Fast two-dimensional grid and transmission X-ray microscopy scanning methods for visualizing and characterizing...

Area of Science:

• Macromolecular Crystallography (MX)
• Structural Biology
• Biophysics

Background:

• Serial crystallography requires efficient methods for locating and centering microcrystals, especially for challenging targets like membrane proteins.
• Traditional methods can be time-consuming and may not be suitable for small or weakly diffracting crystals.

Purpose of the Study:

• To implement and validate a fast continuous grid scan protocol for macromolecular crystallography (MX) at the Swiss Light Source (SLS).
• To enable diffraction-based identification and centering of membrane protein microcrystals in lipid cubic phase (LCP) for *in meso in situ* serial crystallography.
• To demonstrate the complementary utility of scanning transmission X-ray microscopy (STXM) for low-dose crystal detection.

Main Methods:

• Integration of a fast continuous grid scan protocol into the SLS MX beamline data acquisition software.
• Utilizing fast readout single-photon counting hybrid pixel array detectors (PILATUS and EIGER).
• Application of scanning transmission X-ray microscopy (STXM) for near-zero-dose crystal detection on various sample supports at room and cryogenic temperatures.

Main Results:

• Successful implementation of continuous grid scans for identifying diffraction hotspots and locating/centering membrane protein microcrystals in LCP.
• Demonstration of grid scans on large LCP samples (50 nL) with analysis of diffraction images.
• STXM successfully identified flash-cooled crystals in nylon loops and 10 µm thick crystals using integrated phase images with EIGER data.

Conclusions:

• The fast continuous grid scan protocol significantly enhances the efficiency of crystal screening and centering in serial crystallography.
• STXM provides a valuable low-dose method for locating crystals on supports before X-ray diffraction data collection.
• These advancements facilitate the structural determination of challenging proteins, including membrane proteins, using X-ray crystallography.