Protein-Ligand Interactions: Room Temperature or Cryo-Crystallography?

After more than two decades of cryo-crystallography, finding someone with skills in mounting protein crystals for room-temperature X-ray crystallography won’t be easy. It’s safe to say that most young crystallographers today haven’t even seen an X-ray capillary of the type used in X-ray crystallography at room temperature. However, there are shortcomings in cryo-crystallography, and they need to be addressed. Robert Steiner has put together a virtual issue of Acta Cryst. D, Structural Biology, with articles on room-temperature X-ray crystallography.

Why X-ray Crystallography at Room Temperature?

An obvious advantage of room-temperature X-ray crystallography over cryo-crystallography can be found on MiTeGen‘s site, a company that offers products and tools for X-ray crystallography:” Roughly 98% of low-temperature datasets collected on structural biology beamlines do not yield diffraction data of sufficient quality to determine a structure. Room temperature measurements are essential to determine whether the cause of poor diffraction is poor as-grown crystal quality, sample damage caused by ligand and/or cryoprotectant soaks, and/or sample damage caused by the flash cooling process. Far too much effort is wasted searching for cryoprotection conditions for crystals that are poorly ordered to start with.“
Other shortcomings of cryo-crystallography are listed in a review article by Robert E. Thorn in the issue Acta Cryst. mentioned above:

• Regions of ordered density in a tertiary protein structure obtained by cryo-crystallography may be disordered at room temperature (and vice versa). Furthermore, disorder at room temperature may be functionally relevant. However, this is often overlooked in cryo-crystallography, as most structures are refined to a single conformation. Room-temperature X-ray crystallographic data collection can facilitate the identification of functionally important alternative conformations at active sites and elsewhere in the protein’s tertiary structure.
• The cryoprotectant used in cryo-crystallography may perturb side chain conformations and contribute additional electron density, including within the active site region.
• Cryo-cooling may degrade crystal lattice order, even if the thermal disorder is reduced. It may also introduce crystal non-isomorphism and high mosaicity. Variability in cryoprotectant soaks and cooling may also result in non-isomorphism.

These realizations, combined with recent technical developments discussed in Robert E. Thorn’s review, triggered the revival of room-temperature X-ray crystallography.

• The development of high intensity X-ray-free electron laser (XFEL) sources and micro-focus beamlines for serial crystallography at synchrotrons, as well as developing serial-sample delivery and software for processing and modeling data collected from many tiny crystals.
• Improved detector speed and sensitivity; new techniques for time-resolved study of reactions and conformational dynamics within crystals.

Protein-Ligand Interactions: Conformational Diversity At Room Temperature

The new findings demonstrate that room-temperature and multi-temperature X-ray crystallography can be utilized to investigate functionally significant conformations and identify temperature-dependent variations in protein-ligand interactions, including novel binding sites and distinctive binding poses. Such studies may provide a more comprehensive understanding of protein-ligand interactions, making room-temperature X-ray structures a more accurate representation of the functionally relevant conformations of proteins and, thus, more efficient for drug discovery than cryo-crystallography.

An example of a study of ligand-binding discrepancies between cryo-cooled and RT-temperature X-ray crystal structures presented in the work by Huang and co-workers in the same issue. The authors used a temperature-variation approach to examine the binding of TL00150, a 175.15 Da inhibitor of the aspartic proteinase endothiapepsin. The authors report the details of the experimental setup for the temperature-dependent measurements and show that, at 298 K, depending on the DMSO concentration, the fragment TL00150 has two binding positions. In contrast, at cryo-temperature (100 K), it was only observed in a single position at the S1 site of the active site pocket. Subsequently, data were collected for a series of structures with bound ligands to assess ligand occupancy at different temperatures. The data showed different ligand binding states with different occupancies at the S1 and S1′ sites. This was suggested to be an effect of the flexible loop close to the protein’s active site.

The new technical developments, which make room-temperature X-ray crystallography and multi-temperature measurements relatively easily accessible at synchrotrons, provide new opportunities for studying functionally important conformations that may aid the studies of protein-ligand interactions and ultimately, the drug design process. Last but not least, we could start testing crystals directly at synchrotrons at room temperature without all the hassle of finding a suitable cryoprotectant and freezing!

SARomics Biostructures’ X-ray crystallography services are leveraged by two beamlines, BioMAX and MicroMAX, at the MAX IV, a fourth-generation synchrotron just a few kilometers from Medicon Village, where our company offices are located. Contact us for new questions or project discussions.

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