Room-temperature serial synchrotron crystallography of Drosophila cryptochrome

Abstract

Fixed-target serial crystallography allows the high-throughput collection of diffraction data from small crystals at room temperature. This methodology is particularly useful for difficult samples that have sensitivity to radiation damage or intolerance to cryoprotection measures; fixed-target methods also have the added benefit of low sample consumption. Here, this method is applied to the structure determination of the circadian photoreceptor cryptochrome (CRY), previous structures of which have been determined at cryogenic temperature. In determining the structure, several data-filtering strategies were tested for combining observations from the hundreds of crystals that contributed to the final data set. Removing data sets based on the average correlation coefficient among equivalent reflection intensities between a given data set and all others was most effective at improving the data quality and maintaining overall completeness. CRYs are light sensors that undergo conformational photoactivation. Comparisons between the cryogenic and room-temperature CRY structures reveal regions of enhanced mobility at room temperature in loops that have functional importance within the CRY family of proteins. The B factors of the room-temperature structure correlate well with those predicted from molecular-dynamics simulations.

Exploring serial crystallography for drug discovery

“…Interestingly, the B-factors of the residues around the active site are significantly elevated in the room-temperature structures relative to the cryo-structures, suggesting a dynamic behaviour of the active site under ambient conditions that is masked in the frozen state (Figure 7). This type of enhanced loop mobility of functionally important regions was reported recently in a lower-resolution SX study (Schneps et al ., 2022), where it was suggested that information gained from analysing dynamic behaviour could be useful to identify key structural elements in a protein.…”

“…In our experiments, we calculate the radiation dose to ~4.5 × 10 4 Gy at each grid position of the chip (Bury et al, 2018). In a recent study of a cryptochrome using fixed-target SX, it was noted that the refined B-factors correlated well with those predicted molecular-dynamics simulations (Schneps et al, 2022), pointing towards the protein being in a fully solvated, functional state in the microcrystals. The structures of sEH display flexibility in some loop-regions at both temperatures, including those surrounding the active site.…”

“…The crystal lattice of microcrystals may be more resilient to conformational changes thus allowing the dynamics of the protein to be observed. Radiation damage can also be manifested in the form of high B-factors, and care must be taken to stay below the dose limit of 5 × 10 5 Gy when X-ray radiation induced damage is typically observed in data collected at room temperature (Schneps et al ., 2022). In our experiments, we calculate the radiation dose to ∼4.5 × 10 4 Gy at each grid position of the chip (Bury et al ., 2018).…”

Introduction to the virtual thematic issue on room-temperature biological crystallography

“…Using multi-copy ensemble models they highlight conformational heterogeneity at various sites including a key substrate-binding loop that may inspire new strategies for antiviral drug development. Sharma and co-workers further explored the theme of conformational variability and allosteric coupling in protein tyrosine phosphatase 1B (PTP1B) by solving its SSX structure in the unliganded state (Sharma et al, 2023) whilst Schneps and colleagues employed SSX on the circadian photoreceptor cryptochrome (CRY) revealing regions of enhanced mobility in loops that have functional importance within this protein family and whose atomic displacement parameters correlate well with those predicted from molecular-dynamics simulations (Schneps et al, 2022). The theme of ligand-binding discrepancies between cryo-cooled and physiological temperature structures is discussed by Huang and co-workers by looking at the interaction of TL00150, a 175.15 Da fragment, with endothiapepsin using a 'temperature-resolved' approach (Huang et al, 2022).…”

Introduction to the virtual thematic issue on room-temperature biological crystallography

“…Using multi-copy ensemble models they highlight conformational heterogeneity at various sites including a key substrate-binding loop that may inspire new strategies for antiviral drug development. Sharma and co-workers further explored the theme of conformational variability and allosteric coupling in protein tyrosine phosphatase 1B (PTP1B) by solving its SSX structure in the unliganded state (Sharma et al, 2023) whilst Schneps and colleagues employed SSX on the circadian photoreceptor cryptochrome (CRY) revealing regions of enhanced mobility in loops that have functional importance within this protein family and whose atomic displacement parameters correlate well with those predicted from molecular-dynamics simulations (Schneps et al, 2022). The theme of ligand-binding discrepancies between cryo-cooled and physiological temperature structures is discussed by Huang and co-workers by looking at the interaction of TL00150, a 175.15 Da fragment, with endothiapepsin using a 'temperature-resolved' approach (Huang et al, 2022).…”