Researchers have used advanced X-ray crystallography methods to determine the structure of the archaeal rubredoxin protein at unprecedented resolution
Summary
- Researchers have determined the structure of the protein rubredoxin at 0.43 Ångström resolution, the highest resolution ever achieved for a protein structure.
- These findings and methodology could now allow researchers to routinely apply advanced quantum crystallography methods to the investigation of biological macromolecules, given accurate X-ray diffraction data.
- 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.
Proteins are high-performance molecular machines that carry out nearly every biochemical function inside living cells. To understand how they achieve this, scientists often look into their detailed atomic structure, which holds important clues to their activity. The more precision they can achieve while doing this, the deeper the insight they might potentially gain into the chemical principles that underlie protein structure and ultimately function.
Marking a major advancement in X-ray crystallography, scientists headed by Ashwin Chari from the Max Planck Institute for Multidisciplinary Sciences (Göttingen, Germany), Gleb Bourenkov from EMBL Hamburg (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 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," explained Ashwin 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." The results have now been published in Acta Crystallographica Section D, Structural Biology .
A record made possible by advanced instrumentation and protocols
Previous experiments by Chari, Bricogne, and Bourenkov laid the groundwork for this resolution record. There, the scientists investigated how X-rays alter protein structure and how radiation damage interferes with atomic model refinement and interpretation. During these experiments, the researchers came up with a 'resolution-in-dose' approach, which relied on low-dose data collection protocols on large protein crystals bathed in a 'top-hat' beam, which ultimately allowed the scientists to reach sub-Ångström resolutions.
The new study made use of one of the world's brightest X-ray radiation sources: PETRA III at DESY in Hamburg (Germany). To collect the high-quality data that led to the resolution record, the scientists used the beamline P14 provided by EMBL Hamburg. 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 precise control over the X-ray dose delivered to the sample - just as the 'resolution-in-dose' approach suggests.
The data collection and processing were carried out according to advanced protocols created on-the-fly by Global Phasing's workflow software, tailored to each individual sample and designed to produce high data quality.
What X-rays revealed about the protein
The high-resolution X-ray crystallographic data 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," said 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.
"These findings are of paramount importance to our interest in precisely mapping the role of local electric fields in enzymes and visualising their function during reactions," said 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 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.
