Three-dimensional electron crystallography of protein microcrystals.

Area of Science:

• Structural Biology
• Biophysics
• Crystallography

Background:

• Determining high-resolution protein structures is crucial for understanding biological processes.
• Electron cryo-microscopy (CryoEM) has emerged as a powerful tool for structural biology.
• Limitations exist in resolving structures of small or challenging protein crystals.

Purpose of the Study:

• To demonstrate the feasibility of determining high-resolution protein structures using electron crystallography.
• To introduce and validate a novel technique named MicroED (3D electron diffraction).
• To establish a protocol for data collection and structure determination from microcrystals.

Main Methods:

• Utilized electron cryo-microscopy (CryoEM) for data acquisition.
• Collected electron diffraction data from lysozyme microcrystals frozen on an EM grid.
• Developed a data collection protocol for a full-tilt series to atomic resolution with minimal electron dose.

Main Results:

• Achieved data collection to 1.7 Å resolution from a single crystal.
• Indexed diffraction data from three separate crystals.
• Determined the lysozyme structure by molecular replacement and crystallographic refinement to 2.9 Å resolution.

Conclusions:

• Electron crystallography of 3D crystals in CryoEM is a feasible method for high-resolution structure determination.
• The developed MicroED technique shows promise for broad applicability in structural biology.
• This proof of principle opens new avenues for analyzing protein structures, especially from microcrystals.