{"name":"Department of Biochemistry and Biophysics, Stockholm University, 10691 Stockholm, Sweden"},{"name":"Department of Experimental Medical Science, Lund University, 22184 Lund, Sweden"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2021,12,23]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","unstructured":"Williamson, C.H., Sahl, J.W., Smith, T.J., Xie, G., Foley, B.T., Smith, L.A., Fern\u00e1ndez, R.A., Lindstr\u00f6m, M., Korkeala, H., and Keim, P. (2016). Comparative genomic analyses reveal broad diversity in botulinum-toxin-producing Clostridia. BMC Genom., 17.","DOI":"10.1186\/s12864-016-2502-z"},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"14130","DOI":"10.1038\/ncomms14130","article-title":"Identification and characterization of a novel botulinum neurotoxin","volume":"8","author":"Zhang","year":"2017","journal-title":"Nat. Commun."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"811","DOI":"10.1146\/annurev-biochem-013118-111654","article-title":"Botulinum and tetanus neurotoxins","volume":"88","author":"Dong","year":"2019","journal-title":"Annu. Rev. Biochem."},{"key":"ref_4","doi-asserted-by":"crossref","unstructured":"Rossetto, O., and Montecucco, C. (2019). Tables of toxicity of botulinum and tetanus neurotoxins. Toxins, 11.","DOI":"10.3390\/toxins11120686"},{"key":"ref_5","doi-asserted-by":"crossref","unstructured":"Rasetti-Escargueil, C., and Popoff, M.R. (2019). Antibodies and vaccines against botulinum toxins: Available measures and novel approaches. Toxins, 11.","DOI":"10.3390\/toxins11090528"},{"key":"ref_6","doi-asserted-by":"crossref","unstructured":"Stenmark, P., Dupuy, J., Imamura, A., Kiso, M., and Stevens, R.C. (2008). Crystal structure of botulinum neurotoxin type A in complex with the cell surface co-receptor GT1b-insight into the toxin-neuron interaction. PLoS Pathog., 4.","DOI":"10.2210\/pdb2vu9\/pdb"},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"2058","DOI":"10.1038\/ncomms3058","article-title":"Structure of dual receptor binding to botulinum neurotoxin B","volume":"4","author":"Berntsson","year":"2013","journal-title":"Nat. Commun."},{"key":"ref_8","doi-asserted-by":"crossref","unstructured":"Masuyer, G., Davies, J.R., and Stenmark, P. (2021). Mechanism of ganglioside receptor recognition by botulinum neurotoxin serotype E. Int. J. Mol. Sci., 22.","DOI":"10.3390\/ijms22158315"},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"1584","DOI":"10.1111\/j.1471-4159.2009.06093.x","article-title":"Cell entry strategy of clostridial neurotoxins","volume":"109","author":"Binz","year":"2009","journal-title":"J. Neurochem."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"10498","DOI":"10.1016\/S0021-9258(17)34087-5","article-title":"Identification of protein receptor for Clostridium botulinum type B neurotoxin in rat brain synaptosomes","volume":"269","author":"Nishiki","year":"1994","journal-title":"J. Biol. Chem."},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"253","DOI":"10.1016\/0014-5793(95)01471-3","article-title":"The high-affinity binding of Clostridium botulinum type B neurotoxin to synaptotagmin II associated with gangliosides GT1b\/GD1a","volume":"378","author":"Nishiki","year":"1996","journal-title":"FEBS Lett."},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"1293","DOI":"10.1083\/jcb.200305098","article-title":"Synaptotagmins I and II mediate entry of botulinum neurotoxin B into cells","volume":"162","author":"Dong","year":"2003","journal-title":"J. Cell Biol."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"30865","DOI":"10.1074\/jbc.M403945200","article-title":"Synaptotagmins I and II act as nerve cell receptors for botulinum neurotoxin G","volume":"279","author":"Rummel","year":"2004","journal-title":"J. Biol. Chem."},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"1092","DOI":"10.1038\/nature05387","article-title":"Botulinum neurotoxin B recognizes its protein receptor with high affinity and specificity","volume":"444","author":"Jin","year":"2006","journal-title":"Nature"},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"1096","DOI":"10.1038\/nature05411","article-title":"Structural basis of cell surface receptor recognition by botulinum neurotoxin B","volume":"444","author":"Chai","year":"2006","journal-title":"Nature"},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"1511","DOI":"10.1083\/jcb.200707184","article-title":"Mechanism of botulinum neurotoxin B and G entry into hippocampal neurons","volume":"179","author":"Dong","year":"2007","journal-title":"J. Cell Biol."},{"key":"ref_17","first-page":"3233","article-title":"Botulinum neurotoxin D-C uses synaptotagmin I and II as receptors, and human synaptotagmin II is not an effective receptor for type B, D-C and G toxins","volume":"125","author":"Peng","year":"2012","journal-title":"J. Cell Sci."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"591","DOI":"10.1146\/annurev.biochem.051908.125345","article-title":"Botulinum neurotoxin: A marvel of protein design","volume":"79","author":"Montal","year":"2010","journal-title":"Ann. Rev. Biochem."},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"474","DOI":"10.1126\/science.1161748","article-title":"Membrane fusion: Grappling with SNARE and SM proteins","volume":"323","author":"Rothman","year":"2009","journal-title":"Science"},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"717","DOI":"10.1152\/physrev.2000.80.2.717","article-title":"Neurotoxins affecting neuroexocytosis","volume":"80","author":"Schiavo","year":"2000","journal-title":"Physiol. Rev."},{"key":"ref_21","doi-asserted-by":"crossref","unstructured":"Fonfria, E., Maignel, J., Lezmi, S., Martin, V., Splevins, A., Shubber, S., Kalinichev, M., Foster, K., Picaut, P., and Krupp, J. (2018). The expanding therapeutic utility of botulinum neurotoxins. Toxins, 10.","DOI":"10.3390\/toxins10050208"},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"63","DOI":"10.1007\/164_2020_351","article-title":"Novel native and engineered botulinum neurotoxins","volume":"263","author":"Steward","year":"2021","journal-title":"Handb. Exp. Pharmacol."},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"e00446","DOI":"10.1002\/prp2.446","article-title":"A comparison of biological activity of commercially available purified native botulinum neurotoxin serotypes A1 to F1 in vitro, ex vivo, and in vivo","volume":"6","author":"Donald","year":"2018","journal-title":"Pharmacol. Res. Perspect."},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"84","DOI":"10.1016\/j.toxicon.2020.02.020","article-title":"Clinical duration of action of different botulinum toxin types in humans","volume":"179","author":"Eleopra","year":"2020","journal-title":"Toxicon"},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"116516","DOI":"10.1016\/j.jns.2019.116516","article-title":"Safety and pharmacodynamics of a novel recombinant botulinum toxin E (rBoNT-E): Results of a phase 1 study in healthy male subjects compared with abobotulinumtoxinA (Dysport\u00ae)","volume":"407","author":"Pons","year":"2019","journal-title":"J. Neurol. Sci."},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"847e","DOI":"10.1097\/PRS.0000000000005029","article-title":"Safety and efficacy of EB-001, a novel type E botulinum toxin, in subjects with glabellar frown lines: Results of a phase 2, Randomized, Placebo-Controlled, Ascending-Dose Study","volume":"142","author":"Yoelin","year":"2018","journal-title":"Plast. Reconstr. Surg."},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"898","DOI":"10.1038\/2338","article-title":"Crystal structure of botulinum neurotoxin type A and implications for toxicity","volume":"5","author":"Lacy","year":"1998","journal-title":"Nat. Struct. Biol."},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"693","DOI":"10.1038\/78005","article-title":"Structural analysis of the catalytic and binding sites of Clostridium botulinum neurotoxin B","volume":"7","author":"Swaminathan","year":"2000","journal-title":"Nat. Struct. Biol."},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"233","DOI":"10.1016\/j.jmb.2008.12.027","article-title":"Domain organization in Clostridium botulinum neurotoxin type E is unique: Its implication in faster translocation","volume":"386","author":"Kumaran","year":"2009","journal-title":"J. Mol. Biol."},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"16993","DOI":"10.1074\/jbc.M710442200","article-title":"Novel chimeras of botulinum neurotoxins A and E unveil contributions from the binding, translocation, and protease domains to their functional characteristics","volume":"283","author":"Wang","year":"2008","journal-title":"J. Biol. Chem."},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"3997","DOI":"10.1074\/jbc.M707917200","article-title":"Molecular architecture of botulinum neurotoxin E revealed by single particle electron microscopy","volume":"283","author":"Fischer","year":"2008","journal-title":"J. Biol. Chem."},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"1306","DOI":"10.15252\/embr.201744198","article-title":"The structure of the tetanus toxin reveals pH-mediated domain dynamics","volume":"18","author":"Masuyer","year":"2017","journal-title":"EMBO Rep."},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"3022","DOI":"10.1093\/molbev\/msab120","article-title":"MEGA11: Molecular evolutionary genetics analysis version 11","volume":"38","author":"Tamura","year":"2021","journal-title":"Mol. Biol. Evol."},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"W636","DOI":"10.1093\/nar\/gkz268","article-title":"The EMBL-EBI search and sequence analysis tools APIs in 2019","volume":"47","author":"Madeira","year":"2019","journal-title":"Nucleic Acids Res."},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"219","DOI":"10.1016\/S1046-5928(02)00002-5","article-title":"Expression and purification of catalytically active, non-toxic endopeptidase derivatives of Clostridium botulinum toxin type A","volume":"25","author":"Chaddock","year":"2002","journal-title":"Protein Expr. Purif."},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"70","DOI":"10.1002\/pro.3943","article-title":"UCSF ChimeraX: Structure visualization for researchers, educators, and developers","volume":"30","author":"Pettersen","year":"2021","journal-title":"Protein Sci."},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"861","DOI":"10.1107\/S2059798319011471","article-title":"Macromolecular structure determination using X-rays, neutrons and electrons: Recent developments in Phenix","volume":"75","author":"Liebschner","year":"2019","journal-title":"Acta Crystallogr. D Struct. Biol."},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"515","DOI":"10.1111\/j.1742-4658.2011.08444.x","article-title":"Engineering botulinum neurotoxin domains for activation by toxin light chain","volume":"279","author":"Stancombe","year":"2012","journal-title":"FEBS J."},{"key":"ref_39","doi-asserted-by":"crossref","first-page":"925","DOI":"10.1038\/nature03123","article-title":"Substrate recognition strategy for botulinum neurotoxin serotype A","volume":"432","author":"Breidenbach","year":"2004","journal-title":"Nature"},{"key":"ref_40","doi-asserted-by":"crossref","first-page":"1466","DOI":"10.1107\/S1744309111034671","article-title":"Structures of engineered Clostridium botulinum neurotoxin derivatives","volume":"67","author":"Masuyer","year":"2011","journal-title":"Acta Cryst. Sect. F Struct. Biol. Cryst. Commun."},{"key":"ref_41","doi-asserted-by":"crossref","first-page":"1062","DOI":"10.1038\/nprot.2018.018","article-title":"Characterizing metal-binding sites in proteins with X-ray crystallography","volume":"13","author":"Handing","year":"2018","journal-title":"Nat. Protoc."},{"key":"ref_42","doi-asserted-by":"crossref","first-page":"11139","DOI":"10.1073\/pnas.1708609114","article-title":"Detection of isolated protein-bound metal ions by single-particle cryo-STEM","volume":"114","author":"Elad","year":"2017","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_43","doi-asserted-by":"crossref","first-page":"1191","DOI":"10.1371\/journal.ppat.0030113","article-title":"Botulinum neurotoxin heavy chain belt as an intramolecular chaperone for the light chain","volume":"3","author":"Brunger","year":"2007","journal-title":"PLoS Pathog."},{"key":"ref_44","doi-asserted-by":"crossref","first-page":"17795","DOI":"10.1038\/srep17795","article-title":"Molecular Assembly of Clostridium botulinum progenitor M complex of type E","volume":"5","author":"Eswaramoorthy","year":"2015","journal-title":"Sci. Rep."},{"key":"ref_45","first-page":"76","article-title":"Enhanced fold recognition using efficient short fragment clustering","volume":"1","author":"Krissinel","year":"2012","journal-title":"J. Mol. Biochem."},{"key":"ref_46","doi-asserted-by":"crossref","first-page":"2209","DOI":"10.1021\/bi035844k","article-title":"Role of metals in the biological activity of Clostridium botulinum neurotoxins","volume":"43","author":"Eswaramoorthy","year":"2004","journal-title":"Biochemistry"},{"key":"ref_47","doi-asserted-by":"crossref","first-page":"977","DOI":"10.1126\/science.1214270","article-title":"Botulinum neurotoxin is shielded by NTNHA in an interlocked complex","volume":"335","author":"Gu","year":"2012","journal-title":"Science"},{"key":"ref_48","doi-asserted-by":"crossref","first-page":"1240","DOI":"10.1038\/s41467-021-21509-5","article-title":"Local computational methods to improve the interpretability and analysis of cryo-EM maps","volume":"12","author":"Kaur","year":"2021","journal-title":"Nat. Commun."},{"key":"ref_49","doi-asserted-by":"crossref","first-page":"583","DOI":"10.1038\/s41586-021-03819-2","article-title":"Highly accurate protein structure prediction with AlphaFold","volume":"596","author":"Jumper","year":"2021","journal-title":"Nature"},{"key":"ref_50","doi-asserted-by":"crossref","first-page":"290","DOI":"10.1038\/nmeth.4169","article-title":"cryoSPARC: Algorithms for rapid unsupervised cryo-EM structure determination","volume":"14","author":"Punjani","year":"2017","journal-title":"Nat. Methods"},{"key":"ref_51","doi-asserted-by":"crossref","first-page":"12","DOI":"10.1107\/S0907444909042073","article-title":"MolProbity: All-atom structure validation for macromolecular crystallography","volume":"66","author":"Chen","year":"2010","journal-title":"Acta Crystallogr. D"}],"container-title":["Toxins"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/2072-6651\/14\/1\/14\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,11]],"date-time":"2025-10-11T07:52:24Z","timestamp":1760169144000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/2072-6651\/14\/1\/14"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2021,12,23]]},"references-count":51,"journal-issue":{"issue":"1","published-online":{"date-parts":[[2022,1]]}},"alternative-id":["toxins14010014"],"URL":"https:\/\/doi.org\/10.3390\/toxins14010014","relation":{},"ISSN":["2072-6651"],"issn-type":[{"value":"2072-6651","type":"electronic"}],"subject":[],"published":{"date-parts":[[2021,12,23]]}}}