Redetermination of the first unknown protein MicroED structure by high resolution X-ray diffraction

Abstract

Microcrystal electron diffraction (MicroED) has the potential to considerably impact the field of structural biology. Indeed, the method can solve atomic structures of a wide range of molecules, beyond the reach of single particle cryo-electron microscopy, exploiting crystals too small for X-ray diffraction (XRD) even using X-ray free-electron lasers. However, until the first unknown protein structure – a R2-like ligand binding oxidase from Sulfolobus acidocaldarius (SaR2lox) – was recently solved at 3.0 Å resolution, MicroED had only been used to study known protein structures previously obtained by XRD. Here, after adapting sample preparation protocols, the structure of the SaR2lox protein originally solved by MicroED was redetermined by XRD at 2.1 Å resolution. In light of the higher resolution XRD data and taking into account experimental differences of the methods, the quality of the MicroED structure is examined. The analysis demonstrates that MicroED provided an overall accurate model, revealing biologically relevant information specific to SaR2lox, such as the absence of an ether cross-link, but did not allow to detect the presence of a ligand visible by XRD in the protein binding pocket. Furthermore, strengths and weaknesses of MicroED compared to XRD are discussed in the perspective of this real-life protein example. The study provides fundaments to help MicroED become a method of choice for solving novel protein structures.

Comparative structural analysis provides new insights into the function of R2‐like ligand‐binding oxidase

“…Instead, it could represent a mechanism regulating the access of O 2 and/or metal ions allowing the correct maturation of the cofactor under specific conditions. Moreover, the fact that Sa R2loxII did not trap a fatty acid ligand unlike Sa R2loxI although both recombinant proteins were produced under similar conditions, that is, in E. coli cultured in terrific broth media [34], suggests that paralogous R2loxes might have different substrate specificity and thus could be active under different environmental conditions.…”

“…2F). In similar expression conditions in E. coli cultured in rich media, MtR2lox, GkR2loxI and SaR2loxI are copurified with putative fatty acid ligands [8,14,34].…”

Electron Diffraction of 3D Molecular Crystals

“…For lowerquality diffraction data extracted from small molecules, phasing by simulated annealing 124 has also proved a useful approach in 3D ED, often in conjunction with DM. (which was later supplanted by a higher-quality X-ray structure 130 ).…”

“…A substantial fraction originates from studies demonstrating new methodological approaches to 3D ED; this has resulted in well-studied proteins typically used as standards in X-ray crystallography (especially proteinase K, lysozyme, and catalase), accounting for over 40% of macromolecular ED structures deposited in the PDB. Comparatively few de novo structures have been determined by MR; currently, these remain limited to a handful of oligopeptides (with a maximum sequence length of 11 residues; see Table ) and a single novel protein, R2lox (which was later supplanted by a higher-quality X-ray structure).…”

“…An illustrative example is provided by Xu et al ’s multipart investigation of an R2-like ligand-binding oxidase (a metalloenzyme originally isolated from Sulfolobus acidocaldarius). , In 2018, Xu et al disclosed a 3.0 Å 3D ED structure of R2lox, phased by molecular replacement using a homologous X-ray structure with 35% shared sequence identity as a template. Although novel at the time, this structure nevertheless exhibited less-than-ideal completeness (62.8%, despite merging data from 21 crystals, suggesting stark orientation bias) and unusually high R meas (56.1% overall) statistics.…”