Researchers have determined the structure of the protein rubredoxin at 0.43 Ångström resolution. The results of the research in which Prof. Paulina Dominiak from the University of Warsaw took part have been published in the journal „Acta Crystallographica Section D: Structural Biology”.

Researchers have determined the structure of the protein rubredoxin at 0.43 Ångström resolution. The findings and methodology could now allow, with accurate X-ray diffraction data, to routinely apply the advanced methods of quantum crystallography to the investigation of biological macromolecules.

Gaining deep insights into enzyme-catalysed reactions through this approach opens the door to developing drugs to treat human diseases and creating designer enzymes for use in chemistry, material science, and industrial applications.

Enzymes are true high-performance machines. They carry out a variety of chemical reactions within organisms with almost perfect precision and efficiency. While synthetic chemical processes often require extreme temperatures or pressures, enzymes work energy-efficiently and sustainably in aqueous solutions at body temperature. But how do they achieve this?

Uncovering the secrets of high-performing enzymes

To uncover their magic formula, scientists must first understand the chemical principles underlying their architecture and functional parts. Examining the enzyme’s structure using X-ray crystallography at a resolution better than 1.1 Ångström (Å) can reveal these secrets. This level of precision allows the calculation of electron densities, which can be used to determine the position of atoms and the exact nature of the bonds between them within the molecule and to derive processes with single-electron accuracy.

In detail, electron densities reveal electronic properties and quantum mechanical phenomena. These, in turn, provide insights into the quantum state of an enzyme’s active site, substrates, intermediates, and products. “Ultimately, this full experimental observation will enable the development of customized drugs for human diseases, as well as designer enzymes to catalyze chemical reactions,” says Ashwin Chari, head of the Structural Biochemistry and Mechanisms research group at the Max Planck Institute for Multidisciplinary Sciences in Göttingen (Germany).

However, reaching such high resolutions is not without its difficulties, as irradiation by X-rays damages the very structural details the scientists strive to see.

The highest resolution protein structure resolved to date

Scientists headed by Ashwin Chari, Gleb Bourenkov from European Molecular Biology Laboratory Hamburg Unit (Hamburg, Germany), Clemens Schulze-Briese from DECTRIS (Baden, Switzerland), Paulina Maria Dominiak from the University of Warsaw (Poland) and Gérard Bricogne from Global Phasing Ltd. (Cambridge, UK) have now found a way to overcome previous limitations, marking a major advancement in X-ray crystallography: they have determined the structure of the archaeal rubredoxin protein at a resolution of 0.43 Å. “This, to the best of our knowledge, represents the highest resolution protein structure determined yet,” explains Chari. “This has been enabled by a series of technical innovations and streamlined procedures. We have combined cutting-edge X-ray crystallography with advanced quantum-chemical models, bridging the gap between structural biology and quantum chemistry,” he adds. The results have now been published in Acta Crystallographica Section D, Structural Biology.

Experiments on radiation damage paved the way

Previous experiments of Chari, Bricogne, and Bourenkov laid the groundwork for this resolution record. There, the scientists investigated how X-rays alter the protein’s structure and how radiation damage interferes with atomic model refinement and interpretation. In conclusion, Bourenkov says that using “low-dose data collection protocols on large protein crystals bathed in a “top-hat” beam with a uniform fluence profile allows the study macromolecule structures at the sub-Ångström scale”. “Only this setup can deliver the uniform spatial distribution of X-rays necessary to resolve the fine details of progressive radiation damage,” adds Chari. Bricogne describes this approach with the term “resolution in dose”, emphasising that it provides the best opportunity for mitigating the detrimental effects of radiation damage on the experimental data. The details were published in May 2026 in Acta Crystallographica Section D, Structural Biology.

Now, the scientists used these findings to achieve the sub-Ångström analysis of the rubredoxin structure by combining cutting-edge X-ray crystallography and quantum mechanical calculations.

A record made possible by advanced instrumentation and protocols

This entails the use of one of the world’s brightest X-ray radiation sources: a storage-ring-based system named PETRA III at the research center DESY in Hamburg (Germany). To collect the high-quality data that led to the resolution record, the scientists used the beamline P14 provided by the European Molecular Biology Laboratory (EMBL) spearheaded by beamline scientist Bourenkov. This produces a tailored X-ray beam with a uniform radiation intensity, known as a “top-hat” beam. Its advantage: the size and shape can be adjusted before the start of every data collection to match the dimensions of each protein crystal. This allows a precise control over the X-ray dose delivered to the sample – just as the “resolution in dose” approach suggests.

The data collection and processing themselves were carried out according to advanced protocols created on-the-fly by Global Phasing´s workflow software, that are tailored to each individual sample and designed to produce the highest data quality that the sample is capable of delivering.

What X-rays revealed about the protein

The X-ray crystallographic data to 0.43 Ångström resolution allowed the team to determine accurate nuclear positions of atoms within the structure, including all hydrogen atoms. Beyond that, the accuracy of the electron densities derived from these data revealed quantum mechanical phenomena directly from the experimental structure, including electrons in the mid-point of chemical bonds and atomic partial charges.

“This observation indicates that ‘spherical’ scattering factors commonly used to refine atomic models of biological macromolecules are inadequate to describe the structural results at such high resolutions,” says Dominiak. To adequately explain the experimental structure and electron densities, the team made use of so-called “aspherical” scattering factors in order to account for the observed quantum mechanical phenomena. For this the DiSCaMB transferable aspherical atom model (TAAM) library was connected to Global Phasing´s protein model refinement software BUSTER. With this combination the team was able to satisfactorily refine accurate structures with aspherical electron densities against the exceptional diffraction data.

Promising prospects in several fields

“These findings are of paramount importance to our interest in precisely mapping the role of local electric fields in enzymes and visualizing their function during reactions,” says Chari. “Extrapolating our findings and methodology leads us to anticipate that, with accurate X-ray diffraction data, it should now be possible to routinely perform quantum crystallography on biological macromolecules. The long-term goal is to gain deep insights into enzyme-catalysed reactions to support future achievements in chemistry, materials science, and medicine.”

The two studies were the result of long-term collaborations with beamline scientists from EMBL, crystallographic software developers from Global Phasing Ltd., the X-ray detector company DECTRIS, quantum crystallographers from the University of Warsaw, as well as experts in biochemistry, X-ray crystallography and interpretation from the Max Planck Institute for Multidisciplinary Sciences.

This work was funded by the Volkswagen Foundation, Germany, and the National Science Center, Poland.