Hydrogens and hydrogen-bond networks in macromolecular MicroED data

Area of Science:

• Structural Biology
• Biophysics
• Biochemistry

Background:

• Microcrystal electron diffraction (MicroED) is a powerful technique for protein structure determination.
• It is particularly useful for crystalline samples too small for X-ray crystallography.
• Electron scattering provides stronger contrast for hydrogen atoms compared to X-rays.

Purpose of the Study:

• To identify hydrogen atom positions and visualize hydrogen bonding networks using MicroED data.
• To assess the potential of MicroED for drug discovery applications.
• To determine protein structure and function at subatomic resolution.

Main Methods:

• Utilized subatomic resolution MicroED data of triclinic hen egg-white lysozyme (0.87 Å resolution).
• Data were collected under low exposure conditions using an electron-counting detector.
• Analyzed data for difference peaks indicating hydrogen atom positions.

Main Results:

• Successfully identified over a third of all hydrogen atom positions.
• Directly visualized hydrogen bonding interactions and residue charged states.
• Found that hydrogen bond lengths are more accurately described by inter-nuclei distances.

Conclusions:

• MicroED can now uncover hydrogen atoms and hydrogen bonding interactions in proteins.
• This technique opens new avenues for structural biology and drug discovery.
• Ongoing advances in MicroED promise further insights into protein structure and function.