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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.


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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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