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Molecular nucleation mechanisms and control strategies for crystal polymorph selection


The formation of condensed (compacted) protein phases is associated with a wide range of human disorders, such as eye cataracts1, amyotrophic lateral sclerosis2, sickle cell anaemia3 and Alzheimer’s disease4. However, condensed protein phases have their uses: as crystals, they are harnessed by structural biologists to elucidate protein structures5, or are used as delivery vehicles for pharmaceutical applications6. The physiochemical properties of crystals can vary substantially between different forms or structures (‘polymorphs’) of the same macromolecule, and dictate their usability in a scientific or industrial context. To gain control over an emerging polymorph, one needs a molecular-level understanding of the pathways that lead to the various macroscopic states and of the mechanisms that govern pathway selection. However, it is still not clear how the embryonic seeds of a macromolecular phase are formed, or how these nuclei affect polymorph selection. Here we use time-resolved cryo-transmission electron microscopy to image the nucleation of crystals of the protein glucose isomerase, and to uncover at molecular resolution the nucleation pathways that lead to two crystalline states and one gelled state. We show that polymorph selection takes place at the earliest stages of structure formation and is based on specific building blocks for each space group. Moreover, we demonstrate control over the system by selectively forming desired polymorphs through site-directed mutagenesis, specifically tuning intermolecular bonding or gel seeding. Our results differ from the present picture of protein nucleation7,8,9,10,11,12, in that we do not identify a metastable dense liquid as the precursor to the crystalline state. Rather, we observe nucleation events that are driven by oriented attachments between subcritical clusters that already exhibit a degree of crystallinity. These insights suggest ways of controlling macromolecular phase transitions, aiding the development of protein-based drug-delivery systems and macromolecular crystallography.

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Figure 1: Pre-nucleation assemblies of glucose isomerase induced by ammonium sulfate.
Figure 2: Emergence of faceted prismatic and rhombic nanocrystals 15–30 minutes after mixing glucose isomerase and ammonium sulfate.
Figure 3: Cryo-TEM imaging of PEG-induced crystallization of glucose isomerase.
Figure 5: Gel-mediated nucleation of P21212 crystals.
Figure 4: Lattice contact analysis for both space groups of glucose isomerase.
Figure 6: Proposed model for glucose isomerase crystallization.


  1. Siezen, R. J ., Fisch, M. R ., Slingsby, C. & Benedek, G. B. Opacification of gamma-crystallin solutions from calf lens in relation to cold cataract formation. Proc. Natl Acad. Sci. USA 82, 1701–1705 (1985)

    ADS  CAS  Article  Google Scholar 

  2. Patel, A . et al. A liquid-to-solid phase transition of the ALS protein FUS accelerated by disease mutation. Cell 162, 1066–1077 (2015)

    CAS  Article  Google Scholar 

  3. Eaton, W. A. & Hofrichter, J. in Advances in Protein Chemistry (eds Anfinsen, C. B., Edsal, J. T., Richards, F. M. & Eisenberg, D. S. ) 63–279 (Academic Press, 1990)

  4. Ghiso, J. & Frangione, B. Amyloidosis and Alzheimer’s disease. Adv. Drug Deliv. Rev. 54, 1539–1551 (2002)

    CAS  Article  Google Scholar 

  5. Berman, H ., Henrick, K ., Nakamura, H. & Markley, J. L. The worldwide Protein Data Bank (wwPDB): ensuring a single, uniform archive of PDB data. Nucleic Acids Res. 35, D301–D303 (2007)

    CAS  Article  Google Scholar 

  6. Basu, S. K ., Govardhan, C. P ., Jung, C. W. & Margolin, A. L. Protein crystals for the delivery of biopharmaceuticals. Expert Opin. Biol. Ther. 4, 301–317 (2004)

    CAS  Article  Google Scholar 

  7. ten Wolde, P. R. & Frenkel, D. Enhancement of protein crystal nucleation by critical density fluctuations. Science 277, 1975–1978 (1997)

    CAS  Article  Google Scholar 

  8. Pan, W ., Galkin, O ., Filobelo, L ., Nagel, R. L. & Vekilov, P. G. Metastable mesoscopic clusters in solutions of sickle-cell hemoglobin. Biophys. J. 92, 267–277 (2007)

    ADS  CAS  Article  Google Scholar 

  9. Sleutel, M. & Van Driessche, A. E. S. Role of clusters in nonclassical nucleation and growth of protein crystals. Proc. Natl Acad. Sci. USA 111, E546–E553 (2014)

    ADS  CAS  Article  Google Scholar 

  10. Sauter, A . et al. Real-time observation of nonclassical protein crystallization kinetics. J. Am. Chem. Soc. 137, 1485–1491 (2015)

    CAS  Article  Google Scholar 

  11. Sauter, A . et al. Nonclassical pathways of protein crystallization in the presence of multivalent metal ions. Cryst. Growth Des. 14, 6357–6366 (2014)

    CAS  Article  Google Scholar 

  12. Gliko, O . et al. Metastable Liquid Clusters in Super- and Undersaturated Protein Solutions. J. Phys. Chem. B 111, 3106–3114 (2007)

    CAS  Article  Google Scholar 

  13. Sleutel, M ., Lutsko, J ., Van Driessche, A. E. S ., Durán-Olivencia, M. A. & Maes, D. Observing classical nucleation theory at work by monitoring phase transitions with molecular precision. Nat. Commun. 5, 5598 (2014)

    ADS  CAS  Article  Google Scholar 

  14. Chung, S ., Shin, S.-H ., Bertozzi, C. R. & Yoreo, J. J. D. Self-catalyzed growth of S layers via an amorphous-to-crystalline transition limited by folding kinetics. Proc. Natl Acad. Sci. USA 107, 16536–16541 (2010)

    ADS  CAS  Article  Google Scholar 

  15. Yau, S.-T. & Vekilov, P. G. Quasi-planar nucleus structure in apoferritin crystallization. Nature 406, 494–497 (2000)

    ADS  CAS  Article  Google Scholar 

  16. Nielsen, M. H ., Aloni, S. & Yoreo, J. J. D. In situ TEM imaging of CaCO3 nucleation reveals coexistence of direct and indirect pathways. Science 345, 1158–1162 (2014)

    ADS  CAS  Article  Google Scholar 

  17. Yamazaki, T . et al. Two types of amorphous protein particles facilitate crystal nucleation. Proc. Natl Acad. Sci. USA 114, 2154–2159 (2017)

    ADS  CAS  Article  Google Scholar 

  18. Bhosale, S. H ., Rao, M. B. & Deshpande, V. V. Molecular and industrial aspects of glucose isomerase. Microbiol. Rev. 60, 280–300 (1996)

    CAS  PubMed  PubMed Central  Google Scholar 

  19. Gillespie, C. M ., Asthagiri, D. & Lenhoff, A. M. Polymorphic protein crystal growth: influence of hydration and ions in glucose isomerase. Cryst. Growth Des. 14, 46–57 (2014)

    CAS  Article  Google Scholar 

  20. Arakawa, T. & Timasheff, S. N. Preferential interactions of proteins with salts in concentrated solutions. Biochemistry 21, 6545–6552 (1982)

    CAS  Article  Google Scholar 

  21. Melander, W. & Horváth, C. Salt effect on hydrophobic interactions in precipitation and chromatography of proteins: an interpretation of the lyotropic series. Arch. Biochem. Biophys. 183, 200–215 (1977)

    CAS  Article  Google Scholar 

  22. Fudo, S ., Qi, F ., Nukaga, M. & Hoshino, T. Influence of precipitants on molecular arrangement and space group of protein crystals. Cryst. Growth Des. 17, 534–542 (2017)

    CAS  Article  Google Scholar 

  23. Paterová, J . et al. Reversal of the Hofmeister series: specific ion effects on peptides. J. Phys. Chem. B 117, 8150–8158 (2013)

    Article  Google Scholar 

  24. Asakura, S. & Oosawa, F. On interaction between two bodies immersed in a solution of macromolecules. J. Chem. Phys. 22, 1255–1256 (1954)

    ADS  CAS  Article  Google Scholar 

  25. Kraft, D. J . et al. Surface roughness directed self-assembly of patchy particles into colloidal micelles. Proc. Natl Acad. Sci. USA 109, 10787–10792 (2012)

    ADS  CAS  Article  Google Scholar 

  26. Whitelam, S. Control of pathways and yields of protein crystallization through the interplay of nonspecific and specific attractions. Phys. Rev. Lett. 105, 088102 (2010)

    ADS  Article  Google Scholar 

  27. Hedges, L. O. & Whitelam, S. Limit of validity of Ostwald’s rule of stages in a statistical mechanical model of crystallization. J. Chem. Phys. 135, 164902 (2011)

    ADS  Article  Google Scholar 

  28. Russo, J. & Tanaka, H. Crystal nucleation as the ordering of multiple order parameters. J. Chem. Phys. 145, 211801 (2016)

    ADS  Article  Google Scholar 

  29. Vekilov, P. G. Dense liquid precursor for the nucleation of ordered solid phases from solution. Cryst. Growth Des. 4, 671–685 (2004)

    CAS  Article  Google Scholar 

  30. Berne, B. J. & Pecora, R. Dynamic Light Scattering: With Applications to Chemistry, Biology, and Physics (Courier Dover Publications, 1976)

  31. Ortega, A. & García de la Torre, J. Hydrodynamic properties of rodlike and disklike particles in dilute solution. J. Chem. Phys. 119, 9914–9919 (2003)

    ADS  CAS  Article  Google Scholar 

  32. Krall, A. H. & Weitz, D. A. Internal dynamics and elasticity of fractal colloidal gels. Phys. Rev. Lett. 80, 778–781 (1998)

    ADS  CAS  Article  Google Scholar 

  33. Friedrich, H., Frederik, P. M., de With, G. & Sommerdijk, N. A. J. M. Imaging of self-assembled structures: interpretation of TEM and cryo-TEM images. Angew. Chem. Int. Edn 49, 7850–7858 (2010)

    CAS  Article  Google Scholar 

  34. Hohn, M. et al. SPARX, a new environment for cryo-EM image processing. J. Struct. Biol. 157, 47–55 (2007)

    CAS  Article  Google Scholar 

  35. Schneider, C. A., Rasband, W. S. & Eliceiri, K. W. NIH Image to ImageJ: 25 years of image analysis. Nat. Methods 9, 671–675 (2012)

    CAS  Article  Google Scholar 

  36. Potterton, E., Briggs, P., Turkenburg, M. & Dodson, E. A graphical user interface to the CCP4 program suite. Acta Crystallogr. D 59, 1131–1137 (2003)

    Article  Google Scholar 

  37. Vuolanto, A., Uotila, S., Leisola, M. & Visuri, K. Solubility and crystallization of xylose isomerase from Streptomyces rubiginosus. J. Cryst. Growth 257, 403–411 (2003)

    ADS  CAS  Article  Google Scholar 

  38. Sleutel, M., Willaert, R., Wyns, L. & Maes, D. Kinetics and thermodynamics of glucose isomerase crystallization. Cryst. Growth Des. 9, 497–504 (2009)

    CAS  Article  Google Scholar 

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M.S. and N.V.G. acknowledge financial support from the Research Foundation Flanders (FWO) under projects G0H5316N and 1516215N. We thank J. A. Gavira for providing the commercial glucose isomerase sample, S. Van der Verren for assistance with single-particle processing, and H. Remaut for help in designing glucose isomerase mutants.

Author information

Authors and Affiliations



M.S. and A.E.S.V.D. designed the project and carried out the crystallization and light-scattering experiments. N.V.G. cloned the glucose isomerase mutants and optimized recombinant expression. Mutant proteins were produced and purified by M.S. with the help from N.V.G. Cryogenic freezing and cryoTEM imaging was performed by D.G.-C., P.H.H.B. and R.R.M.J. H.F. advised and co-supervised during cryoTEM imaging. M.S., A.E.S.V.D. and N.A.J.M.S. supervised the study. M.S. and A.E.S.V.D. wrote the paper, with contributions from all authors.

Corresponding authors

Correspondence to Alexander E. S. Van Driessche or Mike Sleutel.

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The authors declare no competing financial interests.

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Reviewer Information Nature thanks C. Betzel and the other anonymous reviewer(s) for their contribution to the peer review of this work.

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Extended data figures and tables

Extended Data Figure 1 Phase diagrams for glucose isomerase.

a, b, Wide-field microscopic images of the I222 (a) and P21212 (b) glucose isomerase (GI) polymorphs obtained with 0.8 M and 1.5 M ammonium sulfate (AS). c, Glucose isomerase phase diagram in ammonium sulfate ((NH4)2SO4) at 22 °C (single points represent triplicate measurements), showing the solubility line Ce,avg (dashed line). Smaller diamonds and crosses denote smaller numbers of crystals than larger symbols. Ce,avg is a mathematical average that we calculated by using the solubilities at 19 °C and 25 °C from ref. 9. d, Glucose isomerase phase diagram in PEG1000 at 22 °C, with the Ce,avg solubility line taken from ref. 38. The dotted lines, following the same colour code as the single points, indicate the phase boundaries in PEG1500. The photographs to the right are representative microscopy images at the indicated precipitant concentrations.

Extended Data Figure 2 Induction time measurements.

Induction time, tind, as a function of ammonium sulfate concentration. Values next to data points correspond to calculated supersaturation (lnC/Ce) values, according to ref. 37.

Extended Data Figure 3 Determination of the intermolecular distance along the nanorod axis.

a, Complete image acquired at ×24,000 magnification. b, CryoTEM image of single nanorods. c, Greyscale values along the length of the dotted line in panel a. d, 1D-FFT of panel c, calculated using OriginPro 8.6.0. e, 2D-FFT image calculated using ImageJ 1.50i. f, Radial average of panel e. g, Nanorod length expressed in molecular units as a function of time in 1.5 M ammonium sulfate. h, Intermolecular distance along the nanorod axis compared with the crystallographic distance along the c-axis of the prismatic crystals. The box range shows the 25th to 75th percentiles; the horizontal line is the median; error bars highlight the 10th and 90th percentiles.

Extended Data Figure 4 Nanorod formation at early time points.

CryoTEM images of crystallizing glucose isomerase solutions 20 seconds after protein–precipitant mixing with 1.35 M, 1.50 M or 1.55 M ammonium sulfate.

Extended Data Figure 5 I222/P21212/gel coexistence point.

ac, Crystallization experiments using the microbatch-under-oil set-up, with 86 mg ml−1 glucose isomerase, 50 mM HEPES pH 7.0 and 100 mM MgCl2, and 4% (a), 4.5% (b) or 5% (c) (w/v) PEG1500. a, I222 crystals; b, I222 + P21212, P21212, P21212 and gel; c, gel.

Extended Data Figure 6 Time-resolved DLS of crystallizing glucose isomerase solutions.

a, DLS time series of a crystallizing 48 mg ml−1 glucose isomerase solution with 50 mM HEPES pH 7.0, 100 mM MgCl2, 1.5 M ammonium sulfate, collected at an angle of 90°, ranging from 30 seconds to 14 minutes after protein/precipitant mixing. R, particle radius. Microscopy snapshots at the right were taken ex situ after 30 minutes. b, Time evolution (from dark to light) of the intensity correlation function of a 50 mM HEPES pH 7.0, 100 mM MgCl2, 6% PEG1000 (w/v) solution collected at an angle of 90°. c, Fitting of a pre-gelled (20 seconds; left-hand y-axis) and gelled (30 minutes; right-hand y-axis) sample using equations (1) and (2) respectively. Inset, wide-field microscopy image of the gelled state.

Extended Data Figure 7 Crystallographic modelling of the nanorods.

Models of glucose isomerase nanorods in various directions, based on the unit-cell dimensions of the PDB entries 9XIA and 1OAD, and the crystallographic symmetry elements of space groups I222 and P21212. The numbers designating intermolecular distances are in nanometres. The number in brackets for P21212 (001) is the value that we obtained experimentally. For reference, we compare a magnified cryoTEM image of a single nanorod and a simulated TEM projection based on the P21212 (001) nanorod model.

Extended Data Figure 8 Crystallization screening of glucose isomerase mutants with perturbed lattice contacts.

a, Initial crystallization screening of mutants in 50 mM HEPES pH 7.0, 100 mM MgCl2, 15 mg ml−1 of glucose isomerase mutant and 4% (w/v) PEG1000 or 1.5 M ammonium sulfate. The mutants are S171W (with perturbed C1 interactions), GI_His (perturbed C1), R387A (perturbed C2) and R331A/R340D (perturbed C3). b, Cryo-TEM images of various mutants in 50 mM HEPES pH 7.0, 100 mM MgCl2, 15 mg ml−1 mutant protein and 1.5 M ammonium sulfate, 2 minutes after protein/precipitant mixing.

Extended Data Table 1 Comparison of experimental and theoretical distances for both space groups
Extended Data Table 2 Lattice-contact analysis of both space groups

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Van Driessche, A., Van Gerven, N., Bomans, P. et al. Molecular nucleation mechanisms and control strategies for crystal polymorph selection. Nature 556, 89–94 (2018).

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