{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,1,16]],"date-time":"2026-01-16T20:42:31Z","timestamp":1768596151455,"version":"3.49.0"},"reference-count":83,"publisher":"MDPI AG","issue":"19","license":[{"start":{"date-parts":[[2021,10,8]],"date-time":"2021-10-08T00:00:00Z","timestamp":1633651200000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100001871","name":"Funda\u00e7\u00e3o para a Ci\u00eancia e Tecnologia","doi-asserted-by":"publisher","award":["PTDC\/BII-BBF\/29564\/2017"],"award-info":[{"award-number":["PTDC\/BII-BBF\/29564\/2017"]}],"id":[{"id":"10.13039\/501100001871","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100001871","name":"Funda\u00e7\u00e3o para a Ci\u00eancia e Tecnologia","doi-asserted-by":"publisher","award":["UIDB\/04612\/2020"],"award-info":[{"award-number":["UIDB\/04612\/2020"]}],"id":[{"id":"10.13039\/501100001871","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100001871","name":"Funda\u00e7\u00e3o para a Ci\u00eancia e Tecnologia","doi-asserted-by":"publisher","award":["UIDP\/04612\/2020"],"award-info":[{"award-number":["UIDP\/04612\/2020"]}],"id":[{"id":"10.13039\/501100001871","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100000780","name":"European Union","doi-asserted-by":"publisher","award":["GA 824017"],"award-info":[{"award-number":["GA 824017"]}],"id":[{"id":"10.13039\/501100000780","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["IJMS"],"abstract":"<jats:p>Bacillus subtilis\u00a0BsDyP belongs to class I of the dye-decolorizing peroxidase (DyP) family of enzymes and is an interesting biocatalyst due to its high redox potential, broad substrate spectrum and thermostability. This work reports the optimization of BsDyP using directed evolution for improved oxidation of 2,6-dimethoxyphenol, a model lignin-derived phenolic. After three rounds of evolution, one variant was identified displaying 7-fold higher catalytic rates and higher production yields as compared to the wild-type enzyme. The analysis of X-ray structures of the wild type and the evolved variant showed that the heme pocket is delimited by three long conserved loop regions and a small \u03b1 helix where, incidentally, the mutations were inserted in the course of evolution. One loop in the proximal side of the heme pocket becomes more flexible in the evolved variant and the size of the active site cavity is increased, as well as the width of its mouth, resulting in an enhanced exposure of the heme to solvent. These conformational changes have a positive functional role in facilitating electron transfer from the substrate to the enzyme. However, they concomitantly resulted in decreasing the enzyme\u2019s overall stability by 2 kcal mol\u22121, indicating a trade-off between functionality and stability. Furthermore, the evolved variant exhibited slightly reduced thermal stability compared to the wild type. The obtained data indicate that understanding the role of loops close to the heme pocket in the catalysis and stability of DyPs is critical for the development of new and more powerful biocatalysts: loops can be modulated for tuning important DyP properties such as activity, specificity and stability.<\/jats:p>","DOI":"10.3390\/ijms221910862","type":"journal-article","created":{"date-parts":[[2021,10,10]],"date-time":"2021-10-10T21:23:25Z","timestamp":1633901005000},"page":"10862","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":15,"title":["Loops around the Heme Pocket Have a Critical Role in the Function and Stability of BsDyP from Bacillus subtilis"],"prefix":"10.3390","volume":"22","author":[{"given":"Carolina F.","family":"Rodrigues","sequence":"first","affiliation":[{"name":"Instituto de Tecnologia Qu\u00edmica e Biol\u00f3gica Ant\u00f3nio Xavier, Universidade Nova de Lisboa, Av da Rep\u00fablica, 2780-157 Oeiras, Portugal"}]},{"given":"Patr\u00edcia T.","family":"Borges","sequence":"additional","affiliation":[{"name":"Instituto de Tecnologia Qu\u00edmica e Biol\u00f3gica Ant\u00f3nio Xavier, Universidade Nova de Lisboa, Av da Rep\u00fablica, 2780-157 Oeiras, Portugal"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-8417-6123","authenticated-orcid":false,"given":"Magali F.","family":"Scocozza","sequence":"additional","affiliation":[{"name":"Instituto de Qu\u00edmica F\u00edsica de los Materiales, Medio Ambiente y Energia (INQUIMAE), CONICET\u2014Universidad de Buenos Aires, Buenos Aires 148EHA, Argentina"}]},{"given":"Diogo","family":"Silva","sequence":"additional","affiliation":[{"name":"Instituto de Tecnologia Qu\u00edmica e Biol\u00f3gica Ant\u00f3nio Xavier, Universidade Nova de Lisboa, Av da Rep\u00fablica, 2780-157 Oeiras, Portugal"}]},{"given":"Andr\u00e9","family":"Taborda","sequence":"additional","affiliation":[{"name":"Instituto de Tecnologia Qu\u00edmica e Biol\u00f3gica Ant\u00f3nio Xavier, Universidade Nova de Lisboa, Av da Rep\u00fablica, 2780-157 Oeiras, Portugal"}]},{"given":"V\u00e2nia","family":"Brissos","sequence":"additional","affiliation":[{"name":"Instituto de Tecnologia Qu\u00edmica e Biol\u00f3gica Ant\u00f3nio Xavier, Universidade Nova de Lisboa, Av da Rep\u00fablica, 2780-157 Oeiras, Portugal"}]},{"given":"Carlos","family":"Fraz\u00e3o","sequence":"additional","affiliation":[{"name":"Instituto de Tecnologia Qu\u00edmica e Biol\u00f3gica Ant\u00f3nio Xavier, Universidade Nova de Lisboa, Av da Rep\u00fablica, 2780-157 Oeiras, Portugal"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-0082-9591","authenticated-orcid":false,"given":"L\u00edgia O.","family":"Martins","sequence":"additional","affiliation":[{"name":"Instituto de Tecnologia Qu\u00edmica e Biol\u00f3gica Ant\u00f3nio Xavier, Universidade Nova de Lisboa, Av da Rep\u00fablica, 2780-157 Oeiras, Portugal"}]}],"member":"1968","published-online":{"date-parts":[[2021,10,8]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"614","DOI":"10.1021\/acs.chemrev.7b00588","article-title":"Bright side of lignin depolymerization: Toward new platform chemicals","volume":"118","author":"Sun","year":"2018","journal-title":"Chem. Rev."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"20","DOI":"10.3389\/fbioe.2018.00020","article-title":"Enzymatic processes to unlock the lignin value","volume":"6","author":"Hamalainen","year":"2018","journal-title":"Front. Bioeng. Biotechnol."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"36","DOI":"10.1007\/s41061-018-0214-3","article-title":"Catalytic strategies towards lignin-derived chemicals","volume":"376","author":"Koelewijn","year":"2018","journal-title":"Top. Curr. Chem."},{"key":"ref_4","doi-asserted-by":"crossref","unstructured":"Runeberg, P.A., Brusentsev, Y., Rendon, S.M.K., and Eklund, P.C. (2019). Oxidative transformations of lignans. Molecules, 24.","DOI":"10.3390\/molecules24020300"},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"9","DOI":"10.1002\/marc.201500474","article-title":"From Lignin-derived Aromatic compounds to novel biobased polymers","volume":"37","author":"Llevot","year":"2016","journal-title":"Macromol. Rapid Commun."},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"5143","DOI":"10.1002\/adsc.202000634","article-title":"Synthesis of functional chemicals from lignin-derived monomers by selective organic transformations","volume":"362","author":"Natte","year":"2020","journal-title":"Adv. Synth. Catal."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"3552","DOI":"10.1021\/cr900354u","article-title":"The catalytic valorization of lignin for the production of renewable chemicals","volume":"110","author":"Zakzeski","year":"2010","journal-title":"Chem. Rev."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"26","DOI":"10.1016\/j.cbpa.2019.11.007","article-title":"Bacterial enzymes for lignin depolymerisation: New biocatalysts for generation of renewable chemicals from biomass","volume":"55","author":"Bugg","year":"2020","journal-title":"Curr. Opin. Chem. Biol."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"110","DOI":"10.1016\/j.jbiotec.2016.08.011","article-title":"Bacterial enzymes involved in lignin degradation","volume":"236","author":"Colpa","year":"2016","journal-title":"J. Biotechnol."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"1190","DOI":"10.1111\/febs.13224","article-title":"Lignin-degrading enzymes","volume":"282","author":"Pollegioni","year":"2015","journal-title":"FEBS J."},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"348","DOI":"10.1016\/j.copbio.2009.05.002","article-title":"Enzymatic delignification of plant cell wall: From nature to mill","volume":"20","author":"Martinez","year":"2009","journal-title":"Curr. Opin. Biotechnol."},{"key":"ref_12","first-page":"1400536","article-title":"Understanding molecular enzymology of porphyrin-binding \u03b1 + \u03b2 barrel proteins\u2014One fold, multiple functions","volume":"1869","author":"Hofbauer","year":"2020","journal-title":"BBA Proteins Proteom."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"871","DOI":"10.1007\/s00253-010-2633-0","article-title":"New and classic families of secreted fungal heme peroxidases","volume":"87","author":"Hofrichter","year":"2010","journal-title":"Appl. Microbiol. Biotechnol."},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"1387","DOI":"10.1007\/s00018-008-8651-8","article-title":"DyP-type peroxidases comprise a novel heme peroxidase family","volume":"66","author":"Sugano","year":"2009","journal-title":"Cell Mol. Life Sci."},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1007\/s10295-013-1371-6","article-title":"DyP-type peroxidases: A promising and versatile class of enzymes","volume":"41","author":"Colpa","year":"2014","journal-title":"J. Ind. Microbiol. Biotechnol."},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"49","DOI":"10.1016\/j.abb.2015.01.022","article-title":"A structural and functional perspective of DyP-type peroxidase family","volume":"574","author":"Yoshida","year":"2015","journal-title":"Arch. Biochem. Biophys."},{"key":"ref_17","first-page":"9","article-title":"Bacterial dye-decolorizing peroxidases: Biochemical properties and biotechnological opportunities","volume":"1","author":"Chen","year":"2016","journal-title":"Phys. Sci. Rev."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"56","DOI":"10.1016\/j.abb.2015.01.014","article-title":"The multihued palette of dye-decolorizing peroxidases","volume":"574","author":"Singh","year":"2015","journal-title":"Arch. Biochem. Biophys."},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"3454","DOI":"10.1021\/acscatal.6b03331","article-title":"Engineering a bacterial dyp-type peroxidase for enhanced oxidation of lignin-related phenolics at alkaline pH","volume":"7","author":"Brissos","year":"2017","journal-title":"ACS Catal."},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"112055","DOI":"10.1016\/j.bios.2020.112055","article-title":"Immobilized dye-decolorizing peroxidase (DyP) and directed evolution variants for hydrogen peroxide biosensing","volume":"153","author":"Barbosa","year":"2020","journal-title":"Biosens. Bioelectron."},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"21","DOI":"10.1016\/j.enzmictec.2019.01.002","article-title":"Protein engineering of Pseudomonas fluorescens peroxidase Dyp1B for oxidation of phenolic and polymeric lignin substrates","volume":"123","author":"Pour","year":"2019","journal-title":"Enz. Microb. Technol."},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"2053","DOI":"10.1007\/s00253-013-5041-4","article-title":"New dye-decolorizing peroxidases from Bacillus subtilis and Pseudomonas putida MET94: Towards biotechnological applications","volume":"98","author":"Santos","year":"2014","journal-title":"Appl. Microbiol. Biotechnol."},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"3074","DOI":"10.1021\/bi301630a","article-title":"Distinct structural and redox properties of heme active in bacterial DyP-type peroxidases from two subfamilies: Resonance Raman and electrochemistry study","volume":"52","author":"Sezer","year":"2013","journal-title":"Biochemistry"},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"8245","DOI":"10.1038\/srep08245","article-title":"A dye-decolorizing peroxidase from Bacillus subtilis exhibiting substrate-dependent optimum temperature for dyes and beta-ether lignin dimer","volume":"5","author":"Min","year":"2015","journal-title":"Sci. Rep."},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"5196","DOI":"10.1039\/C5CY00478K","article-title":"Catalytic mechanism of BsDyP an A-type dye-decolourising peroxidase: Neither aspartate nor arginine is individually essential for peroxidase activity","volume":"5","author":"Mendes","year":"2015","journal-title":"Catal. Sci. Technol."},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"36","DOI":"10.1002\/anie.201812717","article-title":"Directed Evolution Empowered Redesign of Natural Proteins for the Sustainable Production of Chemicals and Pharmaceuticals","volume":"58","author":"Bornscheuer","year":"2019","journal-title":"Angew. Chem. Int. Ed."},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"75","DOI":"10.1016\/j.abb.2014.12.016","article-title":"The toolbox of Auricularia auricula-judae dye-decolorizing peroxidase\u2014Identification of three new potential substrate-interaction sites","volume":"574","author":"Strittmatter","year":"2015","journal-title":"Arch. Biochem. Biophys."},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"815","DOI":"10.1016\/j.biotechadv.2017.06.003","article-title":"Oxidoreductases on their way to industrial biotransformations","volume":"35","author":"Martinez","year":"2017","journal-title":"Biotechnol. Adv."},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"1319","DOI":"10.1111\/j.1365-2958.2004.04341.x","article-title":"Two minimal Tat translocases in Bacillus","volume":"54","author":"Jongbloed","year":"2004","journal-title":"Mol. Microbiol."},{"key":"ref_30","doi-asserted-by":"crossref","unstructured":"van der Ploeg, R., Mader, U., Homuth, G., Schaffer, M., Denham, E.L., Monteferrante, C.G., Miethke, M., Marahiel, M.A., Harwood, C.R., and Winter, T. (2011). Environmental salinity determines the specificity and need for Tat-dependent secretion of the YwbN protein in Bacillus subtilis. PLoS ONE, 6.","DOI":"10.1371\/journal.pone.0018140"},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"1419","DOI":"10.1007\/s00253-009-2369-x","article-title":"A robust and extracellular heme-containing peroxidase from Thermobifida fusca as prototype of a bacterial peroxidase superfamily","volume":"86","author":"Winter","year":"2010","journal-title":"Appl. Microbiol. Biotechnol."},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"13972","DOI":"10.1074\/jbc.M511891200","article-title":"YcdB from Escherichia coli reveals a novel class of Tat-dependently translocated hemoproteins","volume":"281","author":"Sturm","year":"2006","journal-title":"J. Biol. Chem."},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"425","DOI":"10.1016\/j.jbiosc.2016.12.001","article-title":"Characterization of a novel DyP-type peroxidase from Streptomyces avermitilis","volume":"123","author":"Sugawara","year":"2017","journal-title":"J. Biosci. Bioeng."},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"1869","DOI":"10.1007\/s00253-009-2173-7","article-title":"DyP-like peroxidases of the jelly fungus Auricularia auricula-judae oxidize nonphenolic lignin model compounds and high-redox potential dyes","volume":"85","author":"Liers","year":"2010","journal-title":"Appl. Microbiol. Biotechnol."},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"108590","DOI":"10.1016\/j.abb.2020.108590","article-title":"Characterization of dye-decolorizing peroxidase from Bacillus subtilis","volume":"693","author":"Dhankhar","year":"2020","journal-title":"Arch. Biochem. Biophys."},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"5108","DOI":"10.1021\/bi200427h","article-title":"Characterization of dye-decolorizing peroxidases from Rhodococcus jostii RHA1","volume":"50","author":"Roberts","year":"2011","journal-title":"Biochemistry"},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"6352","DOI":"10.1021\/acscatal.7b01861","article-title":"Mechanistic insights into dye-decolorizing peroxidase revealed by solvent isotope and viscosity effects","volume":"7","author":"Shrestha","year":"2017","journal-title":"Acs. Catal."},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"6610","DOI":"10.1021\/acs.biochem.5b00952","article-title":"A dye-decolorizing peroxidase from Vibrio cholerae","volume":"54","author":"Uchida","year":"2015","journal-title":"Biochemistry"},{"key":"ref_39","doi-asserted-by":"crossref","first-page":"2387","DOI":"10.1111\/j.1742-4658.2011.08161.x","article-title":"The catalytic mechanism of dye-decolorizing peroxidase DyP may require the swinging movement of an aspartic acid residue","volume":"278","author":"Yoshida","year":"2011","journal-title":"FEBS J."},{"key":"ref_40","doi-asserted-by":"crossref","first-page":"4095","DOI":"10.1074\/jbc.M112.400176","article-title":"First crystal structure of a fungal high-redox potential dye-decolorizing peroxidase: Substrate interaction sites and long-range electron transfer","volume":"288","author":"Strittmatter","year":"2013","journal-title":"J. Biol. Chem."},{"key":"ref_41","doi-asserted-by":"crossref","first-page":"4351","DOI":"10.1016\/j.febslet.2012.10.049","article-title":"Crystal structures of dye-decolorizing peroxidase with ascorbic acid and 2,6-dimethoxyphenol","volume":"586","author":"Yoshida","year":"2012","journal-title":"FEBS Lett."},{"key":"ref_42","doi-asserted-by":"crossref","first-page":"66","DOI":"10.1016\/j.abb.2015.01.018","article-title":"Basidiomycete DyPs: Genomic diversity, structural-functional aspects, reaction mechanism and environmental significance","volume":"574","author":"Linde","year":"2015","journal-title":"Arch. Biochem. Biophys."},{"key":"ref_43","doi-asserted-by":"crossref","first-page":"41","DOI":"10.1016\/j.molcatb.2013.09.025","article-title":"Phenol oxidation by DyP-type peroxidases in comparison to fungal and plant peroxidases","volume":"103","author":"Liers","year":"2014","journal-title":"J. Mol. Cat B Enz."},{"key":"ref_44","doi-asserted-by":"crossref","first-page":"161","DOI":"10.1016\/j.abb.2013.07.007","article-title":"Radical formation on a conserved tyrosine residue is crucial for DyP activity","volume":"537","author":"Strittmatter","year":"2013","journal-title":"Arch. Biochem. Biophys."},{"key":"ref_45","doi-asserted-by":"crossref","first-page":"8927","DOI":"10.1007\/s00253-015-6665-3","article-title":"Description of the first fungal dye-decolorizing peroxidase oxidizing manganese(II)","volume":"99","author":"Linde","year":"2015","journal-title":"Appl. Microbiol. Biotechnol."},{"key":"ref_46","doi-asserted-by":"crossref","first-page":"13583","DOI":"10.1021\/acs.jpcb.5b02961","article-title":"Redox-active sites in Auricularia auricula-judae dye-decolorizing peroxidase and several directed variants: A multifrequency EPR study","volume":"119","author":"Baratto","year":"2015","journal-title":"J. Phys. Chem."},{"key":"ref_47","doi-asserted-by":"crossref","first-page":"253","DOI":"10.1042\/BJ20141211","article-title":"Catalytic surface radical in dye-decolorizing peroxidase: A computational, spectroscopic and site-directed mutagenesis study","volume":"466","author":"Linde","year":"2015","journal-title":"Biochem. J."},{"key":"ref_48","doi-asserted-by":"crossref","first-page":"1226","DOI":"10.1021\/acs.biochem.1c00129","article-title":"On the track of long-range electron transfer in b-type dye-decolorizing peroxidases: Identification of a tyrosyl radical by computational prediction and electron paramagnetic resonance spectroscopy","volume":"60","author":"Nys","year":"2021","journal-title":"Biochemistry"},{"key":"ref_49","doi-asserted-by":"crossref","first-page":"1030","DOI":"10.1126\/science.1218231","article-title":"Linking Crystallographic Model and Data Quality","volume":"336","author":"Karplus","year":"2012","journal-title":"Science"},{"key":"ref_50","doi-asserted-by":"crossref","first-page":"510","DOI":"10.1088\/0022-3735\/1\/5\/303","article-title":"A computer-linked cathode-ray tube microdensitometer for X-ray crystallography","volume":"1","author":"Arndt","year":"1968","journal-title":"J. Phys. E Sci. Instrum."},{"key":"ref_51","doi-asserted-by":"crossref","first-page":"269","DOI":"10.1038\/nsb0497-269","article-title":"Improved R-factors for diffraction data analysis in macromolecular crystallography","volume":"4","author":"Diederichs","year":"1997","journal-title":"Nat. Struct. Biol."},{"key":"ref_52","doi-asserted-by":"crossref","first-page":"130","DOI":"10.1107\/S0021889800018227","article-title":"Global indicators of X-ray data quality","volume":"34","author":"Weiss","year":"2001","journal-title":"J. Appl. Crystallogr."},{"key":"ref_53","doi-asserted-by":"crossref","first-page":"3201","DOI":"10.1021\/cs500325p","article-title":"Engineering of flexible loops in enzymes","volume":"4","author":"Nestl","year":"2014","journal-title":"ACS Catal."},{"key":"ref_54","doi-asserted-by":"crossref","first-page":"1095","DOI":"10.1038\/s41467-021-21328-8","article-title":"Active-site loop variations adjust activity and selectivity of the cumene dioxygenase","volume":"12","author":"Heinemann","year":"2021","journal-title":"Nat. Commun."},{"key":"ref_55","doi-asserted-by":"crossref","first-page":"83","DOI":"10.1016\/j.sbi.2017.10.020","article-title":"Cooperativity and flexibility in enzyme evolution","volume":"48","author":"Pabis","year":"2018","journal-title":"Curr. Opin. Strut. Biol."},{"key":"ref_56","doi-asserted-by":"crossref","first-page":"785","DOI":"10.1038\/nmeth.1701","article-title":"SignalP 4.0: Discriminating signal peptides from transmembrane regions","volume":"8","author":"Petersen","year":"2011","journal-title":"Nat. Methods"},{"key":"ref_57","doi-asserted-by":"crossref","first-page":"343","DOI":"10.1038\/nmeth.1318","article-title":"Enzymatic assembly of DNA molecules up to several hundred kilobases","volume":"6","author":"Gibson","year":"2009","journal-title":"Nat. Methods"},{"key":"ref_58","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1016\/0003-2697(87)90643-9","article-title":"Simultaneous determination of hemes a, b, and c from pyridine hemochrome spectra","volume":"161","author":"Berry","year":"1987","journal-title":"Anal. Biochem."},{"key":"ref_59","doi-asserted-by":"crossref","first-page":"679","DOI":"10.1107\/S160057751400825X","article-title":"Developments in optics and performance at BL13-XALOC, the macromolecular crystallography beamline at the Alba Synchrotron","volume":"21","author":"Juanhuix","year":"2014","journal-title":"J. Synchrotron Radiat."},{"key":"ref_60","doi-asserted-by":"crossref","first-page":"844","DOI":"10.1107\/S1600577520004002","article-title":"ID30A-3 (MASSIF-3)\u2014A beamline for macromolecular crystallography at the ESRF with a small intense beam","volume":"27","author":"Carpentier","year":"2020","journal-title":"J. Synchrotron Radiat."},{"key":"ref_61","doi-asserted-by":"crossref","first-page":"125","DOI":"10.1107\/S0907444909047337","article-title":"Xds","volume":"66","author":"Kabsch","year":"2010","journal-title":"Acta Crystallogr. D"},{"key":"ref_62","doi-asserted-by":"crossref","first-page":"1865","DOI":"10.1110\/ps.0350503","article-title":"Matthews coefficient probabilities: Improved estimates for unit cell contents of proteins, DNA, and protein-nucleic acid complex crystals","volume":"12","author":"Kantardjieff","year":"2003","journal-title":"Protein Sci."},{"key":"ref_63","doi-asserted-by":"crossref","first-page":"491","DOI":"10.1016\/0022-2836(68)90205-2","article-title":"Solvent Content of Protein Crystals","volume":"33","author":"Matthews","year":"1968","journal-title":"J. Mol. Biol."},{"key":"ref_64","doi-asserted-by":"crossref","first-page":"s19","DOI":"10.1107\/S2053273315099672","article-title":"MoRDa, an automatic molecular replacement pipeline","volume":"71","author":"Vagin","year":"2015","journal-title":"Acta Crystallogr. A"},{"key":"ref_65","doi-asserted-by":"crossref","first-page":"658","DOI":"10.1107\/S0021889807021206","article-title":"Phaser crystallographic software","volume":"40","author":"McCoy","year":"2007","journal-title":"J. Appl. Crystallogr."},{"key":"ref_66","doi-asserted-by":"crossref","first-page":"213","DOI":"10.1107\/S0907444909052925","article-title":"PHENIX: A comprehensive Python-based system for macromolecular structure solution","volume":"66","author":"Adams","year":"2010","journal-title":"Acta Crystallogr. D"},{"key":"ref_67","doi-asserted-by":"crossref","first-page":"s15","DOI":"10.1107\/S010876731009968X","article-title":"New tools for structure refinement in Phenix","volume":"66","author":"Adams","year":"2010","journal-title":"Acta Crystallogr. A"},{"key":"ref_68","doi-asserted-by":"crossref","first-page":"2209","DOI":"10.1107\/S0907444913023512","article-title":"Improved estimates of coordinate error for molecular replacement","volume":"69","author":"Oeffner","year":"2013","journal-title":"Acta Crystallogr. D Biol. Crystallogr."},{"key":"ref_69","doi-asserted-by":"crossref","first-page":"352","DOI":"10.1107\/S0907444912001308","article-title":"Towards automated crystallographic structure refinement with phenix.refine","volume":"68","author":"Afonine","year":"2012","journal-title":"Acta Crystallogr. D"},{"key":"ref_70","doi-asserted-by":"crossref","first-page":"61","DOI":"10.1107\/S090744490705024X","article-title":"Iterative model building, structure refinement and density modification with the PHENIX AutoBuild wizard","volume":"64","author":"Terwilliger","year":"2008","journal-title":"Acta Crystallogr. D"},{"key":"ref_71","doi-asserted-by":"crossref","first-page":"439","DOI":"10.1107\/S0907444906005270","article-title":"Optimal description of a protein structure in terms of multiple groups undergoing TLS motion","volume":"62","author":"Painter","year":"2006","journal-title":"Acta Crystallogr. D"},{"key":"ref_72","doi-asserted-by":"crossref","first-page":"392","DOI":"10.1107\/S0108767391001071","article-title":"Accurate Bond and Angle Parameters for X-Ray Protein-Structure Refinement","volume":"47","author":"Engh","year":"1991","journal-title":"Acta Crystallog. A"},{"key":"ref_73","doi-asserted-by":"crossref","first-page":"2126","DOI":"10.1107\/S0907444904019158","article-title":"Coot: Model-building tools for molecular graphics","volume":"60","author":"Emsley","year":"2004","journal-title":"Acta Crystallogr. D"},{"key":"ref_74","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"},{"key":"ref_75","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1007\/978-1-4939-0366-5_1","article-title":"Protein structure modeling with MODELLER","volume":"1137","author":"Webb","year":"2014","journal-title":"Meth. Mol. Biol."},{"key":"ref_76","doi-asserted-by":"crossref","unstructured":"Chovancova, E., Pavelka, A., Benes, P., Strnad, O., Brezovsky, J., Kozlikova, B., Gora, A., Sustr, V., Klvana, M., and Medek, P. (2012). CAVER 3.0: A tool for the analysis of transport pathways in dynamic protein structures. PLoS Comput. Biol., 8.","DOI":"10.1371\/journal.pcbi.1002708"},{"key":"ref_77","unstructured":"DeLano, W.L. (2002). The PyMOL Molecular Graphics System, DeLano Scientific."},{"key":"ref_78","unstructured":"Schrodinger, L.L.C. (2010). The PyMOL Molecular Graphics System, Schr\u00f6dinger, LLC.. Version 1.7.2.2."},{"key":"ref_79","doi-asserted-by":"crossref","first-page":"2074","DOI":"10.1093\/bioinformatics\/bts310","article-title":"DoGSiteScorer: A web server for automatic binding site prediction, analysis and druggability assessment","volume":"28","author":"Volkamer","year":"2012","journal-title":"Bioinformatics"},{"key":"ref_80","doi-asserted-by":"crossref","first-page":"379","DOI":"10.1016\/0022-2836(71)90324-X","article-title":"Interpretation of Protein Structures\u2014Estimation of Static Accessibility","volume":"55","author":"Lee","year":"1971","journal-title":"J. Mol. Biol."},{"key":"ref_81","doi-asserted-by":"crossref","first-page":"5","DOI":"10.1007\/BF03024331","article-title":"Distributing many points on a sphere","volume":"19","author":"Saff","year":"1997","journal-title":"Math. Intell."},{"key":"ref_82","doi-asserted-by":"crossref","first-page":"235","DOI":"10.1093\/nar\/28.1.235","article-title":"The Protein Data Bank","volume":"28","author":"Berman","year":"2000","journal-title":"Nucleic Acids Res."},{"key":"ref_83","doi-asserted-by":"crossref","first-page":"4864","DOI":"10.1021\/bi027184+","article-title":"Temperature Range of Thermodynamic Stability for the Native State of Reversible Two-State Proteins","volume":"42","author":"Kumar","year":"2003","journal-title":"Biochemistry"}],"container-title":["International Journal of Molecular Sciences"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/1422-0067\/22\/19\/10862\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,11]],"date-time":"2025-10-11T07:10:26Z","timestamp":1760166626000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/1422-0067\/22\/19\/10862"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2021,10,8]]},"references-count":83,"journal-issue":{"issue":"19","published-online":{"date-parts":[[2021,10]]}},"alternative-id":["ijms221910862"],"URL":"https:\/\/doi.org\/10.3390\/ijms221910862","relation":{},"ISSN":["1422-0067"],"issn-type":[{"value":"1422-0067","type":"electronic"}],"subject":[],"published":{"date-parts":[[2021,10,8]]}}}