Parameters affecting specimen flatness of two-dimensional crystals for electron crystallography

Area of Science:

• Materials Science
• Biophysics
• Crystallography

Background:

• Crystal flatness on support films is essential for high-resolution protein electron crystallography.
• Variations in carbon support films and grid materials can impact crystal flatness.

Purpose of the Study:

• To investigate how carbon support film preparation and different grid materials affect the flatness of two-dimensional crystals.
• To identify optimal conditions for achieving flat crystals for electron crystallography.

Main Methods:

• Assessed crystal flatness using the sharpness of diffraction spots in electron diffraction patterns of tilted purple membrane.
• Quantitatively compared carbon films prepared with and without sparking.
• Evaluated various grid materials (copper, titanium, molybdenum) and manufacturers.
• Utilized scanning electron microscopy to examine grid morphology.

Main Results:

• Carbon films evaporated without sparking yielded a higher proportion of flat crystals compared to 'sparked' carbon.
• Titanium grids showed superiority over copper grids, likely due to reduced cryo-crinkling.
• Molybdenum grids from Pacific GridTech outperformed titanium grids, yielding more flat crystals.
• Plano molybdenum grids proved unsuitable due to carbon film breakage; Pacific GridTech grids had superior morphology.

Conclusions:

• Non-sparked carbon film evaporation is preferable for achieving flat 2D crystals.
• Grid material and manufacturer significantly influence crystal flatness, with Pacific GridTech molybdenum grids being optimal.
• Grid bar smoothness and surface-to-hole ratio are critical factors for successful electron crystallography sample preparation.