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Molecular nucleation mechanisms and control strategies for crystal polymorph selection

Nature volume 556, pages 8994 (05 April 2018) | Download Citation


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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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


  1. Université Grenoble Alpes, Université Savoie Mont Blanc, CNRS, IRD, IFSTTAR, ISTerre, F-38000 Grenoble, France

    • Alexander E. S. Van Driessche
  2. Laboratory of Materials and Interface Chemistry and Center of Multiscale Electron Microscopy, Department of Chemical Engineering and Chemistry, Eindhoven University of Technology, PO Box 513, 5600MB Eindhoven, The Netherlands

    • Paul H. H. Bomans
    • , Rick R. M. Joosten
    • , Heiner Friedrich
    •  & Nico A. J. M. Sommerdijk
  3. Institute for Complex Molecular Systems, Eindhoven University of Technology, PO Box 513, 5600MB Eindhoven, The Netherlands

    • Paul H. H. Bomans
    • , Rick R. M. Joosten
    • , Heiner Friedrich
    •  & Nico A. J. M. Sommerdijk
  4. Structural Biology Unit, CIC bioGUNE, Parque Tecnológico de Bizkaia, 48160 Derio, Bizkaia, Spain

    • David Gil-Carton
  5. Structural Biology Brussels, Vrije Universiteit Brussel, Pleinlaan 2, 1050 Brussels, Belgium

    • Nani Van Gerven
    •  & Mike Sleutel
  6. Structural and Molecular Microbiology, Structural Biology Research Center, VIB, Pleinlaan 2, 1050 Brussels, Belgium

    • Nani Van Gerven
    •  & Mike Sleutel


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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.

Competing interests

The authors declare no competing financial interests.

Corresponding authors

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

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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