High-throughput automated molecular replacement for small-molecule MicroED data

Area of Science:

• Crystallography and structural biology
• Electron diffraction (ED) applications
• Small molecule structure determination

Background:

• Electron diffraction (ED) is increasingly used for protein and small molecule structural characterization.
• Ab initio phasing is standard for small molecules but struggles with radiation damage and poor crystallinity.
• Molecular replacement (MR) is common for proteins but limited for small molecules due to challenges in generating conformations.

Purpose of the Study:

• To develop a high-throughput automated molecular replacement (MR) workflow for electron diffraction (ED) data.
• To solve the novel ED structure of corilagin, a pharmaceutically relevant macrocyclic gallotannin.
• To validate the MR workflow against known macrocyclic structures at various resolutions.

Main Methods:

• Development of a high-throughput automated molecular replacement workflow.
• Application of the workflow to solve the ED structure of corilagin.
• Validation using three known macrocycles (paritaprevir-α, paritaprevir-β, grazoprevir) at resolutions from 1.0 to 2.0 Å.

Main Results:

• Successfully solved the novel ED structure of corilagin, which was intractable with ab initio phasing.
• The automated MR workflow achieved successful solutions for all validation structures across tested resolutions.
• Obtained R factors and RMSD values within acceptable limits compared to ab initio solved structures.

Conclusions:

• The developed automated MR workflow is effective for solving small molecule ED structures, especially challenging ones.
• This method overcomes limitations of ab initio phasing in electron diffraction.
• The workflow demonstrates broad applicability for structural determination of macrocyclic compounds using ED data.