{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"institution":[{"name":"bioRxiv"}],"indexed":{"date-parts":[[2026,6,24]],"date-time":"2026-06-24T06:05:07Z","timestamp":1782281107425,"version":"3.54.5"},"posted":{"date-parts":[[2025,12,14]]},"group-title":"Bioinformatics","reference-count":56,"publisher":"openRxiv","license":[{"start":{"date-parts":[[2025,12,14]],"date-time":"2025-12-14T00:00:00Z","timestamp":1765670400000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by-nc-nd\/4.0\/"}],"funder":[{"DOI":"10.13039\/100000002","name":"National Institutes of Health","doi-asserted-by":"crossref","award":["1R21AI187928-01A1"],"award-info":[{"award-number":["1R21AI187928-01A1"]}],"id":[{"id":"10.13039\/100000002","id-type":"DOI","asserted-by":"crossref"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"accepted":{"date-parts":[[2026,4,16]]},"abstract":"<jats:title>Abstract<\/jats:title>\n                <jats:p>\n                  At the molecular level, selection pressures often act on protein structural features, yet most evolutionary analyses remain confined to linear sequences. Early structure-informed approaches improved interpretability by mapping single-site metrics onto protein structures, and later methods introduced 3D sliding windows to capture spatially clustered signals missed by linear window approaches. These frameworks, however, are restricted to predefined statistics and narrowly defined 3D window types, limiting the scope of questions that can be addressed. We developed an R package,\n                  <jats:monospace>evo3D<\/jats:monospace>\n                  , as a new framework for structure-informed evolutionary analysis that supports a wide range of downstream statistics and scales from simple to complex structures.\n                  <jats:monospace>evo3D<\/jats:monospace>\n                  extracts structure-informed multiple sequence alignment subsets (spatial haplotypes), making the structure-informed unit of analysis directly available to users. The framework supports fixed-count and fixed-distance spatial windows, introduces residue and codon analysis modes, and extends to multimers, interfaces, and multiple structural models through a single wrapper,\n                  <jats:monospace>run_evo3d()<\/jats:monospace>\n                  . We demonstrate\n                  <jats:monospace>evo3D<\/jats:monospace>\n                  \u2019s utility by performing an epitope-level diversity scan of Hepatitis C virus E1\/E2 complex, identifying conserved spatial neighbourhoods missed by linear sliding windows, and by evaluating\n                  <jats:monospace>evo3D<\/jats:monospace>\n                  \u2019s scalability on the octameric Chikungunya virus E1\/E2 assembly. Importantly,\n                  <jats:monospace>evo3D<\/jats:monospace>\n                  formalises the core components of structure-informed analysis of molecular evolution and removes technical barriers. As a result, the framework streamlines the evaluation of evolutionary patterns directly within 3D structural contexts, and we anticipate its wide application in molecular evolution studies. The package is available at github.com\/bbroyle\/evo3D.\n                <\/jats:p>","DOI":"10.64898\/2025.12.12.694041","type":"posted-content","created":{"date-parts":[[2025,12,15]],"date-time":"2025-12-15T02:05:11Z","timestamp":1765764311000},"source":"Crossref","is-referenced-by-count":0,"title":["evo3D R package: a spatial haplotype framework for structure-informed analysis of molecular evolution"],"prefix":"10.64898","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-3608-4491","authenticated-orcid":false,"given":"Bradley K.","family":"Broyles","sequence":"first","affiliation":[{"name":"Department of Biological Sciences, Purdue University, West Lafayette, IN, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-1696-8203","authenticated-orcid":false,"given":"Qixin","family":"He","sequence":"additional","affiliation":[{"name":"Department of Biological Sciences, Purdue University, West Lafayette, IN, USA"},{"name":"Purdue Institute of Inflammation, Immunology and Infectious Disease, West Lafayette, IN, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"54368","reference":[{"key":"2026041811401175000_2025.12.12.694041v2.1","doi-asserted-by":"publisher","DOI":"10.1038\/S41586-024-07487-W"},{"key":"2026041811401175000_2025.12.12.694041v2.2","doi-asserted-by":"publisher","DOI":"10.1128\/JVI.02070-15"},{"key":"2026041811401175000_2025.12.12.694041v2.3","doi-asserted-by":"publisher","DOI":"10.1006\/jmbi.2000.4474"},{"key":"2026041811401175000_2025.12.12.694041v2.4","doi-asserted-by":"publisher","DOI":"10.1038\/s41586-024-07783-5"},{"key":"2026041811401175000_2025.12.12.694041v2.5","doi-asserted-by":"publisher","DOI":"10.1053\/j.gastro.2018.08.060"},{"key":"2026041811401175000_2025.12.12.694041v2.6","doi-asserted-by":"publisher","DOI":"10.2307\/2346101"},{"key":"2026041811401175000_2025.12.12.694041v2.7","doi-asserted-by":"publisher","DOI":"10.1007\/s00239-004-0223-4"},{"key":"2026041811401175000_2025.12.12.694041v2.8","doi-asserted-by":"publisher","DOI":"10.1093\/bioinformatics\/btv494"},{"key":"2026041811401175000_2025.12.12.694041v2.9","doi-asserted-by":"publisher","DOI":"10.1186\/1471-2105-10-421"},{"key":"2026041811401175000_2025.12.12.694041v2.10","doi-asserted-by":"crossref","unstructured":"Charif D , Lobry JR . 2007. SeqinR 1.0-2: a contributed package to the R project for statistical computing devoted to biological sequences retrieval and analysis. In: Bastolla U , Porto M , Roman HE , Vendruscolo M , editors. Structural approaches to sequence evolution: Molecules, networks, populations. Biological and Medical Physics, Biomedical Engineering. New York: Springer Verlag. p. 207\u2013232.","DOI":"10.1007\/978-3-540-35306-5_10"},{"key":"2026041811401175000_2025.12.12.694041v2.11","doi-asserted-by":"crossref","first-page":"btaf166","DOI":"10.1093\/bioinformatics\/btaf166","article-title":"CONSTRUCT: an algorithmic tool for identifying functional or structurally important regions in protein tertiary structure","volume":"41","year":"2025","journal-title":"Bioinformatics"},{"key":"2026041811401175000_2025.12.12.694041v2.12","doi-asserted-by":"crossref","first-page":"pgae102","DOI":"10.1093\/pnasnexus\/pgae102","article-title":"Cryogenic electron microscopy and tomography reveal imperfect icosahedral symmetry in alphaviruses","volume":"3","year":"2024","journal-title":"PNAS Nexus"},{"key":"2026041811401175000_2025.12.12.694041v2.13","doi-asserted-by":"crossref","first-page":"105227","DOI":"10.1016\/j.ebiom.2024.105227","article-title":"Diversity and selection analyses identify transmission-blocking antigens as the optimal vaccine candidates in Plasmodium falciparum","volume":"106","year":"2024","journal-title":"eBioMedicine"},{"key":"2026041811401175000_2025.12.12.694041v2.14","doi-asserted-by":"publisher","DOI":"10.1038\/nrg.2015.18"},{"key":"2026041811401175000_2025.12.12.694041v2.15","doi-asserted-by":"crossref","unstructured":"Eddelbuettel D . 2013. 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