Towards time-resolved MicroED grid preparation using mix-and-inject gas dynamic virtual nozzles

Area of Science:

• Structural Biology
• Biophysics
• Materials Science

Background:

• Serial X-ray diffraction using gas dynamic virtual nozzles (GDVN) enables visualization of protein dynamics in crystals.
• This method requires numerous crystals and significant beam time, limiting accessibility.
• Cryogenic Microcrystal Electron Diffraction (MicroED) offers a more accessible alternative for nanocrystallography, requiring fewer crystals.

Purpose of the Study:

• To integrate GDVN technology into the sample preparation workflow for cryogenic MicroED.
• To assess the feasibility of using GDVN for depositing protein crystals onto electron microscopy grids for MicroED.
• To lay the groundwork for developing time-resolved MicroED experiments.

Main Methods:

• Incorporation of GDVN technology into the sample freezing process for MicroED.
• Deposition of protein crystals (proteinase K) onto transmission electron microscopy (TEM) grids using GDVNs.
• Vitrification of samples and subsequent MicroED data collection.
• Analysis of deposition efficiency and MicroED data quality.

Main Results:

• Successful deposition of protein crystals onto TEM grids using GDVNs was demonstrated.
• MicroED data collection was successful, yielding high-resolution structural information.
• Limited deposition efficiency of protein crystals using GDVNs was observed.

Conclusions:

• GDVN technology can be utilized for depositing crystals on grids for MicroED.
• This represents a crucial first step towards developing time-resolved MicroED experiments.
• Methodological challenges in optimizing deposition efficiency need to be addressed for a reliable workflow.