Integrating X-ray, neutron, and electron crystallography to reveal charge distributions in Coulomb potential maps

• Applicant: Dr. Alaa Shaikhqasem
• Subject Area: Structural Biology
• Term: since 2025
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 565987258

The remarkable progress in macromolecular crystallography over the last century has enabled protein structure determination at subatomic resolution. Nevertheless, the study of protein protonation states, whether by neutron crystallography or subatomic resolution X-ray crystallography, is often hindered by the need for large, well-ordered crystals, which can be challenging to obtain. Additionally, despite the detailed information revealed by current methods, neither X-ray nor neutron scattering can directly provide insights into the charge states and electrostatic potentials of protein structures. These features are crucial for understanding protein biochemistry and functionality, including protein folding, enzymatic mechanisms, synthetic enzymology, and drug design. Recent advancements in electron crystallography have enabled diffraction data collection from biological macromolecules using electron beams. Unlike X-rays or neutrons, electrons, as charged particles, interact with atoms primarily through electrostatic forces exerted by the positively charged nucleus and negatively charged electrons. This interaction pattern results in a calculated density represented as a Coulomb potential map or electrostatic potential map. While electrons exhibit much higher sensitivity to hydrogen atoms and are strongly affected by charge distribution, this map is widely employed in constructing protein models and is interpreted in a similar way to electron density maps generated by X-ray crystallography. Currently, there is no established interpretation of these maps that allows the experimental assignment of these features to macromolecule structures. This proposal aims to address this gap by applying an integrative approach that combines neutron, X-ray, and electron crystallography to crystals of a selected enzyme, chorismate dehydratase. Neutron crystallography will be used to track protonation and deprotonation patterns at varying pH levels. Thus, providing an experimental reference to evaluate data processing and refinement approaches to be developed and investigated during the project. As a proof of concept, once the methodology is established, it will be applied to electron diffraction data of human transketolase to assign low-barrier hydrogen bonds involved in the enzyme’s cooperativity mechanism. Additionally, to further demonstrate the practical application of the methodology, electron diffraction will be used to investigate the role of electrostatic potential distribution in fine-tuning the redox potential in the blue copper protein pseudo-azurin. This work will be complemented with spectroscopic and biochemical analyses to support the structural data, ensuring a comprehensive understanding of the obtained results. Ultimately, this work aims to establish a reliable interpretation approach for Coulomb potential maps and showcase its practical application by addressing a long-standing biological question.

• DFG Programme: Research Grants