Direct from the seed: an atomic resolution protein structure by ab initio MicroED.

Area of Science:

• Structural Biology
• Biophysics
• Crystallography

Background:

• Protein structure determination is crucial for understanding biological function.
• Microcrystal electron diffraction (MicroED) offers a promising alternative to X-ray crystallography for small or sensitive crystals.
• Challenges remain in achieving high resolution and solving structures ab initio with MicroED.

Purpose of the Study:

• To determine the atomic resolution structure of the seed protein crambin using MicroED.
• To establish a benchmark for ab initio structure solution with MicroED.
• To demonstrate the effectiveness of serial merging and anisotropy correction for MicroED data.

Main Methods:

• Spontaneous formation of protein nanocrystals from ethanolic purification drops.
• Data collection using microcrystal electron diffraction (MicroED) on 58 nanocrystals.
• Serial merging of diffraction data and anisotropy aware truncation.
• Ab initio structure solution using a five-residue helical fragment for phasing.

Main Results:

• An atomic resolution (0.85 Å) MicroED structure of crambin was solved ab initio.
• Automated model building and resolution of individual hydrogen atoms were achieved.
• The study demonstrates successful sub-ångström resolution MicroED without specialized equipment.

Conclusions:

• Protein nanocrystals suitable for MicroED can be readily obtained.
• Serial merging combined with anisotropy correction enables ab initio MicroED structure solution.
• This work provides a benchmark for high-resolution MicroED on standard instrumentation.