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Porous nucleating agents for protein crystallization


Solving the structure of proteins is pivotal to achieving success in rational drug design and in other biotechnological endeavors. The most powerful method for determining the structure of proteins is X-ray crystallography, which relies on the availability of high-quality crystals. However, obtaining such crystals is a major hurdle. Nucleation is the crucial prerequisite step, which requires overcoming an energy barrier. The presence in a protein solution of a nucleant, a solid or a semiliquid substance that facilitates overcoming that barrier allows crystals to grow under ideal conditions, paving the way for the formation of high-quality crystals. The use of nucleants provides a unique means for optimizing the diffraction quality of crystals, as well as for discovering new crystallization conditions. We present a protocol for controlling the nucleation of protein crystals that is applicable to a wide variety of nucleation-inducing substances. Setting up crystallization trials using these nucleating agents takes an additional few seconds compared with conventional setup, and it can accelerate crystallization, which typically takes several days to months.

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Figure 1: Illustration of the 20% success rate in obtaining diffraction quality crystals from purified protein.
Figure 2: Light microscope image of Human Cardiac Myosin Binding Protein C crystals growing from a carbon nanotube nucleant in metastable conditions.
Figure 3: The porous nature of Bioglass and its suitability for protein crystallization.
Figure 4: Light microscope image of spear-shaped human macrophage migration inhibitory factor (MIF) crystals growing from an MIP nucleant surface.
Figure 5: Flow diagram illustrating the procedure involved when conducting crystallization trials.
Figure 6: Different protein sample quality determined by using SDS-PAGE.
Figure 7: Monodisperse and polydisperse size distribution plots obtained using DLS when characterizing protein samples.
Figure 8: Schematic illustration of a protein crystallization phase diagram.
Figure 9: A working phase diagram.
Figure 10: Technique used to add nucleant grains to crystallization drops.


  1. 1

    McPherson, A. & Shlichta, P. Heterogeneous and epitaxial nucleation of protein crystals on mineral surfaces. Science 239, 385–387 (1988).

    Article  CAS  Google Scholar 

  2. 2

    Falini, G., Fermani, S., Conforti, G. & Ripamonti, A. Protein crystallisation on chemically modified mica surfaces. Acta Crystallogr. D Biol. Crystallogr. 58, 1649–1652 (2002).

    Article  CAS  Google Scholar 

  3. 3

    Nanev, C.N. On the slow kinetics of protein crystallization. Cryst. Growth Des. 7, 1533–1540 (2007).

    Article  CAS  Google Scholar 

  4. 4

    Pechkova, E. & Nicolini, C. Protein nucleation and crystallization by homologous protein thin film template. J. Cell. Biochem. 85, 243–251 (2002).

    Article  CAS  Google Scholar 

  5. 5

    Pechkova, E. & Nicolini, C. Atomic structure of a CK-α human kinase by microfocus diffraction of extra-small microcrystals grown with nanobiofilm template. J. Cell. Biochem. 91, 1010–1020 (2004).

    Article  CAS  Google Scholar 

  6. 6

    Pechkova, E. et al. μ GISAXS and protein nanotemplate crystallization: methods and instrumentation. J. Synchrotron Radiat. 12, 713–716 (2005).

    Article  CAS  Google Scholar 

  7. 7

    D'Arcy, A., Mac Sweeney, A. & Haber, A. Using natural seeding material to generate nucleation in protein crystallization experiments. Acta Crystallogr. D Biol. Crystallogr. 59, 1343–1346 (2003).

    Article  Google Scholar 

  8. 8

    Georgieva, D.G., Kuil, M.E., Oosterkamp, T.H., Zandbergen, H.W. & Abrahams, J.P. Heterogeneous nucleation of three-dimensional protein nanocrystals. Acta Crystallogr. D Biol. Crystallogr. 63, 564–570 (2007).

    Article  CAS  Google Scholar 

  9. 9

    Chayen, N.E., Saridakis, E., El-Bahar, R. & Nemirovsky, Y. Porous silicon: an effective nucleation-inducing material for protein crystallization. J. Mol. Biol. 312, 591–595 (2001).

    Article  CAS  Google Scholar 

  10. 10

    Chayen, N.E., Saridakis, E. & Sear, R.P. Experiment and theory for heterogeneous nucleation of protein crystals in a porous medium. Proc. Natl. Acad Sci. USA 103, 597–601 (2006).

    Article  CAS  Google Scholar 

  11. 11

    Asanithi, P. et al. Carbon-nanotube-based materials for protein crystallization. ACS App. Mater. Interfaces 1, 1203–1210 (2009).

    Article  CAS  Google Scholar 

  12. 12

    Kertis, F. et al. Heterogeneous nucleation of protein crystals using nanoporous gold nucleants. J. Mater. Chem. 22, 21928–21934 (2012).

    Article  CAS  Google Scholar 

  13. 13

    Saridakis, E. et al. Protein crystallization facilitated by molecularly imprinted polymers. Proc. Natl. Acad. Sci. USA 108, 11081–11086 (2011).

    Article  PubMed  PubMed Central  Google Scholar 

  14. 14

    Sepulveda, P., Jones, J.R. & Hench, L.L. Characterization of melt-derived 45S5 and sol-gel-derived 58S bioactive glasses. J. Biomed. Mater. Res. 58, 734–740 (2001).

    Article  CAS  Google Scholar 

  15. 15

    Reddy, S.M. et al. Protein crystallization and biosensor applications of hydrogel-based molecularly imprinted polymers. Biomacromolecules 13, 3959–3965 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. 16

    Sugahara, M., Asada, Y., Morikawa, Y., Kageyama, Y. & Kunishima, N. Nucleant-mediated protein crystallization with the application of microporous synthetic zeolites. Acta Crystallogr. D Biol. Crystallogr. 64, 686–695 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. 17

    Rong, L., Komatsu, H. & Yoda, S. Control of heterogeneous nucleation of lysozyme crystals by using poly-L-lysine modified substrate. J. Cryst. Growth 235, 489–493 (2002).

    Article  CAS  Google Scholar 

  18. 18

    Di Profio, G., Curcio, E., Ferraro, S., Stabile, C. & Drioli, E. Effect of supersaturation control and heterogeneous nucleation on porous membrane surfaces in the crystallization of L-glutamic acid polymorphs. Cryst. Growth Des. 9, 2179–2186 (2009).

    Article  CAS  Google Scholar 

  19. 19

    Kallio, J.M. et al. The contribution of polystyrene nanospheres towards the crystallization of proteins. PLoS ONE 4, e4198 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. 20

    Ino, K. et al. Heterogeneous nucleation of protein crystals on fluorinated layered silicate. PLoS ONE 6, e22582 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. 21

    Page, A.J. & Sear, R.P. Heterogeneous nucleation in and out of pores. Phys. Rev. Lett. 97, 065701 (2006).

    Article  CAS  Google Scholar 

  22. 22

    Cacciuto, A., Auer, S. & Frenkel, D. Onset of heterogeneous crystal nucleation in colloidal suspensions. Nature 428, 404–406 (2004).

    Article  CAS  Google Scholar 

  23. 23

    Fermani, S. et al. Heterogeneous crystallization of proteins: is it a prenucleation clusters mediated process? Cryst Growth Des. 13, 3110–3115 (2013).

    Article  CAS  Google Scholar 

  24. 24

    Saridakis, E. & Chayen, N.E. Towards a 'universal' nucleant for protein crystallization. Trends Biotechnol. 27, 99–106 (2009).

    Article  CAS  Google Scholar 

  25. 25

    Chayen, N.E. Smart materials for protein crystallization (lecture abstract no. MS1-04, European Crystallography Meeting, Bergen, Norway, 2012).

  26. 26

    Newby, Z.E.R. et al. A general protocol for the crystallization of membrane proteins for X-ray structural investigation. Nat. Protoc. 4, 619–637 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. 27

    Caffrey, M. & Cherezov, V. Crystallizing membrane proteins using lipidic mesophases. Nat. Protoc. 4, 706–731 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. 28

    Benvenuti, M. & Mangani, S. Crystallization of soluble proteins in vapor diffusion for X-ray crystallography. Nat. Protoc. 2, 1633–1651 (2007).

    Article  CAS  Google Scholar 

  29. 29

    Bergfors, T. Seeds to crystals. J. Struct. Biol. 142, 66–76 (2003).

    Article  CAS  Google Scholar 

  30. 30

    D'Arcy, A., Villard, F. & Marsh, M. An automated microseed matrix-screening method for protein crystallization. Acta Crystallogr. D Biol. Crystallogr. 63, 550–554 (2007).

    Article  CAS  Google Scholar 

  31. 31

    Saridakis, E.E., Stewart, P.D., Lloyd, L.F. & Blow, D.M. Phase diagram and dilution experiments in the crystallization of carboxypeptidase G2. Acta Crystallogr. D Biol. Crystallogr. 50, 293–297 (1994).

    Article  CAS  Google Scholar 

  32. 32

    Saridakis, E. & Chayen, N.E. Improving protein crystal quality by decoupling nucleation and growth in vapor diffusion. Protein Sci. 9, 755–757 (2000).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. 33

    Nanev, C.N. & Hodzhaoglu, F.V. Temperature control of protein crystal nucleation. Cryst. Res. Technol. 47, 1195–1200 (2012).

    Article  CAS  Google Scholar 

  34. 34

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

    Article  CAS  Google Scholar 

  35. 35

    Laemmli, U.K. Cleavage of structural proteins during assembly of head of bacteriophage-T4. Nature 227, 680–685 (1970).

    Article  CAS  Google Scholar 

  36. 36

    D'Arcy, A. Crystallizing proteins: a rational approach? Acta Crystallogr. D Biol. Crystallogr. 50, 469–471 (1994).

    Article  CAS  Google Scholar 

  37. 37

    Oberthuer, D. et al. Monitoring and scoring counter-diffusion protein crystallization experiments in capillaries by in situ dynamic light scattering. PLoS ONE 7, e33545 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. 38

    Geerlof, A. et al. The impact of protein characterization in structural proteomics. Acta Crystallogr. D 62, 1125–1136 (2006).

    Article  CAS  Google Scholar 

  39. 39

    Bergfors, T. Handling the protein sample in Protein Crystallization 2nd edn. (ed. Bergfors, T.) Chapter 14, 271 (International University Line, 2009).

  40. 40

    Yuan, Y.R., Martsinkevich, O. & Hunt, J.F. Structural characterization of an MJ1267 ATP-binding cassette crystal with a complex pattern of twinning caused by promiscuous fiber packing. Acta Crystallogr. D 59, 225–238 (2003).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. 41

    Chayen, N.E. Rigorous filtration for protein crystallization. J. Appl. Crystallogr. 42, 743–744 (2009).

    Article  CAS  Google Scholar 

  42. 42

    Rupp, B. Maximum-likelihood crystallization. J. Struct. Biol. 142, 162–169 (2003).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. 43

    Gorrec, F., Palmer, C.M., Lebon, G. & Warne, T. Pi sampling: a methodical and flexible approach to initial macromolecular crystallization screening. Acta Crystallogr. D Biol. Crystallogr. 67, 463–470 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  44. 44

    Newman, J. et al. Towards rationalization of crystallization screening for small- to medium-sized academic laboratories: the PACT/JCSG+ strategy. Acta Crystallogr. D Biol. Crystallogr. 61, 1426–1431 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  45. 45

    Cudney, B., Patel, S., Weisgraber, K. & Newhouse, Y. Screening and optimization strategies for macromolecular crystal-growth. Acta Crystallogr. D 50, 414–4123 (1994).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  46. 46

    McPherson, A. & Cudney, B. Searching for silver bullets: an alternative strategy for crystallizing macromolecules. J. Struct. Biol. 156, 387–406 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  47. 47

    Gorrec, F. The MORPHEUS protein crystallization screen. J. Appl. Crystallogr. 42, 1035–1042 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  48. 48

    Bergfors, T. in Protein Crystallization Strategies for Structural Genomics (ed. Chayen, N.E.) Chapter 3, 59–73 (International University Line, 2007).

  49. 49

    Chayen, N.E. Crystallization with oils: a new dimension in macromolecular crystal growth. J. Cryst. Growth 196, 434–441 (1999).

    Article  CAS  Google Scholar 

  50. 50

    Garcia-Ruiz, J.M., Gonzalez-Ramirez, L.A., Gavira, J.A. & Otalora, F. Granada crystallisation box: a new device for protein crystallisation by counter-diffusion techniques. Acta Crystallogr. D 58, 1638–1642 (2002).

    Article  CAS  Google Scholar 

  51. 51

    Sauter, C., Dhouib, K. & Lorber, B. From macrofluidics to microfluidics for the crystallization of biological macromolecules. Cryst. Growth Des. 7, 2247–2250 (2007).

    Article  CAS  Google Scholar 

  52. 52

    Haas, C. & Drenth, J. Understanding protein crystallization on the basis of the phase diagram. J. Cryst. Growth 196, 388–394 (1999).

    Article  CAS  Google Scholar 

  53. 53

    Chayen, N.E. Turning protein crystallisation from an art into a science. Curr. Opin. Struct. Biol. 14, 577–583 (2004).

    Article  CAS  Google Scholar 

  54. 54

    Stewart, P.D.S., Kolek, S.A., Briggs, R.A., Chayen, N.E. & Baldock, P.F.M. Random microseeding: a theoretical and practical exploration of seed stability and seeding techniques for successful protein crystallization. Cryst. Growth Design 11, 3432–3441 (2011).

    Article  CAS  Google Scholar 

  55. 55

    Graslund, S. et al. Protein production and purification. Nat. Methods 5, 135–146 (2008).

    Article  Google Scholar 

  56. 56

    Smith, C. Striving for purity: advances in protein purification. Nat. Methods 2, 71–77 (2005).

    Article  CAS  Google Scholar 

  57. 57

    Doublie, S. Preparation and Crystallization of Macromolecules Vol. 1 Chapters 1–5, 1–108 (Humana Press, 2007).

  58. 58

    Bergfors, T. (ed.). Protein Crystallization 2nd edn. (International University Line, 2009).

  59. 59

    McPherson, A. Crystallization of Biological Macromolecules (Cold Spring Harbor Laboratory Press, 1999).

  60. 60

    Ducruix, A. & Giege, R. Crystallization of Nucleic Acids and Proteins: A Practical Approach 2nd edn. (Oxford University Press, 1999).

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Author information




S.K. and E.S. designed and performed the research, and wrote the paper. L.G. designed and performed the research. N.E.C. designed the research, coordinated the research and wrote the paper.

Corresponding author

Correspondence to Naomi E Chayen.

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Competing interests

The authors declare that separate patents for the use of Bioglass and MIPs for protein crystallization have been granted. Bioglass is available as a commercial product called 'Naomi's Nucleant'. In the case of MIPs, the patent protects commercial use but does not preclude its use for scientific research.

Supplementary information

Insertion of a solid nucleant into a crystallization trial.

A single grain of a solid nucleant, Bioglass, is introduced using forceps into a vessel containing the components of a crystallization trial. (MP4 3748 kb)

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Khurshid, S., Saridakis, E., Govada, L. et al. Porous nucleating agents for protein crystallization. Nat Protoc 9, 1621–1633 (2014).

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