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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.


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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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