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Sample Preparation for Time-Resolved Serial Crystallography: Practical Considerations

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Advanced Methods in Structural Biology

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2652))

Abstract

Time-resolved serial crystallography is an emerging method to elucidate the structure-function relationship of biomolecular systems at up to atomic resolution. However, to make this demanding method a success, a number of experimental requirements have to be met. In this chapter, we summarize general guidelines and protocols towards performing time-resolved crystallography experiments, with a particular emphasis on sample requirements and preparation but also a brief excursion into reaction initiation.

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References

  1. Moffat K (1989) Time-resolved macromolecular crystallography. Annu Rev Biophys Bioeng 18:309–332

    Article  CAS  Google Scholar 

  2. Colletier J-P, Schirò G, Weik M (2018) X-ray free electron lasers, a revolution in structural biology. pp 331–356

    Google Scholar 

  3. Moffat K (1989) Time-resolved macromolecular crystallography. Annu Rev Biophys 18:309–332

    Article  CAS  Google Scholar 

  4. Pai EF (1992) Time-resolved macromolecular crystallography. Curr Opin Struct Biol 2:821–827

    Article  CAS  Google Scholar 

  5. Schmidt M (2008) Ultrashort laser pulses in biology and medicine. Biol Med Phys Biomed:201–241

    Google Scholar 

  6. Mehrabi P, Sung S, von Stetten D, et al. (2022) Millisecond cryo-trapping by the spitrobot crystal plunger simplifies time-resolved crystallography

    Google Scholar 

  7. Halle B (2004) Biomolecular cryocrystallography: structural changes during flash-cooling. Proc Natl Acad Sci USA 101:4793–4798

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Mehrabi P, von Stetten D, Leimkohl J-P, et al. (2021) An environmental control box for serial crystallography enables multi-dimensional experiments. https://doi.org/10.1101/2021.11.07.467596

  9. Schmidt M, Srajer V, Henning R et al (2013) Protein energy landscapes determined by five-dimensional crystallography. Acta Crystallogr D Biol Crystallogr 69:2534–2542

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Moffat K, Szebenyi D, Bilderback D (1984) X-ray Laue diffraction from protein crystals. Science 223:1423–1425

    Article  CAS  PubMed  Google Scholar 

  11. Ren Z, Bourgeois D, Helliwell JR et al (1999) Laue crystallography: coming of age. J Synchrotron Radiat 6:891–917

    Article  CAS  Google Scholar 

  12. Neutze R, Wouts R, van der Spoel D et al (2000) Potential for biomolecular imaging with femtosecond X-ray pulses. Nature 406:752–757

    Article  CAS  PubMed  Google Scholar 

  13. Schlichting I, Miao J (2012) Emerging opportunities in structural biology with X-ray free-electron lasers. Curr Opin Struct Biol 22:613–626

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Boutet S, Lomb L, Williams GJ et al (2012) High-resolution protein structure determination by serial femtosecond crystallography. Science 337:362–364

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Aquila A, Hunter MS, Doak RB et al (2012) Time-resolved protein nanocrystallography using an X-ray free-electron laser. Opt Express 20:2706–2716

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Botha S, Nass K, Barends TRM et al (2015) Room-temperature serial crystallography at synchrotron X-ray sources using slowly flowing free-standing high-viscosity microstreams. Acta Crystallogr D Biol Crystallogr 71(Pt 2):387–397

    Article  CAS  PubMed  Google Scholar 

  17. Schlichting I (2015) Serial femtosecond crystallography: the first five years. IUCrJ 2:246–255

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Chapman HN, Caleman C, Timneanu N (2014) Diffraction before destruction. Philos Trans R Soc Lond Ser B Biol Sci 369:20130313

    Article  Google Scholar 

  19. Barends TRM, Foucar L, Botha S et al (2014) De novo protein crystal structure determination from X-ray free-electron laser data. Nature 505:244–247

    Article  CAS  PubMed  Google Scholar 

  20. Chapman HN, Fromme P, Barty A et al (2011) Femtosecond X-ray protein nanocrystallography. Nature 470:73–78

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Redecke L, Nass K, DePonte DP et al (2013) Natively inhibited Trypanosoma brucei cathepsin B structure determined by using an X-ray laser. Science 339:227–230

    Article  CAS  PubMed  Google Scholar 

  22. Neutze R, Moffat K (2012) Time-resolved structural studies at synchrotrons and X-ray free electron lasers: opportunities and challenges. Curr Opin Struct Biol 22:651–659

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Kabsch W (2014) Processing of X-ray snapshots from crystals in random orientations. Acta Crystallogr D Biol Crystallogr 70:2204–2216

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. White TA, Kirian RA, Martin AV et al (2012) CrystFEL: A software suite for snapshot serial crystallography. J Appl Crystallogr 45:335–341

    Article  CAS  Google Scholar 

  25. Winter G, Beilsten-Edmands J, Devenish N et al (2022) DIALS as a toolkit. Protein Sci 31:232–250

    Article  CAS  PubMed  Google Scholar 

  26. Sauter NK, Hattne J, Brewster AS et al (2014) Improved crystal orientation and physical properties from single-shot XFEL stills. Acta Crystallogr D Biol Crystallogr 70:3299–3309

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Brewster AS, Sawaya MR, Rodriguez J et al (2015) Indexing amyloid peptide diffraction from serial femtosecond crystallography: new algorithms for sparse patterns. Acta Crystallogr D Biol Crystallogr 71(Pt 2):357–366

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Uervirojnangkoorn M, Zeldin OB, Lyubimov AY et al (2015) Enabling X-ray free electron laser crystallography for challenging biological systems from a limited number of crystals. elife 4:e05421

    Article  PubMed  PubMed Central  Google Scholar 

  29. Zeldin OB, Brewster AS, Hattne J et al (2015) Data Exploration Toolkit for serial diffraction experiments. Acta Crystallogr D Biol Crystallogr 71:352–356

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Martiel I, Müller-Werkmeister HM, Cohen AE (2019) Strategies for sample delivery for femtosecond crystallography. Acta Crystallogr Sect D 75:160–177

    Article  CAS  Google Scholar 

  31. Pearson AR, Mehrabi P (2020) Serial synchrotron crystallography for time-resolved structural biology. Curr Opin Struct Biol 65:168–174

    Article  CAS  PubMed  Google Scholar 

  32. Schulz EC, Yorke BA, Pearson AR et al (2022) Best practices for time-resolved serial synchrotron crystallography. Acta Crystallogr D Struct Biol 78:14–29

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Zhao F, Zhang B, Yan E et al (2019) A guide to sample delivery systems for serial crystallography. FEBS J 286:4402–4417

    Article  CAS  PubMed  Google Scholar 

  34. Grunbein ML, Kovacs GN (2019) Sample delivery for serial crystallography at free-electron lasers and synchrotrons. Acta Crystallogr D Struct Biol 75:178–191

    Article  PubMed  PubMed Central  Google Scholar 

  35. Moreno-Chicano T, Ebrahim A, Axford D et al (2019) High-throughput structures of protein-ligand complexes at room temperature using serial femtosecond crystallography. IUCrJ 6:1074–1085

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Mehrabi P, Bücker R, Bourenkov G et al (2021) Serial femtosecond and serial synchrotron crystallography can yield data of equivalent quality: a systematic comparison. Sci Adv 7:eabf1380

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Weinert T, Olieric N, Cheng R et al (2017) Serial millisecond crystallography for routine room-temperature structure determination at synchrotrons. Nat Commun 8:542

    Article  PubMed  PubMed Central  Google Scholar 

  38. Chapman HN (2019) X-ray free-electron lasers for the structure and dynamics of macromolecules. Annu Rev Biochem 88:35–58

    Article  CAS  PubMed  Google Scholar 

  39. Bar-Even A, Noor E, Savir Y et al (2011) The moderately efficient enzyme: evolutionary and physicochemical trends shaping enzyme parameters. Biochemistry 50:4402–4410

    Article  CAS  PubMed  Google Scholar 

  40. Fisher SZ, Schantz LV, Håkansson M et al (2015) Neutron crystallographic studies reveal hydrogen bond and water-mediated interactions between a carbohydrate-binding module and its bound carbohydrate ligand. Biochemistry 54:6435–6438

    Article  CAS  PubMed  Google Scholar 

  41. Kono F, Tamada T (2021) Neutron crystallography for the elucidation of enzyme catalysis. Curr Opin Struct Biol 71:36–42

    Article  CAS  PubMed  Google Scholar 

  42. Kukic P, Lundström P, Camilloni C et al (2016) Structural insights into the calcium-mediated allosteric transition in the C-terminal domain of calmodulin from nuclear magnetic resonance measurements. Biochemistry 55:19–28

    Article  CAS  PubMed  Google Scholar 

  43. Granata D, Camilloni C, Vendruscolo M et al (2013) Characterization of the free-energy landscapes of proteins by NMR-guided metadynamics. Proc Natl Acad Sci USA 110:6817–6822

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  44. Wang Y, Martins JM, Lindorff-Larsen K (2017) Biomolecular conformational changes and ligand binding: from kinetics to thermodynamics. Chem Sci 8:6466–6473

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  45. Brotzakis ZF, Vendruscolo M, Bolhuis PG (2021) A method of incorporating rate constants as kinetic constraints in molecular dynamics simulations. Proc Natl Acad Sci USA 118:e2012423118

    Article  CAS  PubMed  Google Scholar 

  46. Boulton S, Melacini G (2016) Advances in NMR methods to map allosteric sites: from models to translation. Chem Rev 116:6267–6304

    Article  CAS  PubMed  Google Scholar 

  47. Fogarty AC, Laage D (2014) Water dynamics in protein hydration shells: the molecular origins of the dynamical perturbation. J Phys Chem B 118:7715–7729

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  48. Woodhouse J, Kovacs GN, Coquelle N et al (2020) Photoswitching mechanism of a fluorescent protein revealed by time-resolved crystallography and transient absorption spectroscopy. Nat Commun 11:741

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  49. Bücker R, Hogan-Lamarre P, Mehrabi P et al (2020) Serial protein crystallography in an electron microscope. Nat Commun 11:996

    Article  PubMed  PubMed Central  Google Scholar 

  50. Martin RW, Zilm KW (2003) Preparation of protein nanocrystals and their characterization by solid state NMR. J Magn Reson 165:162–174

    Article  CAS  PubMed  Google Scholar 

  51. Kupitz C, Grotjohann I, Conrad CE et al (2014) Microcrystallization techniques for serial femtosecond crystallography using photosystem II from Thermosynechococcus elongatus as a model system. Philos Trans R Soc Lond B Biol Sci 369:20130316

    Article  PubMed  PubMed Central  Google Scholar 

  52. Norton-Baker B, Mehrabi P, Boger J et al (2021) A simple vapor-diffusion method enables protein crystallization inside the HARE serial crystallography chip. Acta Crystallogr Sect D 77:1–15

    Article  Google Scholar 

  53. Lieske J, Cerv M, Kreida S et al (2019) On-chip crystallization for serial crystallography experiments and on-chip ligand-binding studies. IUCrJ 6:714–728

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  54. Ren Z, Ayhan M, Bandara S et al (2018) Crystal-on-crystal chips for: in situ serial diffraction at room temperature. Lab Chip 18:2246–2256

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  55. Lee D, Park S, Lee K et al (2020) Application of a high-throughput microcrystal delivery system to serial femtosecond crystallography. J Appl Crystallogr 53:477–485

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  56. Opara N, Martiel I, Arnold SA et al (2017) Direct protein crystallization on ultrathin membranes for diffraction measurements at X-ray free-electron lasers. J Appl Crystallogr 50:909–918

    Article  CAS  Google Scholar 

  57. Beale JH, Bolton R, Marshall SA et al (2019) Successful sample preparation for serial crystallography experiments. J Appl Crystallogr 52:1385–1396

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  58. Stohrer C, Horrell S, Meier S et al (2021) Homogeneous batch micro-crystallization of proteins from ammonium sulfate. Acta Crystallogr D Struct Biol 77:194–204

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  59. Cherezov V (2011) Lipidic cubic phase technologies for membrane protein structural studies. Curr Opin Struct Biol 21:559–566

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  60. Suga M, Akita F, Yamashita K et al (2019) An oxyl/oxo mechanism for oxygen-oxygen coupling in PSII revealed by an x-ray free-electron laser. Science 366:334–338

    Article  CAS  PubMed  Google Scholar 

  61. Suga M, Akita F, Sugahara M et al (2017) Light-induced structural changes and the site of O=O bond formation in PSII caught by XFEL. Nature 543:131–135

    Article  CAS  PubMed  Google Scholar 

  62. Liu W, Wacker D, Gati C et al (2013) Serial femtosecond crystallography of G protein-coupled receptors. Science 342:1521–1524

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  63. Steyaert J, Kobilka BK (2011) Nanobody stabilization of G protein-coupled receptor conformational states. Curr Opin Struct Biol 21:567–572

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  64. Rasmussen SGF, Choi H-J, Fung JJ et al (2011) Structure of a nanobody-stabilized active state of the β(2) adrenoceptor. Nature 469:175–180

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  65. Mehrabi P, Muller-Werkmeister HM, Leimkohl JP et al (2020) The HARE chip for efficient time-resolved serial synchrotron crystallography. J Synchrotron Radiat 27:360–370

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  66. Kissick DJ, Wanapun D, Simpson GJ (2011) Second-order nonlinear optical imaging of chiral crystals. Annu Rev Anal Chem (Palo Alto, Calif) 4:419–437

    Article  CAS  PubMed  Google Scholar 

  67. Dierks K, Meyer A, Einspahr H et al (2008) Dynamic light scattering in protein crystallization droplets: adaptations for analysis and optimization of crystallization processes. Cryst Growth Des 8:1628–1634

    Article  CAS  Google Scholar 

  68. Oberthuer D, Melero-García E, Dierks K et al (2012) Monitoring and scoring counter-diffusion protein crystallization experiments in capillaries by in situ dynamic light scattering. PLoS One 7:e33545–e33546

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  69. Stevenson HP, Makhov AM, Calero M et al (2014) Use of transmission electron microscopy to identify nanocrystals of challenging protein targets. Proc Natl Acad Sci USA 111:8470–8475

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  70. Weiss S, Vergara S, Lin G et al (2020) cryoEM, methods and protocols. Methods Mol Biol 2215:299–307

    Article  Google Scholar 

  71. Grünbein ML, Stricker M, Kovacs GN et al (2020) Illumination guidelines for ultrafast pump-probe experiments by serial femtosecond crystallography. Nat Methods 17:681–684

    Article  PubMed  Google Scholar 

  72. Mehrabi P, Schulz EC, Agthe M et al (2019) Liquid application method for time-resolved analyses by serial synchrotron crystallography. Nat Methods 16:979

    Article  CAS  PubMed  Google Scholar 

  73. Schmidt M (2013) Mix and inject: Reaction initiation by diffusion for time-resolved macromolecular crystallography. Adv Cond Matter Phys 2013:1–10

    Article  Google Scholar 

  74. Bowler MW, Montgomery MG, Leslie AGW et al (2006) Reproducible improvements in order and diffraction limit of crystals of bovine mitochondrial F1-ATPase by controlled dehydration. Acta Crystallogr D Biol Crystallogr 62:991–995

    Article  PubMed  Google Scholar 

  75. Sanchez-Weatherby J, Bowler MW, Huet J et al (2009) Improving diffraction by humidity control: a novel device compatible with X-ray beamlines. Acta Crystallogr D Biol Crystallogr 65:1237–1246

    Article  CAS  PubMed  Google Scholar 

  76. Bowler MW, Mueller U, Weiss MS et al (2015) Automation and experience of controlled crystal dehydration: results from the European synchrotron hc1 collaboration. Cryst Growth Des 15:1043–1054

    Article  CAS  Google Scholar 

  77. Emsley P, Cowtan K (2004) Coot: model-building tools for molecular graphics. Acta Crystallogr D Biol Crystallogr 60:2126–2132

    Article  PubMed  Google Scholar 

  78. DeLano WL The PyMol molecular viewer. DeLano Scientific, San Carlos, California, USA. www.pymol.org

  79. Pettersen EF, Goddard TD, Huang CC et al (2004) UCSF Chimera – a visualization system for exploratory research and analysis. J Comput Chem 25:1605–1612

    Article  CAS  PubMed  Google Scholar 

  80. Mehrabi P, Schulz EC, Dsouza R et al (2019) Time-resolved crystallography reveals allosteric communication aligned with molecular breathing. Science 365:1167–1170

    Article  CAS  PubMed  Google Scholar 

  81. Pande K, Hutchison CDM, Groenhof G et al (2016) Femtosecond structural dynamics drives the trans/cis isomerization in photoactive yellow protein. Science 352:725–729

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  82. Pandey S, Bean R, Sato T et al (2020) Time-resolved serial femtosecond crystallography at the European XFEL. Nat Methods 17:73–78

    Article  PubMed  Google Scholar 

  83. Monteiro DCF, Amoah E, Rogers C et al (2021) Using photocaging for fast time-resolved structural biology studies. Acta Crystallogr Sect D 77:1218–1232

    Article  CAS  Google Scholar 

  84. Keedy DA, Kenner LR, Warkentin M et al (2015) Mapping the conformational landscape of a dynamic enzyme by multitemperature and XFEL crystallography. elife 4:1–26

    Article  Google Scholar 

  85. Keedy DA, Hill ZB, Biel JT et al (2018) An expanded allosteric network in PTP1B by multitemperature crystallography, fragment screening, and covalent tethering. elife 7:1–36

    Article  Google Scholar 

  86. Thompson MC, Barad BA, Wolff AM et al (2019) Temperature-jump solution X-ray scattering reveals distinct motions in a dynamic enzyme. Nat Chem 11:1058–1066

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  87. Keedy DA (2019) Journey to the center of the protein: allostery from multitemperature multiconformer X-ray crystallography. Acta Crystallogr Sect D 75:123–137

    Article  CAS  Google Scholar 

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Acknowledgements

The authors gratefully acknowledge the support provided by the Max Planck Society. PM acknowledges support from the Deutsche Forschungsgemeinschaft (DFG) via grant No. 451079909 and from a Joachim Herz Stiftung add-on fellowship. ES acknowledges support by the DFG via grant No. 458246365, and by the Federal Ministry of Education and Research, Germany, under grant number 01KI2114.

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Authors and Affiliations

  1. Institute for Nanostructure and Solid State Physics, Universität Hamburg, Hamburg, Germany

    Pedram Mehrabi

  2. Max Planck Institute for Structure and Dynamics of Matter, Hamburg, Germany

    Pedram Mehrabi & Eike C. Schulz

  3. University Medical Center Hamburg-Eppendorf (UKE), Hamburg, Germany

    Eike C. Schulz

Authors
  1. Pedram Mehrabi
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  2. Eike C. Schulz
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Correspondence to Pedram Mehrabi or Eike C. Schulz .

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Editors and Affiliations

  1. Universidade da Beira Interior, CICS-UBI, Covilhã, Portugal

    Ângela Sousa

  2. University of Beira Interior, Covilhã, Portugal

    Luis Passarinha

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Mehrabi, P., Schulz, E.C. (2023). Sample Preparation for Time-Resolved Serial Crystallography: Practical Considerations. In: Sousa, Â., Passarinha, L. (eds) Advanced Methods in Structural Biology. Methods in Molecular Biology, vol 2652. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-3147-8_21

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  • DOI: https://doi.org/10.1007/978-1-0716-3147-8_21

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