Researchers showed the structure and growth of protein crystals by using Cryo-electron microscopy. They allow to control crystallization and could be used to aid drug formulation. Crystallizing a protein can be a shot in the dark. No one quite knows how protein crystals form, so researchers frequently must use trial and error to find the right crystallization conditions. Scientists have used cryo-electron microscopy (cryo-EM), the technique that took home the 2017 Nobel Prize in Chemistry, to watch the earliest stages of protein crystal formation.

Protein Molecules

Crystallization starts with tiny clusters of protein molecules that act as seeds. The clusters are too small to be seen with a light microscope, and they are unstable. To address both challenges, they opted to rapidly freeze their protein samples to halt molecular motion, and to get snapshots at various time points during crystallization with cryo-EM.

Other techniques have provided a partial glimpse of crystallization, but nobody had tried cryo-EM on these types of samples before. There were doubts we would even see anything. They observed the protein glucose isomerase, which Sleutel has been studying for a decade after they added reagents for initiating crystallization.

Protein's Structure

Researchers know this protein’s structure and that it can crystallize into a diamondlike rhombic form or a rectangular prismatic form.

Through their cryo-EM studies, Sleutel’s team learned that the path to each polymorph is different. The path to the rhombic form follows classic crystal formation theory; the smallest seeds detected were miniature versions of the full-sized crystals. First, protein molecules assemble into linear nanorods. The nanorods group into fibers and eventually yield prismatic crystals. Seeing nanorods “made our jaws drop.


The samples had formed a gel, which is a dead end to a crystallographer, much like black tar at the bottom of a flask is to an organic chemist. However, when the researchers transferred a piece of the gel into a solution that had previously elicited rhombic crystals, prism-shaped crystals sprouted.

The fact that the team got prisms instead of rhombic crystals suggests that gels can be used as seeding agents specific to a given polymorph. The team found another way to generate one polymorph selectively.

By examining the cryo-EM images and the protein’s known crystal structure, they determined which amino acids on the protein’s surface make intermolecular contacts in each of the two crystal forms. By mutating these amino acids, they could destabilize one form and promote the formation of the other. The study was limited to just one protein, but the team hopes to apply the technique to others.

Protein Crystals

They suggest that protein crystals form from an amorphous, liquid-like state. For instance, sometimes researchers want to get a structure of a protein bound to a given molecule and one crystal form blocks access to the site at which the molecule would bind. Scientists already tweak protein surfaces to enhance crystallization, “but nudging crystals toward a specific form has not been reasonably approachable.”

The team’s mutation strategy for polymorph control was feasible because the team already knew glucose isomerase’s three-dimensional structure. Sleutel acknowledges that the gel strategy is more universal because it doesn’t require prior knowledge of the protein’s structure.

The findings could be “hugely valuable” to drugmakers seeking one specific polymorph, and the insights in this work could prevent valuable material from being wasted on trial-and-error screens of myriad crystallization conditions.