{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"institution":[{"name":"Research Square"}],"indexed":{"date-parts":[[2024,3,3]],"date-time":"2024-03-03T13:25:17Z","timestamp":1709472317725},"posted":{"date-parts":[[2023,5,9]]},"group-title":"In Review","reference-count":36,"publisher":"Research Square Platform LLC","license":[{"start":{"date-parts":[[2023,5,9]],"date-time":"2023-05-09T00:00:00Z","timestamp":1683590400000},"content-version":"unspecified","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"accepted":{"date-parts":[[2023,4,23]]},"abstract":"<jats:title>Abstract<\/jats:title>\n        <jats:p>The crystal structure of orthorhombic Bovine Pancreatic Ribonuclease A has been determined to 0.85\u00c5 resolution using low temperature, 100K, synchrotron X-ray data collected at 16000keV (\u03bb\u2009=\u20090.77\u00c5). This is the first ultra-high-resolution structure of a native form of Ribonuclease A to be reported. Refinement carried out with anisotropic displacement parameters, stereochemical restraints, inclusion of H atoms in calculated positions, five SO<jats:sub>4<\/jats:sub><jats:sup>2\u2212<\/jats:sup> moieties, eleven ethanol molecules and 293 water molecules, converged with final R values of R1(Free)\u2009=\u20090.129 (4279 reflections) and R1\u2009=\u20090.112 (85346 reflections). The refined structure was deposited in the Protein Data Bank as structure 7p4r. Conserved waters, using four high resolution structures, have been investigated. Cluster analysis identified clusters of water molecules that are associated with the active site of Bovine Ribonuclease A. Particular attention has been paid to making detailed comparisons between the present structure and other high quality Bovine Pancreatic Ribonuclease A X-ray crystal structures with special reference to the deposited classic monoclinic structure 3RN3 Howlin et al [1]. Detailed studies of various aspects of hydrogen bonding and conformation have been carried out with particular reference to active site residues Lys-1, Lys-7, Gln-11, His-12, Lys-41, Asn-44, Thr-45, Lys-66, His-119 and Ser-123. For the two histidine residues in the active site the initial electron density map gives a clear confirmation that the position of His-12 is very similar in the orthorhombic structure to that in 3RN3. In 3RN3 His-119 exhibited poor electron density which was modelled and refined as two distinct sites, A (65%) and B (35%) but with respect to His-119 in the present ultra-high resolution orthorhombic structure there is clear electron density which was modelled and refined as a single conformation distinct from either conformation A or B in 3RN3. Other points of interest include Serine-32 which is disordered at the end of the sidechain in the present orthorhombic form but has been modelled as a single form in 3RN3. Lysine-66: there is density indicating a possible conformation for this residue. However, the density is relatively weak, and the conformation is unclear. Three types of amino acid representation in the ultra-high resolution electron density are examined: (i) sharp with very clearly resolved features, for example Lys-37; (ii) well resolved but clearly divided into two conformations which are well behaved in the refinement, both having high quality geometry, for example Tyr-76; (iii) poor density and difficult or impossible to model, an example is Lys-31 for which density is missing except for C\u03b2. The side chains of Gln-11, His-12, Lys-41, Thr-45 and His-119 are generally recognised as being closely involved in the enzyme activity. It has also been suggested that Lys-7, Asp-44, Lys-66, Phe-120, Asp-121 and Ser-123 may also have possible roles in this mechanism. A molecular dynamics study on both structures has investigated the conformations of His-119 which was modelled as two conformations in 3RN3 but is observed to have a single clearly defined conformation in the present orthorhombic structure. MD has also been used to investigate Lys-31, Lys-41 and Ser32. The form of the Ribonuclease A enzyme used in both the present study and in 3RN3 [1] includes a sulphate anion which occupies approximately the same location as the PO<jats:sub>4<\/jats:sub><jats:sup>2\u2212<\/jats:sup> phosphate group in protein nucleotide complexes [2]. The present structure contains 5 SO<jats:sub>4<\/jats:sub><jats:sup>2\u2212<\/jats:sup> groups SO41151 \u2013 SO41155 two of which, SO41152 and SO41153 are disordered, SO41152 being in the active site, and 11 EtOH molecules, EOH A 201 \u2013 EOH A 211 all of which have good geometry. H atoms were built into the EtOH molecules geometrically. Illustrations of these features in the present structure are included here. The sulphates are presumably present in the material purchased for use in the present study. 293 water molecules are included in the present structure compared to 134 in 3RN3[1].<\/jats:p>","DOI":"10.21203\/rs.3.rs-2852137\/v1","type":"posted-content","created":{"date-parts":[[2023,5,9]],"date-time":"2023-05-09T13:40:19Z","timestamp":1683639619000},"source":"Crossref","is-referenced-by-count":0,"title":["Ultra-High Resolution X-ray Structure of Orthorhombic Bovine Pancreatic Ribonuclease A at 100K"],"prefix":"10.21203","author":[{"given":"David. R","family":"Lisgarten","sequence":"first","affiliation":[{"name":"Canterbury Christ Church University"}]},{"given":"Rex. A","family":"Palmer","sequence":"additional","affiliation":[{"name":"Birkbeck, University of London"}]},{"given":"Jon","family":"Cooper","sequence":"additional","affiliation":[{"name":"University College London"}]},{"given":"Claire. E","family":"Naylor","sequence":"additional","affiliation":[{"name":"Kiasco"}]},{"given":"Rosemary. C","family":"Talbert","sequence":"additional","affiliation":[{"name":"Canterbury Christ Church University"}]},{"given":"Brenden. J","family":"Howlin","sequence":"additional","affiliation":[{"name":"University of Surrey"}]},{"given":"John. N","family":"Lisgarten","sequence":"additional","affiliation":[{"name":"University of Greenwich"}]},{"given":"Janez.","family":"Konc","sequence":"additional","affiliation":[{"name":"National Institute of Chemistry"}]},{"given":"Shabir.","family":"Najmudin","sequence":"additional","affiliation":[{"name":"King's College London"}]},{"given":"Carina. M.C.","family":"Lobley","sequence":"additional","affiliation":[{"name":"European Spallation Source ERIC"}]}],"member":"8761","reference":[{"key":"ref1","doi-asserted-by":"crossref","first-page":"851","DOI":"10.1107\/S0108767389009177","article-title":"Segmented anisotropic refinement of bovine ribonuclease A by the application of the rigid-body TLS model","volume":"45","author":"Howlin B","year":"1989","unstructured":"Howlin B, Moss DS, Harris GW. Segmented anisotropic refinement of bovine ribonuclease A by the application of the rigid-body TLS model. Acta Crystallogr A. 1989;45:851\u201361.","journal-title":"Acta Crystallogr A"},{"key":"ref2","first-page":"743","article-title":"Specificity of Pancreatic Ribonuclease-A \u2013 An X-ray Study of a Protein-Nucleotide Complex","volume":"169","author":"Borkakoti N","year":"1983","unstructured":"Borkakoti N, Palmer RA, Haneef I, Moss DS. Specificity of Pancreatic Ribonuclease-A \u2013 An X-ray Study of a Protein-Nucleotide Complex. J Mol Biol. 1983;169:743\u201355.","journal-title":"J Mol Biol"},{"key":"ref3","doi-asserted-by":"crossref","first-page":"862","DOI":"10.1038\/213862a0","article-title":"Tertiary Structure of Ribonuclease","volume":"213","author":"Kartha G","year":"1967","unstructured":"Kartha G, Bello J, Harker D. Tertiary Structure of Ribonuclease. Nat (London). 1967;213:862\u20135.","journal-title":"Nat (London)"},{"key":"ref4","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1016\/0022-2836(74)90125-9","article-title":"The Structure of Ribuclease at 2.5\u00c5 Resolution","volume":"85","author":"Carlisle CH","year":"1974","unstructured":"Carlisle CH, Palmer RA, Mazumdar SK, Gorinsky BA, Yeates DGR. The Structure of Ribuclease at 2.5\u00c5 Resolution. J Mol Biol. 1974;85:1\u201318.","journal-title":"J Mol Biol"},{"key":"ref5","doi-asserted-by":"crossref","first-page":"2210","DOI":"10.1107\/S0567740882008346","article-title":"Ribonuclease-A: least-squares refinement of the structure at 1.45 \u00c5 resolution","volume":"B38","author":"Borkakoti N","year":"1982","unstructured":"Borkakoti N, Moss DS, Palmer RA. Ribonuclease-A: least-squares refinement of the structure at 1.45 \u00c5 resolution. Acta Crystallogr. 1982;B38:2210\u20137.","journal-title":"Acta Crystallogr"},{"key":"ref6","doi-asserted-by":"crossref","first-page":"1826","DOI":"10.1107\/S0567740880007315","article-title":"Studies of Ribonuclease-A by X-ray and Neutron Diffraction","volume":"B36","author":"Wlodawer A","year":"1980","unstructured":"Wlodawer A. Studies of Ribonuclease-A by X-ray and Neutron Diffraction. Acta Cryst. 1980;B36:1826\u201331.","journal-title":"Acta Cryst"},{"key":"ref7","doi-asserted-by":"crossref","first-page":"1194","DOI":"10.1016\/j.ijbiomac.2018.01.135","article-title":"Raman-markers of X-ray radiation damage of proteins","volume":"111","author":"Vergara A","year":"2018","unstructured":"Vergara A, Caterino M, Merlino A. Raman-markers of X-ray radiation damage of proteins. Int J Biol Macromol. 2018;111:1194\u2013205.","journal-title":"Int J Biol Macromol"},{"issue":"Pt3","key":"ref8","doi-asserted-by":"crossref","first-page":"441","DOI":"10.1107\/S0907444901021758","article-title":"Atomic resolution structures of ribonuclease A at six pH values","volume":"58","author":"Berisio R","year":"2002","unstructured":"Berisio R, Sica F, Lamzin VS, Wilson KS, Zagari A, Mazzarella L. Atomic resolution structures of ribonuclease A at six pH values. 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J Synchrotron Rad. 2009;16:872\u201379. 10.1107\/S0909049509036681].","journal-title":"J Synchrotron Rad"},{"key":"ref11","unstructured":"https:\/\/www.diamond.ac.uk\/Instruments\/Mx\/Common\/Common-Manual\/Using-ISPyB\/Data-management.html."},{"key":"ref12","author":"Kabsch W","year":"2010","unstructured":"Kabsch W. (2010) XDS Acta Crystallogr D Bio Crystallogr 66(Pt 2):125\u2013132."},{"issue":"Pt 1","key":"ref13","doi-asserted-by":"crossref","first-page":"72","DOI":"10.1107\/S0907444905036693","article-title":"Scaling and assessment of data quality","volume":"66","author":"Evans PR","year":"2006","unstructured":"Evans PR. Scaling and assessment of data quality. Acta Crystallogr D Bio Crystallogr. 2006;66(Pt 1):72\u201382.","journal-title":"Acta Crystallogr D Bio Crystallogr"},{"key":"ref14","doi-asserted-by":"crossref","first-page":"1022","DOI":"10.1107\/S0021889897006766","article-title":"MOLREP: an automated programme for molecular replacement","volume":"30","author":"Vagin A","year":"1997","unstructured":"Vagin A, Teplyakov A. MOLREP: an automated programme for molecular replacement. J Appl Cryst. 1997;30:1022\u20135.","journal-title":"J Appl Cryst"},{"key":"ref15","first-page":"355","article-title":"REFMACS for the refinement of macromolecular crystal structures","volume":"D67","author":"Murshdov GN","year":"2011","unstructured":"Murshdov GN, Skubak P, Lebedev AA, Pannu NS, Steiner RA, Nicholls RA, Winn MD, Long F, Vagin AA. REFMACS for the refinement of macromolecular crystal structures. Acta Cryst. 2011;D67:355\u2013367.","journal-title":"Acta Cryst"},{"key":"ref16","first-page":"68","article-title":"CCP4i2: the new graphical user interface to the CCP4 program suite","volume":"D74","author":"Potterton L","year":"2018","unstructured":"Potterton L, Agirre J, Ballard C, Cowtan K, Dodson E, Evans PR, Jenkins HT, Keegan R, Krissinel E, Stevenson K, Lebedev A, McNicholas SJ, Nicholls RA, Noble M, Pannu NS, Roth C, Sheldrick G, Skubak P, Turkenburg J, Uski V, von Delft F, Waterman D, Wilson K, Winn M, Wojdyr M. CCP4i2: the new graphical user interface to the CCP4 program suite. Acta Cryst. 2018;D74:68\u201384.","journal-title":"Acta Cryst"},{"key":"ref17","first-page":"486","volume":"D66","author":"Emsley P","year":"2010","unstructured":"Emsley P, Lohkamp B, Scott WG, Cowtan K. WinCoot Acta Cryst. 2010;D66:486\u2013501.","journal-title":"WinCoot Acta Cryst"},{"key":"ref18","author":"Joosten RP","year":"2014","unstructured":"Joosten RP, Long F, Murshudov GN, Perrakis A. (2014) The PDB_REDO server for macromolecular structure model optimization. IUCrJ. May 30;1(Pt 4):213 \u2013 20"},{"key":"ref19","first-page":"12","volume":"D66","author":"Chen VB","year":"2010","unstructured":"Chen VB, Arendall WB, Headd JJ, Keedy DA, Immormino RM, Kapral GJ, Murray LW, Richardson JS, Richardson DC. MolProbity Acta Cryst. 2010;D66:12\u201321.","journal-title":"MolProbity Acta Cryst"},{"key":"ref20","unstructured":"Biovia: Accelerys Corporation:. (2012) Discovery Studio Software version 3.5.http:\/\/accelrys.com\/products\/datasheets\/whats-new-in-discovery-studio.pdf)."},{"key":"ref21","doi-asserted-by":"crossref","first-page":"389","DOI":"10.1107\/S0108768102003324","article-title":"New software for searching the Cambridge Structural Database and visualizing crystal structures","volume":"B58","author":"Bruno IJ","year":"2002","unstructured":"Bruno IJ, Cole JC, Edgington PR, Kessler MK, Macrae CF, McCabe P, Pearson J, Taylor R. New software for searching the Cambridge Structural Database and visualizing crystal structures. Acta Cryst. 2002;B58:389\u201397.","journal-title":"Acta Cryst"},{"key":"ref22","doi-asserted-by":"crossref","first-page":"474","DOI":"10.1107\/S0907444902023685","article-title":"The structure of T6 human insulin at 1.0 \u00c5 resolution","volume":"59","author":"Smith GD","year":"2003","unstructured":"Smith GD, Pangborn WA, Blessing RH. The structure of T6 human insulin at 1.0 \u00c5 resolution. Acta Cryst D. 2003;59:474\u201382.","journal-title":"Acta Cryst D"},{"key":"ref23","doi-asserted-by":"crossref","first-page":"649","DOI":"10.1107\/S0907444901003468","article-title":"Ultra- high-resolution structure of a BPTI mutant","volume":"57","author":"Addlagatta A","year":"2001","unstructured":"Addlagatta A, Czapinska H, Otleski J, Jaskolski M. Ultra- high-resolution structure of a BPTI mutant. Acta Cryst D. 2001;57:649\u201363.","journal-title":"Acta Cryst D"},{"key":"ref24","doi-asserted-by":"crossref","first-page":"73","DOI":"10.1186\/s13065-017-0296-y","article-title":"Ultra\u2013high resolution X\u2013ray structures of two forms of human recombinant insulin at 100 K","volume":"11","author":"Lisgarten R","year":"2017","unstructured":"Lisgarten R, Palmer RA, Lobley CMC, Naylor CE, Chowdhry BZ, Al-Kurdi ZI, Badwan AA, Howlin BJ, Gibbons CJN, Saldanha JW, Lisgarten JN, Basak AK. Ultra\u2013high resolution X\u2013ray structures of two forms of human recombinant insulin at 100 K. Chem Cent J. 2017;11:73\u201399.","journal-title":"Chem Cent J"},{"issue":"12","key":"ref25","doi-asserted-by":"crossref","first-page":"3094","DOI":"10.1021\/acs.jcim.7b00443","article-title":"Identification of Conserved Water Sites in Protein Structures for Drug Design","volume":"57","author":"Juki\u010d M","year":"2017","unstructured":"Juki\u010d M, Konc J, Gobec S, Jane\u017ei\u010d D. Identification of Conserved Water Sites in Protein Structures for Drug Design. J Chem Inf Model. 2017;57(12):3094\u2013103.","journal-title":"J Chem Inf Model"},{"issue":"5","key":"ref26","doi-asserted-by":"crossref","first-page":"1631","DOI":"10.1021\/acs.jpcb.8b00453","article-title":"What Gives an Insulin Hexamer Its Unique Shape and Stability? Role of Ten Confined Water Molecules","volume":"122","author":"Mukherjee S","year":"2018","unstructured":"Mukherjee S, Mondal S, Deshmukh AA, Gopal B, Bagchi B. What Gives an Insulin Hexamer Its Unique Shape and Stability? Role of Ten Confined Water Molecules. J Phys Chem B. 2018;122(5):1631\u20137.","journal-title":"J Phys Chem B"},{"key":"ref27","author":"Zegers I","year":"1994","unstructured":"Zegers I, Maes D, Minh-Hoa D-T, Poortmans F, Palmer R, Wyns L. (1994) The structures of RNase A complexed with 3\u2019-CMP and d(CpA): Active site conformation and conserved water molecules. Protein Science. 3:2322\u20132339. Cambridge University Press."},{"issue":"19","key":"ref28","doi-asserted-by":"crossref","first-page":"10018","DOI":"10.1073\/pnas.93.19.10018","article-title":"On the mechanism of action of ribonuclease A: Relevance of enzymatic studies with a p-nitrophenylphosphate ester and a thiophosphate ester","volume":"93","author":"Breslow R","year":"1996","unstructured":"Breslow R, Chapman WH Jr. On the mechanism of action of ribonuclease A: Relevance of enzymatic studies with a p-nitrophenylphosphate ester and a thiophosphate ester. PNAS. 1996;93(19):10018\u201321.","journal-title":"PNAS"},{"key":"ref29","author":"Canada","year":"2020","unstructured":"Canada. H3A 2R7 (2020)."},{"issue":"8","key":"ref30","doi-asserted-by":"crossref","first-page":"3696","DOI":"10.1021\/acs.jctc.5b00255","article-title":"ff14SB: Improving the Accuracy of Protein Side Chain and Backbone Parameters from ff99SB","volume":"11","author":"Maier JA","year":"2015","unstructured":"Maier JA, Martinez C, Kasavajhala K, Wickstrom L, Hauser KE, Simmerling C. ff14SB: Improving the Accuracy of Protein Side Chain and Backbone Parameters from ff99SB. J Chem Theory Comput. 2015;11(8):3696\u2013713.","journal-title":"J Chem Theory Comput"},{"issue":"3","key":"ref31","doi-asserted-by":"crossref","first-page":"251","DOI":"10.1007\/BF00124456","article-title":"MAB, a generally applicable molecular force field for structure modelling in medicinal chemistry","volume":"9","author":"Gerber PR","year":"1995","unstructured":"Gerber PR, M\u00fcller K. MAB, a generally applicable molecular force field for structure modelling in medicinal chemistry. J Comput Aided Mol Des. 1995;9(3):251\u201368.","journal-title":"J Comput Aided Mol Des"},{"key":"ref32","volume-title":"Symplectic Algorithm for Constant Pressure Molecular Dynamics Using a Nos\u00e9","author":"Sturgeon JB","year":"2002","unstructured":"Sturgeon JB, Laird BB. Symplectic Algorithm for Constant Pressure Molecular Dynamics Using a Nos\u00e9. -Poincar\u00e9 Thermostat; University of Kansas Technical Paper; 2002."},{"key":"ref33","doi-asserted-by":"crossref","first-page":"114","DOI":"10.1006\/jcph.1998.6171","article-title":"The Nos\u00e9\u2013Poincar\u00e9 Method for Constant Temperature Molecular Dynamics","volume":"151","author":"Bond SD","year":"1999","unstructured":"Bond SD, Benedict JL, Laird BB. The Nos\u00e9\u2013Poincar\u00e9 Method for Constant Temperature Molecular Dynamics. J Comp Phys. 1999;151:114\u201334.","journal-title":"J Comp Phys"},{"key":"ref34","doi-asserted-by":"crossref","first-page":"3684","DOI":"10.1063\/1.448118","article-title":"Molecular dynamics with coupling to an external bath","volume":"81","author":"Berendsen HJC","year":"1984","unstructured":"Berendsen HJC, Postma JPM, van Gunsteren WF, Di Nola A, Haak JR. Molecular dynamics with coupling to an external bath. J Chem Phys. 1984;81:3684\u201390.","journal-title":"J Chem Phys"},{"issue":"51","key":"ref35","first-page":"34134","volume":"273","author":"Fisher BM","year":"1998","unstructured":"Fisher BM, Grilley EJ, Raines RT. J Bichem. 1998;273(51):34134\u20138.","journal-title":"J Bichem"},{"key":"ref36","unstructured":"A New Remote. Subsite in Ribonuclease A by Barbra M.Fisher."}],"container-title":[],"original-title":[],"link":[{"URL":"https:\/\/www.researchsquare.com\/article\/rs-2852137\/v1","content-type":"text\/html","content-version":"vor","intended-application":"text-mining"},{"URL":"https:\/\/www.researchsquare.com\/article\/rs-2852137\/v1.html","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2023,10,17]],"date-time":"2023-10-17T17:28:21Z","timestamp":1697563701000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.researchsquare.com\/article\/rs-2852137\/v1"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2023,5,9]]},"references-count":36,"URL":"https:\/\/doi.org\/10.21203\/rs.3.rs-2852137\/v1","relation":{"is-preprint-of":[{"id-type":"doi","id":"10.1186\/s13065-023-00959-6","asserted-by":"subject"}]},"subject":[],"published":{"date-parts":[[2023,5,9]]},"subtype":"preprint"}}