A Robust Method for Collecting X-ray Diffraction Data from Protein Crystals across Physiological Temperatures

Abstract

Traditional X-ray diffraction data collected at cryo-temperatures have delivered invaluable insights into the three-dimensional structures of proteins, providing the backbone of structurefunction studies. While cryo-cooling mitigates radiation damage, cryo-temperatures can alter protein conformational ensembles and solvent structure. Further, conformational ensembles underlie protein function and energetics, and recent advances in room-temperature X-ray crystallography have delivered conformational heterogeneity information that is directly related to biological function. The next challenge is to develop a robust and broadly applicable method to collect single-crystal X-ray diffraction data at and above room temperatures and was addressed herein. This approach provides complete diffraction datasets with total collection times as short as ~5 sec from single protein crystals, dramatically increasing the amount of data that can be collected within allocated synchrotron beam time. Its applicability was demonstrated by collecting 1.09-1.54 Å resolution data over a temperature range of 293-363 K for proteinase K, thaumatin, and lysozyme crystals. Our analyses indicate that the diffraction data is of high-quality and do not suffer from excessive dehydration or damage.

Macromolecular room temperature crystallography

“…Temperature changes have also been used to analyze flavodoxin oxidation states (Watt et al, 1991), and the in crystallo thermodynamics of conformational states of a redox quinone co-factor in bacterial copper amine oxidase during the catalytic reaction (Murakawa et al, 2019). Advances in collecting high-temperature datasets at 293-363 K promise future temperature explorations of the conformational landscape beyond proteinase K, thaumatin, and lysozyme (Doukov et al, 2020).…”

qFit 3: Protein and ligand multiconformer modeling for X‐ray crystallographic and single‐particle cryo‐EM density maps

“…For example, crystallographic data sets collected across multiple temperatures-as opposed to at a single cryogenic temperature-often reveal ensembles with more conformational diversity, [4][5][6][7][8] including at dynamic enzyme active sites. 9 High-throughput crystallographic protein:ligand screening can identify otherwise undetectable low-occupancy ligand-bound protein states.…”

qFit 3: Protein and ligand multiconformer modeling for X-ray crystallographic and single-particle cryo-EM density maps

“…For example, crystallographic datasets collected across multiple temperatures -as opposed to at a single cryogenic temperature -often reveal ensembles with more conformational diversity [4][5][6][7][8] , including at dynamic enzyme active sites 9 . High-throughput crystallographic protein:ligand screening can identify otherwise undetectable low-occupancy ligand-bound protein states 7,10,11 .…”