{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,6,5]],"date-time":"2026-06-05T21:54:54Z","timestamp":1780696494333,"version":"3.54.1"},"reference-count":71,"publisher":"MDPI AG","issue":"7","license":[{"start":{"date-parts":[[2024,3,25]],"date-time":"2024-03-25T00:00:00Z","timestamp":1711324800000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"MIUR, Italy","award":["PRIN-201744NR8S"],"award-info":[{"award-number":["PRIN-201744NR8S"]}]},{"name":"PON \u201cRicerca e innovazione\u201d 2014\u20132020 (Italy)","award":["PRIN-201744NR8S"],"award-info":[{"award-number":["PRIN-201744NR8S"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["IJMS"],"abstract":"<jats:p>The advent of deep learning algorithms for protein folding opened a new era in the ability of predicting and optimizing the function of proteins once the sequence is known. The task is more intricate when cofactors like metal ions or small ligands are essential to functioning. In this case, the combined use of traditional simulation methods based on interatomic force fields and deep learning predictions is mandatory. We use the example of [FeFe] hydrogenases, enzymes of unicellular algae promising for biotechnology applications to illustrate this situation. [FeFe] hydrogenase is an iron\u2013sulfur protein that catalyzes the chemical reduction of protons dissolved in liquid water into molecular hydrogen as a gas. Hydrogen production efficiency and cell sensitivity to dioxygen are important parameters to optimize the industrial applications of biological hydrogen production. Both parameters are related to the organization of iron\u2013sulfur clusters within protein domains. In this work, we propose possible three-dimensional structures of Chlorella vulgaris 211\/11P [FeFe] hydrogenase, the sequence of which was extracted from the recently published genome of the given strain. Initial structural models are built using: (i) the deep learning algorithm AlphaFold; (ii) the homology modeling server SwissModel; (iii) a manual construction based on the best known bacterial crystal structure. Missing iron\u2013sulfur clusters are included and microsecond-long molecular dynamics of initial structures embedded into the water solution environment were performed. Multiple-walkers metadynamics was also used to enhance the sampling of structures encompassing both functional and non-functional organizations of iron\u2013sulfur clusters. The resulting structural model provided by deep learning is consistent with functional [FeFe] hydrogenase characterized by peculiar interactions between cofactors and the protein matrix.<\/jats:p>","DOI":"10.3390\/ijms25073663","type":"journal-article","created":{"date-parts":[[2024,3,25]],"date-time":"2024-03-25T12:47:11Z","timestamp":1711370831000},"page":"3663","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":5,"title":["Predicting the Structure of Enzymes with Metal Cofactors: The Example of [FeFe] Hydrogenases"],"prefix":"10.3390","volume":"25","author":[{"given":"Simone","family":"Botticelli","sequence":"first","affiliation":[{"name":"Department of Physics, University of Roma Tor Vergata, 00133 Rome, Italy"},{"name":"Section of Roma Tor Vergata, National Institute of Nuclear Physics, 00133 Rome, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-8619-4867","authenticated-orcid":false,"given":"Giovanni","family":"La Penna","sequence":"additional","affiliation":[{"name":"Section of Roma Tor Vergata, National Institute of Nuclear Physics, 00133 Rome, Italy"},{"name":"Institute of Chemistry of Organometallic Compounds, National Research Council, 50019 Florence, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-9851-9932","authenticated-orcid":false,"given":"Velia","family":"Minicozzi","sequence":"additional","affiliation":[{"name":"Department of Physics, University of Roma Tor Vergata, 00133 Rome, Italy"},{"name":"Section of Roma Tor Vergata, National Institute of Nuclear Physics, 00133 Rome, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-8102-7982","authenticated-orcid":false,"given":"Francesco","family":"Stellato","sequence":"additional","affiliation":[{"name":"Department of Physics, University of Roma Tor Vergata, 00133 Rome, Italy"},{"name":"Section of Roma Tor Vergata, National Institute of Nuclear Physics, 00133 Rome, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Silvia","family":"Morante","sequence":"additional","affiliation":[{"name":"Department of Physics, University of Roma Tor Vergata, 00133 Rome, Italy"},{"name":"Section of Roma Tor Vergata, National Institute of Nuclear Physics, 00133 Rome, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-9688-9454","authenticated-orcid":false,"given":"Giancarlo","family":"Rossi","sequence":"additional","affiliation":[{"name":"Department of Physics, University of Roma Tor Vergata, 00133 Rome, Italy"},{"name":"Section of Roma Tor Vergata, National Institute of Nuclear Physics, 00133 Rome, Italy"},{"name":"Museo Storico della Fisica e Centro Studi e Ricerche E. Fermi, 00184 Rome, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Cecilia","family":"Faraloni","sequence":"additional","affiliation":[{"name":"Institute of Bioeconomy, National Research Council, 50019 Florence, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2024,3,25]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","unstructured":"Seibert, M., and Torzillo, G. (2018). Microalgal Hydrogen Production, The Royal Society of Chemistry.","DOI":"10.1039\/9781849737128"},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"4081","DOI":"10.1021\/cr4005814","article-title":"Hydrogenases","volume":"114","author":"Lubitz","year":"2014","journal-title":"Chem. Rev."},{"key":"ref_3","doi-asserted-by":"crossref","unstructured":"Morra, S. (2022). Fantastic [FeFe]-Hydrogenases and Where to Find Them. Front. Microbiol., 13.","DOI":"10.3389\/fmicb.2022.853626"},{"key":"ref_4","unstructured":"G\u00f3mez Castro, F.I., and Guti\u00e9rrez-Antonio, C. (2022). Biofuels and Biorefining, Elsevier."},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"1289","DOI":"10.1111\/tpj.14508","article-title":"Chlorella vulgaris genome assembly and annotation reveals the molecular basis for metabolic acclimation to high light conditions","volume":"100","author":"Cecchin","year":"2019","journal-title":"Plant J."},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"102303","DOI":"10.1016\/j.algal.2021.102303","article-title":"Changes in photosynthesis, growth and biomass composition in outdoor Chlorella g120 culture during the metabolic shift from heterotrophic to phototrophic cultivation regime","volume":"56","author":"Rearte","year":"2021","journal-title":"Algal Res."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"3684","DOI":"10.1016\/j.ijhydene.2020.10.257","article-title":"Sustained photobiological hydrogen production by Chlorella vulgaris without nutrient starvation","volume":"46","author":"Touloupakis","year":"2021","journal-title":"Int. J. Hydrogen Energy"},{"key":"ref_8","unstructured":"Lennarz, W.J., and Lane, M.D. (2013). Encyclopedia of Biological Chemistry, Academic Press. [2nd ed.]."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"214191","DOI":"10.1016\/j.ccr.2021.214191","article-title":"The catalytic cycle of [FeFe] hydrogenase: A tale of two sites","volume":"449","author":"Birrell","year":"2021","journal-title":"Coord. Chem. Rev."},{"key":"ref_10","unstructured":"Schr\u00f6dinger, L. (2015). The PyMOL Molecular Graphics System, Version 1.8, Schr\u00f6dinger, LLC."},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"829","DOI":"10.1007\/s00425-011-1431-y","article-title":"Evolutionary significance of an algal gene encoding an [FeFe]-hydrogenase with F-domain homology and hydrogenase activity in Chlorella variabilis NC64A","volume":"234","author":"Meuser","year":"2011","journal-title":"Planta"},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"1227","DOI":"10.1021\/jacs.9b08756","article-title":"Tuning Catalytic Bias of Hydrogen Gas Producing Hydrogenases","volume":"142","author":"Artz","year":"2020","journal-title":"J. Am. Chem. Soc."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"13","DOI":"10.1016\/S0969-2126(99)80005-7","article-title":"Desulfovibrio desulfuricans iron hydrogenase: The structure shows unusual coordination to an active site Fe binuclear center","volume":"7","author":"Nicolet","year":"1999","journal-title":"Structure"},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"248","DOI":"10.1038\/nature08993","article-title":"Stepwise [FeFe]-hydrogenase H-cluster assembly revealed in the structure of HydA Delta EFG","volume":"465","author":"Mulder","year":"2010","journal-title":"Nature"},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"W500","DOI":"10.1093\/nar\/gkh429","article-title":"STRIDE: A web server for secondary structure assignment from known atomic coordinates of proteins","volume":"32","author":"Heinig","year":"2004","journal-title":"Nucleic Acids Res."},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"4206","DOI":"10.1021\/cr050196r","article-title":"Occurrence, Classification, and Biological Function of Hydrogenases: An Overview","volume":"107","author":"Vignais","year":"2007","journal-title":"Chem. Rev."},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"1809","DOI":"10.1021\/ja510169s","article-title":"[FeFe]-Hydrogenase Oxygen Inactivation Is Initiated at the H Cluster 2Fe Subcluster","volume":"137","author":"Swanson","year":"2015","journal-title":"J. Am. Chem. Soc."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"410","DOI":"10.1016\/j.bbabio.2008.02.002","article-title":"Isolation and first EPR characterization of the [FeFe]-hydrogenases from green algae","volume":"1777","author":"Kamp","year":"2008","journal-title":"Biochim. Biophys. Acta Bioenerg."},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"W296","DOI":"10.1093\/nar\/gky427","article-title":"SWISS-MODEL: Homology modelling of protein structures and complexes","volume":"46","author":"Waterhouse","year":"2018","journal-title":"Nucleic Acids Res."},{"key":"ref_20","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_21","doi-asserted-by":"crossref","first-page":"2237","DOI":"10.1016\/j.jmb.2017.12.007","article-title":"A Completely Reimplemented MPI Bioinformatics Toolkit with a New HHpred Server at its Core","volume":"430","author":"Zimmermann","year":"2018","journal-title":"J. Mol. Biol."},{"key":"ref_22","doi-asserted-by":"crossref","unstructured":"Binbay, F.A., Rathod, D.C., George, A.A.P., and Imhof, D. (2023). Quality Assessment of Selected Protein Structures Derived from Homology Modeling and AlphaFold. Pharmaceuticals, 16.","DOI":"10.3390\/ph16121662"},{"key":"ref_23","doi-asserted-by":"crossref","unstructured":"Liu, D., Yuan, C., Guo, C., Huang, M., and Lin, D. (2023). Structural and Functional Insights into the Stealth Protein CpsY of Mycobacterium tuberculosis. Biomolecules, 13.","DOI":"10.3390\/biom13111611"},{"key":"ref_24","doi-asserted-by":"crossref","unstructured":"Nagy, V., Podmaniczki, A., Vidal-Meireles, A., Teng\u00f6lics, R., Kov\u00e1cs, L., R\u00e1khely, G., Scoma, A., and T\u00f3th, S.Z. (2018). Water-splitting-based, sustainable and efficient H2 production in green algae as achieved by substrate limitation of the Calvin-Benson-Bassham cycle. Biotechnol. Biofuels, 11.","DOI":"10.1186\/s13068-018-1069-0"},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"37","DOI":"10.1111\/pre.12500","article-title":"Bioinformatic analysis and relative expression of hyd and fdx during H2 production in microalgae","volume":"71","year":"2023","journal-title":"Phycol. Res."},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"4608","DOI":"10.1039\/D0SC00512F","article-title":"Spectroscopic investigations under whole-cell conditions provide new insight into the metal hydride chemistry of [FeFe]-hydrogenase","volume":"11","author":"Ceccaldi","year":"2020","journal-title":"Chem. Sci."},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"345","DOI":"10.1007\/s00775-022-01928-5","article-title":"Stability of the H-cluster under whole-cell conditions\u2014Formation of an Htrans-like state and its reactivity towards oxygen","volume":"27","author":"Lorenzi","year":"2022","journal-title":"J. Biol. Inorg. Chem."},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"D523","DOI":"10.1093\/nar\/gkac1052","article-title":"UniProt: The Universal Protein Knowledgebase in 2023","volume":"51","author":"Consortium","year":"2022","journal-title":"Nucleic Acids Res."},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"17738","DOI":"10.1016\/j.ijhydene.2012.09.068","article-title":"Solar-to-bioH2 production enhanced by homologous overexpression of hydrogenase in green alga Chlorella sp. DT","volume":"37","author":"Chien","year":"2012","journal-title":"Int. J. Hydrogen Energy"},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"761","DOI":"10.1038\/ismej.2015.153","article-title":"Genomic and metagenomic surveys of hydrogenase distribution indicate H2 is a widely utilised energy source for microbial growth and survival","volume":"10","author":"Greening","year":"2016","journal-title":"ISME J."},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"34212","DOI":"10.1038\/srep34212","article-title":"HydDB: A web tool for hydrogenase classification and analysis","volume":"6","author":"Pedersen","year":"2016","journal-title":"Sci. Rep."},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"2105","DOI":"10.1038\/sj.emboj.7600216","article-title":"The hydrogenase-like Nar1p is essential for maturation of cytosolic and nuclear iron\u2013sulphur proteins","volume":"23","author":"Balk","year":"2004","journal-title":"EMBO J."},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"4946","DOI":"10.1021\/bi900312x","article-title":"Crucial Role of Conserved Cysteine Residues in the Assembly of Two Iron-Sulfur Clusters on the CIA Protein Nar1","volume":"48","author":"Urzica","year":"2009","journal-title":"Biochemistry"},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"2577","DOI":"10.1002\/bip.360221211","article-title":"Dictionary of protein secondary structure\u2014Pattern-recognition of hydrogen-bonded and geometrical features","volume":"22","author":"Kabsch","year":"1983","journal-title":"Biopolymers"},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"D439","DOI":"10.1093\/nar\/gkab1061","article-title":"AlphaFold Protein Structure Database: Massively expanding the structural coverage of protein-sequence space with high-accuracy models","volume":"50","author":"Varadi","year":"2021","journal-title":"Nucleic Acids Res."},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"e79361","DOI":"10.7554\/eLife.79361","article-title":"Structural insight on the mechanism of an electron-bifurcating [FeFe] hydrogenase","volume":"11","author":"Furlan","year":"2022","journal-title":"eLife"},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"403","DOI":"10.1021\/acs.jpcb.1c08124","article-title":"Fast Proton Transport in FeFe Hydrogenase via a Flexible Channel and a Proton Hole Mechanism","volume":"126","author":"Puthenkalathil","year":"2022","journal-title":"J. Phys. Chem. B"},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"1740","DOI":"10.1126\/science.1334572","article-title":"Electron-Tunneling Pathways in Proteins","volume":"258","author":"Beratan","year":"1992","journal-title":"Science"},{"key":"ref_39","doi-asserted-by":"crossref","first-page":"20170916","DOI":"10.1098\/rsif.2017.0916","article-title":"Long-range proton-coupled electron transfer in biological energy conversion: Towards mechanistic understanding of respiratory complex I","volume":"15","author":"Kaila","year":"2018","journal-title":"J. R. Soc. Interface"},{"key":"ref_40","doi-asserted-by":"crossref","first-page":"756","DOI":"10.1038\/s41467-020-20861-2","article-title":"A safety cap protects hydrogenase from oxygen attack","volume":"12","author":"Winkler","year":"2021","journal-title":"Nat. Commun."},{"key":"ref_41","doi-asserted-by":"crossref","first-page":"856","DOI":"10.1021\/acscatal.2c04031","article-title":"Increasing the O2 Resistance of the [FeFe]-Hydrogenase CbA5H through Enhanced Protein Flexibility","volume":"13","author":"Rutz","year":"2023","journal-title":"ACS Catal."},{"key":"ref_42","doi-asserted-by":"crossref","first-page":"17331","DOI":"10.1073\/pnas.0905343106","article-title":"How oxygen attacks [FeFe] hydrogenases from photosynthetic organisms","volume":"106","author":"Stripp","year":"2009","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_43","doi-asserted-by":"crossref","first-page":"3234","DOI":"10.1038\/ncomms4234","article-title":"Photoautotrophic hydrogen production by eukaryotic microalgae under aerobic conditions","volume":"5","author":"Hwang","year":"2014","journal-title":"Nat. Commun."},{"key":"ref_44","doi-asserted-by":"crossref","first-page":"4726","DOI":"10.1038\/s41467-018-07140-x","article-title":"Crystallographic and spectroscopic assignment of the proton transfer pathway in [FeFe]-hydrogenases","volume":"9","author":"Duan","year":"2018","journal-title":"Nat. Commun."},{"key":"ref_45","doi-asserted-by":"crossref","first-page":"206","DOI":"10.1038\/nchembio0407-206","article-title":"Iron-sulfur clusters as oxygen-responsive molecular switches","volume":"3","author":"Outten","year":"2007","journal-title":"Nat. Chem. Biol."},{"key":"ref_46","doi-asserted-by":"crossref","first-page":"D20","DOI":"10.1093\/nar\/gkab1112","article-title":"Database resources of the national center for biotechnology information","volume":"50","author":"Sayers","year":"2021","journal-title":"Nucleic Acids Res."},{"key":"ref_47","doi-asserted-by":"crossref","first-page":"403","DOI":"10.1016\/S0022-2836(05)80360-2","article-title":"Basic local alignment search tool","volume":"215","author":"Altschul","year":"1990","journal-title":"J. Mol. Biol."},{"key":"ref_48","doi-asserted-by":"crossref","first-page":"10915","DOI":"10.1073\/pnas.89.22.10915","article-title":"Amino acid substitution matrices from protein blocks","volume":"89","author":"Henikoff","year":"1992","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_49","first-page":"345","article-title":"Chapter: A Model of Evolutionary Change in Proteins","volume":"Volume 5","author":"Dayhoff","year":"1978","journal-title":"Atlas of Protein Sequence and Structure"},{"key":"ref_50","unstructured":"Schwarz, R., and Dayhoff, M. (1979). Atlas of Protein Sequence and Structure, National Biomedical Research Foundation."},{"key":"ref_51","doi-asserted-by":"crossref","first-page":"W276","DOI":"10.1093\/nar\/gkac240","article-title":"Search and sequence analysis tools services from EMBL-EBI in 2022","volume":"50","author":"Madeira","year":"2022","journal-title":"Nucleic Acids Res."},{"key":"ref_52","doi-asserted-by":"crossref","first-page":"135","DOI":"10.1002\/pro.3290","article-title":"Clustal Omega for making accurate alignments of many protein sequences","volume":"27","author":"Sievers","year":"2018","journal-title":"Protein Sci."},{"key":"ref_53","doi-asserted-by":"crossref","first-page":"871","DOI":"10.1126\/science.abj8754","article-title":"Accurate prediction of protein structures and interactions using a three-track neural network","volume":"373","author":"Baek","year":"2021","journal-title":"Science"},{"key":"ref_54","doi-asserted-by":"crossref","first-page":"1137","DOI":"10.1021\/ct800342w","article-title":"Density Functional Theory Calculation of Bonding and Charge Parameters for Molecular Dynamics Studies on [FeFe] Hydrogenases","volume":"5","author":"Chang","year":"2009","journal-title":"J. Chem. Theory Comput."},{"key":"ref_55","doi-asserted-by":"crossref","first-page":"3257","DOI":"10.1021\/ct300400x","article-title":"Optimization of the Additive CHARMM All-Atom Protein Force Field Targeting Improved Sampling of the Backbone phi, psi and Side-Chain chi1 and chi2 Dihedral Angles","volume":"8","author":"Best","year":"2012","journal-title":"J. Chem. Theory Comput."},{"key":"ref_56","doi-asserted-by":"crossref","first-page":"2641","DOI":"10.1016\/j.cpc.2013.06.003","article-title":"A flexible algorithm for calculating pair interactions on SIMD architectures","volume":"184","author":"Hess","year":"2013","journal-title":"Comput. Phys. Commun."},{"key":"ref_57","doi-asserted-by":"crossref","first-page":"10089","DOI":"10.1063\/1.464397","article-title":"Particle Mesh Ewald: An N-log(N) Method for Ewald Sums in Large Systems","volume":"98","author":"Darden","year":"1993","journal-title":"J. Chem. Phys."},{"key":"ref_58","doi-asserted-by":"crossref","first-page":"952","DOI":"10.1002\/jcc.540130805","article-title":"Settle: An analytical version of the SHAKE and RATTLE algorithm for rigid water models","volume":"13","author":"Miyamoto","year":"1992","journal-title":"J. Comput. Chem."},{"key":"ref_59","doi-asserted-by":"crossref","first-page":"1463","DOI":"10.1002\/(SICI)1096-987X(199709)18:12<1463::AID-JCC4>3.0.CO;2-H","article-title":"LINCS: A linear constraint solver for molecular simulations","volume":"18","author":"Hess","year":"1997","journal-title":"J. Comput. Chem."},{"key":"ref_60","doi-asserted-by":"crossref","first-page":"125103","DOI":"10.1063\/1.4978939","article-title":"Multisystem altruistic metadynamics- Well-tempered variant","volume":"146","author":"Spiwok","year":"2017","journal-title":"J. Chem. Phys."},{"key":"ref_61","doi-asserted-by":"crossref","unstructured":"Nobili, G., Botticelli, S., La Penna, G., Morante, S., Rossi, G., and Salina, G. (2023). Probing protein stability: Towards a computational atomistic, reliable, affordable, and improvable model. Front. Mol. Biosci., 10.","DOI":"10.3389\/fmolb.2023.1122269"},{"key":"ref_62","unstructured":"Bussi, G., Laio, A., and Tiwary, P. (2018). Handbook of Materials Modeling: Methods: Theory and Modeling, Springer International Publishing."},{"key":"ref_63","doi-asserted-by":"crossref","first-page":"031910-10","DOI":"10.1103\/PhysRevE.65.031910","article-title":"Convergence of sampling in protein simulations","volume":"65","author":"Hess","year":"2002","journal-title":"Phys. Rev. E"},{"key":"ref_64","doi-asserted-by":"crossref","first-page":"273","DOI":"10.1002\/jcc.540160303","article-title":"The Double Cubic Lattice Method: Efficient Approaches to Numerical Integration of Surface Area and Volume and to Dot Surface Contouring of Molecular Assemblies","volume":"16","author":"Eisenhaber","year":"1995","journal-title":"J. Comput. Chem."},{"key":"ref_65","doi-asserted-by":"crossref","first-page":"441","DOI":"10.1021\/j100785a001","article-title":"van der Waals Volumes and Radii","volume":"68","author":"Bondi","year":"1964","journal-title":"J. Phys. Chem."},{"key":"ref_66","doi-asserted-by":"crossref","first-page":"044130","DOI":"10.1063\/5.0014475","article-title":"Scalable molecular dynamics on CPU and GPU architectures with NAMD","volume":"153","author":"Phillips","year":"2020","journal-title":"J. Chem. Phys."},{"key":"ref_67","doi-asserted-by":"crossref","first-page":"43","DOI":"10.1016\/0010-4655(95)00042-E","article-title":"GROMACS: A message-passing parallel molecular dynamics implementation","volume":"91","author":"Berendsen","year":"1995","journal-title":"Comput. Phys. Commun."},{"key":"ref_68","doi-asserted-by":"crossref","first-page":"19","DOI":"10.1016\/j.softx.2015.06.001","article-title":"GROMACS: High performance molecular simulations through multi-level parallelism from laptops to supercomputers","volume":"1\u20132","author":"Abraham","year":"2015","journal-title":"SoftwareX"},{"key":"ref_69","doi-asserted-by":"crossref","first-page":"33","DOI":"10.1016\/0263-7855(96)00018-5","article-title":"VMD visual molecular dynamics","volume":"14","author":"Humphrey","year":"1996","journal-title":"J. Mol. Graph."},{"key":"ref_70","doi-asserted-by":"crossref","first-page":"1961","DOI":"10.1016\/j.cpc.2009.05.011","article-title":"PLUMED: A Portable Plugin for Free-Energy Calculations with Molecular Dynamics","volume":"180","author":"Bonomi","year":"2009","journal-title":"J. Comput. Phys."},{"key":"ref_71","doi-asserted-by":"crossref","first-page":"1112","DOI":"10.1021\/acs.jcim.6b00103","article-title":"TopoGromacs: Automated Topology Conversion from CHARMM to GROMACS within VMD","volume":"56","author":"Vermaas","year":"2016","journal-title":"J. Chem. Inform. Model."}],"container-title":["International Journal of Molecular Sciences"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/1422-0067\/25\/7\/3663\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,10]],"date-time":"2025-10-10T14:18:28Z","timestamp":1760105908000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/1422-0067\/25\/7\/3663"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2024,3,25]]},"references-count":71,"journal-issue":{"issue":"7","published-online":{"date-parts":[[2024,4]]}},"alternative-id":["ijms25073663"],"URL":"https:\/\/doi.org\/10.3390\/ijms25073663","relation":{},"ISSN":["1422-0067"],"issn-type":[{"value":"1422-0067","type":"electronic"}],"subject":[],"published":{"date-parts":[[2024,3,25]]}}}