In situ serial crystallography facilitates 96-well plate structural analysis at low symmetry

Area of Science:

• Structural Biology
• Biophysics
• Crystallography

Background:

• Serial crystallography enables room-temperature X-ray structure determination, revealing protein dynamics.
• Challenging samples like membrane proteins and viruses often yield fragile crystals unsuitable for cryo-crystallography.
• Traditional serial crystallography requires numerous microcrystals, often produced via batch crystallization, which is difficult for low-yield mammalian cell line expressed proteins.

Purpose of the Study:

• To develop and validate an in situ serial crystallography (iSX) approach for direct data collection from crystallization plates.
• To enable structure determination under ambient conditions and identify optimal crystallization conditions.
• To overcome sample quantity limitations for serial crystallography using proteins expressed in mammalian cell lines.

Main Methods:

• Developed an in situ serial crystallography (iSX) approach using a universal plate holder at a microfocus beamline (ID23-2, ESRF).
• Collected data directly from 96-well crystallization plates.
• Applied iSX to determine the structure of autotaxin, a therapeutic target, in the P1 space group at 3.0 Å resolution.

Main Results:

• The iSX approach facilitated direct measurement and structure determination from crystallization plates.
• A complete dataset was obtained for autotaxin structure determination, screening multiple crystallization conditions.
• The iSX approach proved highly efficient at a microfocus beamline, improving throughput.

Conclusions:

• In situ serial crystallography (iSX) offers an efficient alternative for presenting samples in serial crystallography experiments at synchrotrons.
• The iSX method expands the applicability of serial crystallography to challenging samples and low-yield protein expression systems.
• Crystallization plates can be routinely utilized for sample presentation in serial crystallography.