Innovative technique boosts room-temperature crystallography

Cryogenic macromolecular crystallography (cryo-MX) has traditionally been used to determine the crystal structures of biomolecules at near-atomic resolution due to its advantages in reducing radiation damage to proteins and trapping them in transient states. However, because cryo-MX requires freezing biomolecules, it doesn’t allow researchers to observe how these molecules behave in their natural, flexible state at room temperature.

Room-temperature macromolecular crystallography can capture biomolecules in their physiological states, which is crucial for understanding how they function and interact in living organisms, and for observing processes such as enzymatic reactions or how proteins change over time. Room-temperature MX therefore holds great promise for improving drug discovery, as it helps researchers obtain accurate, physiologically relevant structures, yet several technical challenges remain unresolved.

Recent advancements in serial crystallography have tackled traditional challenges by enabling protein structure determination at ambient temperatures. The development of synchrotron serial crystallography has mitigated radiation damage to crystals, while the ESRF’s upgraded Extremely Brilliant Source provides high-resolution, detailed structural insights. However, traditional data collection methods are time-consuming and lack high-throughput capabilities, making them less practical for routine use. This challenge is compounded by the fact that many important drug targets can only be produced in small quantities, limiting the size and number of crystals available for diffraction experiments. 

To address this, researchers from the ESRF and the EMBL have developed a new experimental method called in-situ serial crystallography (iSX) at beamline ID23-2. iSX enables faster and more efficient data collection directly from microcrystals grown in 96-well crystallization plates. By raster-scanning each drop, in-situ data collection is converted into a high-throughput serial crystallography technique. A key feature of this approach is the ability to quickly switch between a standard goniometer head used for cryogenic experiments and a specialized plate holder for 96-well plates (Figure 1). This allows for seamless transitions between different experimental setups in under 15 minutes.
 

Fig. 1: Schematic of the in-situ experimental setup on beamline ID23-2 with the plate manipulator installed on a high-precision diffractometer and a loaded 96-well crystallization plate. It also illustrates the fast switching from the conventional mini-kappa goniometer head to the in-situ setup (diffractometer drawing provided by Arinax, Moirans, France).

As a test case to demonstrate the potential of this method, iSX was employed to determine the structure of a challenging protein, autotaxin, which plays a key role in producing lysophosphatidic acid, a molecule involved in biological processes including vascular development, pain, fibrosis, and cancer. The structure of autotaxin has been studied before using cryo-MX, but the new iSX method offers a way to study it under more natural conditions. Using iSX, it was possible to determine the protein’s structure to 3 Å at room temperature in under two hours.

The iSX method, now available at beamline ID23-2, offers a streamlined pipeline from crystallization to structure determination (Figure 2), enabling efficient and high-throughput data collection directly from 96-well crystallization plates. This approach simplifies crystal handling, allows for optimal sampling, and facilitates complete data collection within a single eight-hour shift while screening various crystallization conditions.
 

Fig. 2: Schematic of the iSX pipeline as implemented on ID23-2.

iSX is particularly effective for challenging macromolecules with low expression yields or small crystals that do not diffract well. The method requires minimal sample quantities and offers faster data collection compared to other SSX-based approaches. Its ease and efficiency make it suitable for use across multiple MX beamlines, promoting crystallization plates as a natural and effective sample delivery method for serial crystallography.

The development of iSX not only enhances room-temperature crystallography but also provides a powerful tool for studying biomolecules, protein and ligand dynamics, potentially improving drug discovery by delivering more accurate structural data. Additionally, it opens up new possibilities for time-resolved synchrotron serial crystallography experiments.

Principal publication and authors 
In situ serial crystallography facilities 96-well plate structural analysis at low symmetry, N. Foos (a), J-B. Florial (a), M. Eymery (a), J. Sinoir (a), F. Felisaz (a), M. Oscarsson (b), A. Beteva (b), M.W. Bowler (a), D. Nurizzo (b), G. Papp (a), M. Soler-Lopez (b), M. Nanao (b), S. Basu (a), A.A. McCarthy (a), IUCrJ 11(5), 780-791 (2024); https://doi.org/10.1107/S2052252524005785
(a) European Molecular Biology Laboratory, Grenoble Outstation, Grenoble (France) 
(b) ESRF