Research Article

Exploring the Complexity of Protein Structure Determination Through X-ray Diffraction

Sarah Otun* and Ikechukwu Achilonu

Published: 21 November, 2023 | Volume 7 - Issue 3 | Pages: 124-132

The determination of a protein structure by using X-ray diffraction encompasses a series of sequential steps (including gene identification and cloning, protein expression and purification, crystallization, phasing model building, refinement, and validation), which need the application of several methodologies derived from molecular biology, bioinformatics, and physical sciences. This article thoroughly examines the complicated procedure of elucidating protein structures within plant biology, using X-ray diffraction as the primary methodology. Commencing with the gene identification process and progressing toward crystallography, this article explores the many obstacles and achievements in acquiring diffraction pictures and their subsequent conversion into electron density maps. The ensuing phases of model construction, refinement, and structural validation are thoroughly examined, providing insight into the inherent complexity associated with each stage. The paper also discusses the critical component of understanding the resultant model and scrutinizing its biological significance. By comprehensively examining these stages, this article presents a nuanced comprehension of the intricate procedure in ascertaining protein structures within plant biology. It offers valuable insights into the obstacles encountered and the biological importance of the acquired structural data.

Read Full Article HTML DOI: 10.29328/journal.jpsp.1001117 Cite this Article Read Full Article PDF


  1. PERUTZ MF, ROSSMANN MG, CULLIS AF, MUIRHEAD H, WILL G, NORTH AC. Structure of haemoglobin: a three-dimensional Fourier synthesis at 5.5-A. resolution, obtained by X-ray analysis. Nature. 1960 Feb 13;185(4711):416-22. doi: 10.1038/185416a0. PMID: 18990801.
  2. Seeman JI, Restrepo G. The Mutation of the “Nobel Prize in Chemistry” into the “Nobel Prize in Chemistry or Life Sciences”: Several Decades of Transparent and Opaque Evidence of Change within the Nobel Prize Program. Angewandte Chemie. 2020 Feb 17;132(8):2962–81.
  3. Fetisov GV. X-ray diffraction methods for structural diagnostics of materials: progress and achievements. Physics-Uspekhi. 2020 Jan 1;63(1):2–32.
  4. Wlodawer A, Hodgson KO, Shooter EM. Crystallization of nerve growth factor from mouse submaxillary glands. Proc Natl Acad Sci U S A. 1975 Mar;72(3):777-9. doi: 10.1073/pnas.72.3.777. PMID: 1055377; PMCID: PMC432402.
  5. McDonald NQ, Lapatto R, Murray-Rust J, Gunning J, Wlodawer A, Blundell TL. New protein fold revealed by a 2.3-A resolution crystal structure of nerve growth factor. Nature. 1991 Dec 5;354(6352):411-4. doi: 10.1038/354411a0. PMID: 1956407.
  6. Yonath A, Müssig J, Wittmann HG. Parameters for crystal growth of ribosomal subunits. J Cell Biochem. 1982;19(2):145-55. doi: 10.1002/jcb.240190205. PMID: 7174745.
  7. Ban N, Nissen P, Hansen J, Moore PB, Steitz TA. The complete atomic structure of the large ribosomal subunit at 2.4 A resolution. Science. 2000 Aug 11;289(5481):905-20. doi: 10.1126/science.289.5481.905. PMID: 10937989.
  8. Kim Y, Babnigg G, Jedrzejczak R, Eschenfeldt WH, Li H, Maltseva N, Hatzos-Skintges C, Gu M, Makowska-Grzyska M, Wu R, An H, Chhor G, Joachimiak A. High-throughput protein purification and quality assessment for crystallization. Methods. 2011 Sep;55(1):12-28. doi: 10.1016/j.ymeth.2011.07.010. Epub 2011 Aug 31. PMID: 21907284; PMCID: PMC3690762.
  9. Komar AA. The Art of Gene Redesign and Recombinant Protein Production: Approaches and Perspectives. In 2016; 161–77.
  10. Papageorgiou AC, Poudel N, Mattsson J. Protein Structure Analysis and Validation with X-Ray Crystallography. Methods Mol Biol. 2021;2178:377-404. doi: 10.1007/978-1-0716-0775-6_25. PMID: 33128762.
  11. Kurpiewska K, Borowski T. Seven quick tips for beginners in protein crystallography. Acta Biochim Pol. 2021 Aug 11;68(4):535-546. doi: 10.18388/abp.2020_5589. PMID: 34379378.
  12. Liu H, Chen Q. Computational protein design with data‐driven approaches: Recent developments and perspectives. WIREs Computational Molecular Science. 2023 May15;13(3).
  13. Ye H, Wu J, Liang Z, Zhang Y, Huang Z. Protein S-Nitrosation: Biochemistry, Identification, Molecular Mechanisms, and Therapeutic Applications. J Med Chem. 2022 Apr 28;65(8):5902-5925. doi: 10.1021/acs.jmedchem.1c02194. Epub 2022 Apr 12. PMID: 35412827.
  14. Macek B, Forchhammer K, Hardouin J, Weber-Ban E, Grangeasse C, Mijakovic I. Protein post-translational modifications in bacteria. Nat Rev Microbiol. 2019 Nov;17(11):651-664. doi: 10.1038/s41579-019-0243-0. Epub 2019 Sep 4. PMID: 31485032.
  15. Ma Y, Lee CJ, Park JS. Strategies for Optimizing the Production of Proteins and Peptides with Multiple Disulfide Bonds. Antibiotics (Basel). 2020 Aug 26;9(9):541. doi: 10.3390/antibiotics9090541. PMID: 32858882; PMCID: PMC7558204.
  16. Yamaguchi H, Miyazaki M. Refolding techniques for recovering biologically active recombinant proteins from inclusion bodies. Biomolecules. 2014 Feb 20;4(1):235-51. doi: 10.3390/biom4010235. PMID: 24970214; PMCID: PMC4030991.
  17. Fontana A, de Laureto PP, Spolaore B, Frare E, Picotti P, Zambonin M. Probing protein structure by limited proteolysis. Acta Biochim Pol. 2004;51(2):299-321. PMID: 15218531.
  18. Giegé R. A historical perspective on protein crystallization from 1840 to the present day. FEBS J. 2013 Dec;280(24):6456-97. doi: 10.1111/febs.12580. Epub 2013 Nov 25. PMID: 24165393.
  19. Jahn TR, Radford SE. Folding versus aggregation: polypeptide conformations on competing pathways. Arch Biochem Biophys. 2008 Jan 1;469(1):100-17. doi: 10.1016/j.abb.2007.05.015. Epub 2007 Jun 8. PMID: 17588526; PMCID: PMC2706318.
  20. Mishra V. Affinity Tags for Protein Purification. Curr Protein Pept Sci. 2020;21(8):821-830. doi: 10.2174/1389203721666200606220109. PMID: 32504500.
  21. Skelly JV, Madden CB. Overexpression, Isolation, and Crystallization of Proteins. In: Crystallographic Methods and Protocols. New Jersey: Humana Press; 23–54.
  22. Alias FL, Nezhad NG, Normi YM, Ali MSM, Budiman C, Leow TC. Recent Advances in Overexpression of Functional Recombinant Lipases. Mol Biotechnol. 2023 Nov;65(11):1737-1749. doi: 10.1007/s12033-023-00725-y. Epub 2023 Mar 27. PMID: 36971996.
  23. Karabchevsky A, Katiyi A, Ang AS, Hazan A. On-chip nanophotonics and future challenges. Nanophotonics. 2020 Sep 11; 9(12):3733–53.
  24. Zhang N, Ashikuzzaman M, Rivaz H. Clutter suppression in ultrasound: performance evaluation and review of low-rank and sparse matrix decomposition methods. Biomed Eng Online. 2020 May 28;19(1):37. doi: 10.1186/s12938-020-00778-z. PMID: 32466753; PMCID: PMC7254711.
  25. Qin W, Xie S, Zhang J, Zhao D, He C, Li H. An Analysis on Commercial Screening Kits and Chemical Components in Biomacromolecular Crystallization Screening. Crystal Research and Technology. 2019 Sep 7;54(9).
  26. Zigon N, Duplan V, Wada N, Fujita M. Crystalline Sponge Method: X-ray Structure Analysis of Small Molecules by Post-Orientation within Porous Crystals-Principle and Proof-of-Concept Studies. Angew Chem Int Ed Engl. 2021 Nov 22;60(48):25204-25222. doi: 10.1002/anie.202106265. Epub 2021 Aug 3. PMID: 34109717.
  27. Zhou H, Sabino J, Yang Y, Ward MD, Shtukenberg AG, Kahr B. Tailor-Made Additives for Melt-Grown Molecular Crystals: Why or Why Not? Annu Rev Mater Res. 2023 Jul 3;53(1):143–64.
  28. Liu Z, Zhou Y, Liu J, Chen J, Heck AJR, Wang F. Reductive methylation labeling, from quantitative to structural proteomics. TrAC Trends in Analytical Chemistry. 2019 Sep; 118:771–8.
  29. Derewenda ZS, Godzik A. The “Sticky Patch” Model of Crystallization and Modification of Proteins for Enhanced Crystallizability. In 2017; 77–115.
  30. Naderi-Azad S, Croitoru D, Khalili S, Eder L, Piguet V. Research Techniques Made Simple: Experimental Methodology for Imaging Mass Cytometry. Journal of Investigative Dermatology. 2021 Mar; 141(3):467-473.e1.
  31. Kunachowicz D, Ściskalska M, Jakubek M, Kizek R, Kepinska M. Structural changes in selected human proteins induced by exposure to quantum dots, their biological relevance and possible biomedical applications. NanoImpact. 2022 Apr;26:100405. doi: 10.1016/j.impact.2022.100405. Epub 2022 May 1. PMID: 35560289.
  32. Barends TRM, Stauch B, Cherezov V, Schlichting I. Serial femtosecond crystallography. Nat Rev Methods Primers. 2022 Aug 4;2:59. doi: 10.1038/s43586-022-00141-7. PMID: 36643971; PMCID: PMC9833121.
  33. Candoni N, Grossier R, Lagaize M, Veesler S. Advances in the Use of Microfluidics to Study Crystallization Fundamentals. Annu Rev Chem Biomol Eng. 2019 Jun 7;10:59-83. doi: 10.1146/annurev-chembioeng-060718-030312. Epub 2019 Apr 24. PMID: 31018097.
  34. Deresz KA, Łaski P, Kamiński R, Jarzembska KN. Advances in Diffraction Studies of Light-Induced Transient Species in Molecular Crystals and Selected Complementary Techniques. Crystals (Basel). 2021 Nov 3;11(11):1345.
  35. Burley SK, Berman HM, Duarte JM, Feng Z, Flatt JW, Hudson BP, Lowe R, Peisach E, Piehl DW, Rose Y, Sali A, Sekharan M, Shao C, Vallat B, Voigt M, Westbrook JD, Young JY, Zardecki C. Protein Data Bank: A Comprehensive Review of 3D Structure Holdings and Worldwide Utilization by Researchers, Educators, and Students. Biomolecules. 2022 Oct 4;12(10):1425. doi: 10.3390/biom12101425. PMID: 36291635; PMCID: PMC9599165.
  36. Mingos DMP. Early History of X-Ray Crystallography. 2020; 1-41.
  37. Abdollahi H, Prestegard JH, Valafar H. Computational modeling multiple conformational states of proteins with residual dipolar coupling data. Curr Opin Struct Biol. 2023 Oct;82:102655. doi: 10.1016/j.sbi.2023.102655. Epub 2023 Jul 14. PMID: 37454402.
  38. Kermani AA. A guide to membrane protein X-ray crystallography. FEBS J. 2021 Oct;288(20):5788-5804. doi: 10.1111/febs.15676. Epub 2020 Dec 31. PMID: 33340246.
  39. Zheng H, Hou J, Zimmerman MD, Wlodawer A, Minor W. The future of crystallography in drug discovery. Expert Opin Drug Discov. 2014 Feb;9(2):125-37. doi: 10.1517/17460441.2014.872623. Epub 2013 Dec 28. PMID: 24372145; PMCID: PMC4106240.
  40. Chapuis G. An elementary treatment on the diffraction of crystalline structures. Crystallogr Rev. 2021 Oct 2;27(3–4):146–77.
  41. Collaborative Computational Project, Number 4. The CCP4 suite: programs for protein crystallography. Acta Crystallogr D Biol Crystallogr. 1994 Sep 1;50(Pt 5):760-3. doi: 10.1107/S0907444994003112. PMID: 15299374.
  42. Adams PD, Grosse-Kunstleve RW, Hung LW, Ioerger TR, McCoy AJ, Moriarty NW, Read RJ, Sacchettini JC, Sauter NK, Terwilliger TC. PHENIX: building new software for automated crystallographic structure determination. Acta Crystallogr D Biol Crystallogr. 2002 Nov;58(Pt 11):1948-54. doi: 10.1107/s0907444902016657. Epub 2002 Oct 21. PMID: 12393927.
  43. Minor W, Cymborowski M, Otwinowski Z, Chruszcz M. HKL-3000: the integration of data reduction and structure solution--from diffraction images to an initial model in minutes. Acta Crystallogr D Biol Crystallogr. 2006 Aug;62(Pt 8):859-66. doi: 10.1107/S0907444906019949. Epub 2006 Jul 18. PMID: 16855301.
  44. Chen B, Zheng W, Chun F, Xu X, Zhao Q, Wang F. Synthesis and hybridization of CuInS2nanocrystals for emerging applications. Chem Soc Rev. 2023 Nov 10. doi: 10.1039/d3cs00611e. Epub ahead of print. PMID: 37947021.
  45. Perrakis A, Morris R, Lamzin VS. Automated protein model building combined with iterative structure refinement. Nat Struct Biol. 1999 May;6(5):458-63. doi: 10.1038/8263. PMID: 10331874.
  46. Terwilliger T. SOLVE and RESOLVE: automated structure solution, density modification and model building. J Synchrotron Radiat. 2004 Jan 1;11(Pt 1):49-52. doi: 10.1107/s0909049503023938. Epub 2003 Nov 28. PMID: 14646132.
  47. Turk D. Towards automatic macromolecular crystal structure determination. Nato Science Series Sub Series I Life and Behavioural Sciences. 2001;325:148–55.
  48. Jones TA, Zou JY, Cowan SW, Kjeldgaard M. Improved methods for building protein models in electron density maps and the location of errors in these models. Acta Crystallogr A. 1991 Mar 1;47 ( Pt 2):110-9. doi: 10.1107/s0108767390010224. PMID: 2025413.
  49. Emsley P, Cowtan K. Coot: model-building tools for molecular graphics. Acta Crystallogr D Biol Crystallogr. 2004 Dec;60(Pt 12 Pt 1):2126-32. doi: 10.1107/S0907444904019158. Epub 2004 Nov 26. PMID: 15572765.
  50. Chruszcz M, Wlodawer A, Minor W. Determination of protein structures--a series of fortunate events. Biophys J. 2008 Jul;95(1):1-9. doi: 10.1529/biophysj.108.131789. Epub 2008 Apr 25. PMID: 18441029; PMCID: PMC2426657.
  51. Murshudov GN, Vagin AA, Dodson EJ. Refinement of macromolecular structures by the maximum-likelihood method. Acta Crystallogr D Biol Crystallogr. 1997 May 1;53(Pt 3):240-55. doi: 10.1107/S0907444996012255. PMID: 15299926.
  52. Brünger AT, Adams PD, Clore GM, DeLano WL, Gros P, Grosse-Kunstleve RW, Jiang JS, Kuszewski J, Nilges M, Pannu NS, Read RJ, Rice LM, Simonson T, Warren GL. Crystallography & NMR system: A new software suite for macromolecular structure determination. Acta Crystallogr D Biol Crystallogr. 1998 Sep 1;54(Pt 5):905-21. doi: 10.1107/s0907444998003254. PMID: 9757107.
  53. Patel JR, Joshi H V., A. Shah U, K. Patel J. A Review on Computational Software Tools for Drug Design and Discovery. Indo Global Journal of Pharmaceutical Sciences. 2022; 12:53–81.
  54. Liebschner D, Afonine PV, Baker ML, Bunkóczi G, Chen VB, Croll TI, Hintze B, Hung LW, Jain S, McCoy AJ, Moriarty NW, Oeffner RD, Poon BK, Prisant MG, Read RJ, Richardson JS, Richardson DC, Sammito MD, Sobolev OV, Stockwell DH, Terwilliger TC, Urzhumtsev AG, Videau LL, Williams CJ, Adams PD. Macromolecular structure determination using X-rays, neutrons and electrons: recent developments in Phenix. Acta Crystallogr D Struct Biol. 2019 Oct 1;75(Pt 10):861-877. doi: 10.1107/S2059798319011471. Epub 2019 Oct 2. PMID: 31588918; PMCID: PMC6778852.
  55. Laskowski RA, MacArthur MW, Moss DS, Thornton JM. PROCHECK: a program to check the stereochemical quality of protein structures. J Appl Crystallogr. 1993; 26(2): 283-91.
  56. Hooft RW, Vriend G, Sander C, Abola EE. Errors in protein structures. Nature. 1996 May 23;381(6580):272. doi: 10.1038/381272a0. PMID: 8692262.
  57. Lovell SC, Davis IW, Arendall WB 3rd, de Bakker PI, Word JM, Prisant MG, Richardson JS, Richardson DC. Structure validation by Calpha geometry: phi,psi and Cbeta deviation. Proteins. 2003 Feb 15;50(3):437-50. doi: 10.1002/prot.10286. PMID: 12557186.
  58. Aguilar FJ, Mills JP, Delgado J, Aguilar MA, Negreiros JG, Pérez JL. Modelling vertical error in LiDAR-derived digital elevation models. ISPRS Journal of Photogrammetry and Remote Sensing. 2010 Jan;65(1):103–10.
  59. Xie W, Wang F, Li Y, Lai L, Pei J. Advances and Challenges in De Novo Drug Design Using Three-Dimensional Deep Generative Models. J Chem Inf Model. 2022 May 23;62(10):2269-2279. doi: 10.1021/acs.jcim.2c00042. Epub 2022 May 11. PMID: 35544331.
  60. Wlodawer A, Minor W, Dauter Z, Jaskolski M. Protein crystallography for non-crystallographers, or how to get the best (but not more) from published macromolecular structures. FEBS J. 2008 Jan;275(1):1-21. doi: 10.1111/j.1742-4658.2007.06178.x. Epub 2007 Nov 23. PMID: 18034855; PMCID: PMC4465431.
  61. Šrajer V, Schmidt M. Watching Proteins Function with Time-resolved X-ray Crystallography. J Phys D Appl Phys. 2017 Sep 20;50(37):373001. doi: 10.1088/1361-6463/aa7d32. Epub 2017 Aug 22. PMID: 29353938; PMCID: PMC5771432.
  62. Wlodawer A, Sekula B, Gustchina A, Rotanova TV. Structure and the Mode of Activity of Lon Proteases from Diverse Organisms. J Mol Biol. 2022 Apr 15;434(7):167504. doi: 10.1016/j.jmb.2022.167504. Epub 2022 Feb 17. PMID: 35183556; PMCID: PMC9013511.
  63. Rotanova TV, Botos I, Melnikov EE, Rasulova F, Gustchina A, Maurizi MR, Wlodawer A. Slicing a protease: structural features of the ATP-dependent Lon proteases gleaned from investigations of isolated domains. Protein Sci. 2006 Aug;15(8):1815-28. doi: 10.1110/ps.052069306. PMID: 16877706; PMCID: PMC2242575.
  64. Wlodawer A, Sekula B, Gustchina A, Rotanova TV. Structure and the Mode of Activity of Lon Proteases from Diverse Organisms. J Mol Biol. 2022 Apr 15;434(7):167504. doi: 10.1016/j.jmb.2022.167504. Epub 2022 Feb 17. PMID: 35183556; PMCID: PMC9013511.
  65. Rotanova TV, Botos I, Melnikov EE, Rasulova F, Gustchina A, Maurizi MR, Wlodawer A. Slicing a protease: structural features of the ATP-dependent Lon proteases gleaned from investigations of isolated domains. Protein Sci. 2006 Aug;15(8):1815-28. doi: 10.1110/ps.052069306. PMID: 16877706; PMCID: PMC2242575.


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