Measurement of charges and chemical bonding in a cryo-EM structure

Understanding the role of hydrogen bonding, bond polarity, and charge distribution within protein molecules is essential for comprehending protein structure and function. These factors significantly influence processes such as enzymatic catalysis, electron transfer, and ligand binding. While X-ray crystallography has provided valuable insights, resolving hydrogen atoms and their associated charge characteristics remains challenging due to their low scattering power. Cryogenic electron microscopy (cryo-EM), particularly at high resolution, offers a potential alternative. Recent advancements in cryo-EM technology, including the use of cold field emission (CFE) guns and monochromators, have led to significant improvements in spatial resolution, enabling the visualization of individual atoms, including hydrogen atoms. However, extracting chemical information beyond atomic coordinates from cryo-EM data presents significant challenges, including phase errors, contrast transfer function (CTF) effects, and uncertainties in Euler angle assignments. This study aims to investigate the feasibility of using high-resolution single-particle cryo-EM to measure detailed chemical properties, including charge distribution, within a protein complex. Previous studies using techniques like convergent-beam electron diffraction (CBED) and rotation 3D electron diffraction (3D ED) have shown promise in measuring charge distributions in materials, but their application to single-particle cryo-EM is less developed. Apoferritin, a well-characterized protein complex, is used as a model system to explore this potential.

The literature extensively documents the importance of hydrogen bonding and charge distribution in protein structure and function. Studies have highlighted the role of low-barrier hydrogen bonds in enzymatic activity and electron transfer. The challenges of visualizing hydrogen atoms using X-ray techniques and electron microscopy are well-established. Previous work has explored the use of electron crystallography to study charges in proteins, but this approach has limitations in terms of sample preparation and data interpretation. High-resolution cryo-EM has emerged as a powerful technique for protein structure determination. However, extracting information beyond atomic positions requires careful analysis of the cryo-EM maps and consideration of the inherent limitations of the technique. The study builds upon this existing body of work by attempting to extract detailed chemical information, including charges, from high-resolution single-particle cryo-EM data.

Apoferritin was expressed in *E. coli*, purified, and prepared for cryo-EM using standard techniques. Cryo-EM data were collected using a CRYO ARM 300 electron microscope equipped with a cold field emission gun, operated at 300 kV. Dose-fractionated images were recorded on a K3 camera in super-resolution mode. Two datasets (A and B) were collected with and without JAFIS Tool (JEOL UK) for axial coma aberration and astigmatism correction. Image processing was performed using RELION-3.1, including motion correction, CTF estimation, particle picking, 2D classification, and 3D auto-refinement. The final map for Dataset A reached 1.19 Å resolution. An atomic model of apoferritin was fitted to the map and refined using ISOLDE and REFMAC5. Weighted *F_o*–*F_c* difference maps were calculated between experimental data and models omitting hydrogen atoms using Servalcat. Hydrogen atoms with density levels ≥2σ were picked and manually selected. Spatial resolution selection (limiting data to 2.5 Å resolution) and a dose-dependent analysis using frame series (Frames 2, 3, 20, and 40) were performed to investigate the origin of observed densities. Statistical analysis of hydrogen bond lengths and their uncertainties was conducted.

A high-resolution (1.19 Å) cryo-EM map of apoferritin was obtained. This map revealed clear densities for individual atoms, including hydrogen atoms, particularly within the protein core. A weighted difference map identified positive densities for most hydrogen atoms in the core region, with these densities enabling distinction between the amino- and oxo-termini of asparagine and glutamine side chains. Negative densities were observed around the carboxyl termini of aspartate and glutamate side chains, consistent with negative charges. Analysis using different resolution ranges confirmed that these negative densities were prominent at lower resolutions (≤2.5 Å), consistent with the known behavior of electron scattering factors for charged atoms. The dose-dependent frame series analysis showed that these negative densities were not primarily due to radiation damage. Analysis of hydrogen atom positions showed that the average distance of hydrogen peak positions from their parent atoms was dependent on the bond type, which is supported by the calculated standard uncertainties in bond lengths. Statistical analysis showed that hydrogen peaks in polar bonds (N-H) were closer to their parent atoms than in other C-H bonds.

This study demonstrates that high-resolution single-particle cryo-EM can provide detailed insights into the chemical bonding properties and charge distribution within a protein complex. The ability to differentiate between amino- and oxo-termini of asparagine and glutamine, as well as to observe densities indicative of negative charges on acidic residues, showcases the sensitivity of this technique. The observed dependency of hydrogen atom position on the bonded atom type further highlights the potential for extracting detailed chemical information. The results obtained are consistent with theoretical expectations regarding electron scattering and charge distribution, validating the methodology. While the analysis of charges remains qualitative, this study lays a foundation for more quantitative approaches. Future work may involve improved modeling of electron scattering factors, more sophisticated algorithms for handling partial charges, and potentially, combination with other techniques.

This study successfully measured charges and chemical bond types of hydrogen atoms in a protein structure using high-resolution single-particle cryo-EM. The high accuracy achieved in differentiating the average hydrogen positions based on bond type or polarity, combined with the observation of charge-related densities, validates the method's potential for analyzing chemical properties. This technology holds promise for investigating a range of biological systems, providing a powerful tool for studying protein function and dynamics at a molecular level. Future studies could focus on improving the quantitative aspects of charge measurement and expanding the technique's application to larger and more complex macromolecular assemblies.

The current analysis of charges remains largely qualitative. While the negative densities strongly suggest negative charges, further development of methods is needed to obtain quantitative charge assignments. The sample used, apoferritin, is a well-behaved protein; application to more dynamic or heterogeneous systems might present additional challenges. The observed hydrogen atoms represent approximately 70% of the total, reflecting potential limitations in hydrogen atom visibility. The study focuses on a specific protein, and generalizability to other systems needs further investigation.