Eliminating the missing cone challenge through innovative approaches.

Area of Science:

• Structural biology
• Biophysics
• Crystallography

Background:

• Microcrystal Electron Diffraction (MicroED) enables molecular structure determination from small crystals.
• Plate-like crystals often exhibit preferred orientation on microscopy grids, leading to a 'missing cone' of data.
• This limitation restricts the completeness of structural information obtainable via MicroED.

Purpose of the Study:

• To demonstrate the efficacy of suspended drop crystallization in overcoming preferred orientation in MicroED.
• To validate the suspended drop method for samples with systematic preferred orientations.
• To enable complete structure determination for challenging crystal habits.

Main Methods:

• Implementation of a novel suspended drop crystallization technique.
• Application of Microcrystal Electron Diffraction (MicroED) to plate-like crystals.
• Analysis of crystal orientation data to assess the 'missing cone' effect.

Main Results:

• The suspended drop crystallization strategy successfully eliminated the missing cone of information.
• Complete data collection was achieved for bovine liver catalase and SARS‑CoV‑2 main protease (Mpro) crystals.
• The method proved effective for samples with inherent preferred orientations.

Conclusions:

• Suspended drop crystallization is a powerful tool to address preferred orientation challenges in MicroED.
• This technique significantly enhances the scope and completeness of molecular structure determination by MicroED.
• The approach is particularly valuable for crystals with systematic preferred orientations, such as thin plates.