Quantum refinement can be used to improve time-resolved crystallographic structures
Abstract
Time-resolved serial crystallography has been shown to be a powerful technique to study enzyme reactions. In such studies, a reaction is triggered within the crystal and data are collected at different timepoints. A problem with this approach is that the synchronization is never perfect and therefore the structures at different timepoints typically contain a mixture of states in the active site, for example involving unreacted states, intermediates and products. Interpreting the electron density of mixtures is a challenge for crystallographic modelling and the resulting structures depend strongly on the quality of the empirical restraints employed. In this study, we investigate the potential of using quantum refinement for time-resolved structures, i.e. replacing the empirical restraints with more accurate quantum-mechanical calculations for the active site. We studied three different proteins. The first is a structure of bacteriorhodopsin containing a mixture of Schiff bases of all- trans and 13- cis retinal (PDB entry 5b6w). We show that quantum refinement gives similar structures to the original study, but the strain energies (indicating how much the structures are distorted from the optimum geometry) are much smaller: 36 and 71 kJ mol ⁻¹ for the all- trans and 13- cis conformations, respectively, compared with 366 and 473 kJ mol ⁻¹ for the deposited structure. Next, we studied a reaction intermediate of xylose isomerase (PDB entry 6qni), showing that the structure probably contains a mixture of the substrate-free state, as well as both the ring-closed and ring-opened glucose substrate, in contrast to the deposited structure, which was interpreted as 100% ring-closed glucose. Third, we studied a structure of fluoroacetate dehalogenase (PDB entry 6qhp), which was interpreted as a mixture of a covalent reaction intermediate and two conformations of the fluoroacetate substrate. Our best interpretation (with much less difference density) contains no intermediate and only a single conformation of the substrate, as well as a water molecule. We also studied all of the other structures in the time-resolved study of this enzyme to show that quantum refinement gives consistent structures throughout the reaction. We show that the suggestion of two conformations of the intermediate in the other chain of the protein is also questionable and point out that it is hard to discriminate between the fluoroacetate substrate and the glycolate product. For all structures, we also perform standard crystallographic refinement, with empirical restraints instead of the quantum-mechanical calculations. These often give similar results, but since electrostatic interactions are ignored they cannot properly describe hydrogen bonds, which are key to provide reliable positions of water molecules and other polar groups, especially in the poorly defined electron density of mixed states in time-resolved data.
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BibTeX
@article{yuvaraj2026quantum,
title = {{Quantum refinement can be used to improve time-resolved crystallographic structures}},
author = {Gayathri Yuvaraj and Kristoffer J. M. Lundgren and Esko Oksanen and Ulf Ryde},
journal = {IUCrJ},
year = {2026},
month = nov,
doi = {10.1107/s2052252526009486},
url = {https://journals.iucr.org/paper?S2052252526009486},
note = {CC BY 4.0},
}
RIS
TY - JOUR
TI - Quantum refinement can be used to improve time-resolved crystallographic structures
AU - Gayathri Yuvaraj
AU - Kristoffer J. M. Lundgren
AU - Esko Oksanen
AU - Ulf Ryde
PY - 2026
DA - 2026/11/05
JO - IUCrJ
DO - 10.1107/s2052252526009486
UR - https://journals.iucr.org/paper?S2052252526009486
AB - Time-resolved serial crystallography has been shown to be a powerful technique to study enzyme reactions. In such studies, a reaction is triggered within the crystal and data are collected at different timepoints. A problem with this approach is that the synchronization is never perfect and therefore the structures at different timepoints typically contain a mixture of states in the active site, for example involving unreacted states, intermediates and products. Interpreting the electron density of mixtures is a challenge for crystallographic modelling and the resulting structures depend strongly on the quality of the empirical restraints employed. In this study, we investigate the potential of using quantum refinement for time-resolved structures, i.e. replacing the empirical restraints with more accurate quantum-mechanical calculations for the active site. We studied three different proteins. The first is a structure of bacteriorhodopsin containing a mixture of Schiff bases of all- trans and 13- cis retinal (PDB entry 5b6w). We show that quantum refinement gives similar structures to the original study, but the strain energies (indicating how much the structures are distorted from the optimum geometry) are much smaller: 36 and 71 kJ mol ⁻¹ for the all- trans and 13- cis conformations, respectively, compared with 366 and 473 kJ mol ⁻¹ for the deposited structure. Next, we studied a reaction intermediate of xylose isomerase (PDB entry 6qni), showing that the structure probably contains a mixture of the substrate-free state, as well as both the ring-closed and ring-opened glucose substrate, in contrast to the deposited structure, which was interpreted as 100% ring-closed glucose. Third, we studied a structure of fluoroacetate dehalogenase (PDB entry 6qhp), which was interpreted as a mixture of a covalent reaction intermediate and two conformations of the fluoroacetate substrate. Our best interpretation (with much less difference density) contains no intermediate and only a single conformation of the substrate, as well as a water molecule. We also studied all of the other structures in the time-resolved study of this enzyme to show that quantum refinement gives consistent structures throughout the reaction. We show that the suggestion of two conformations of the intermediate in the other chain of the protein is also questionable and point out that it is hard to discriminate between the fluoroacetate substrate and the glycolate product. For all structures, we also perform standard crystallographic refinement, with empirical restraints instead of the quantum-mechanical calculations. These often give similar results, but since electrostatic interactions are ignored they cannot properly describe hydrogen bonds, which are key to provide reliable positions of water molecules and other polar groups, especially in the poorly defined electron density of mixed states in time-resolved data.
SN - 2052-2525
ER -