Standard Sample Preparation for Serial Femtosecond Crystallography.

Area of Science:

• Structural Biology
• Biophysics
• Crystallography

Background:

• Serial crystallography (SX), encompassing serial synchrotron crystallography (SSX) and serial femtosecond crystallography (SFX), enables high-resolution biomolecular structure determination from microcrystals at room temperature.
• Standardized protein samples are crucial for validating new X-ray Free Electron Laser (XFEL) instruments and experimental setups.

Purpose of the Study:

• To review commonly used standard proteins for SX experiments.
• To present optimized microcrystal preparation workflows for four model systems: lysozyme, myoglobin, iq-mEmerald, and photoactive yellow protein (PYP).
• To provide practical guidance for the serial crystallography community to enhance reproducibility and experimental consistency.

Main Methods:

• Literature review of established methodologies for standard protein sample preparation.
• Description of microcrystal preparation workflows based on experimental experience at the European XFEL.
• Consolidation of refined and optimized protocols for lysozyme, myoglobin, iq-mEmerald, and PYP.

Main Results:

• Identification and summary of widely adopted standard proteins for SX.
• Detailed workflows for preparing microcrystals of lysozyme, myoglobin, iq-mEmerald, and PYP.
• Integration of practical insights and optimized protocols for routine SX applications.

Conclusions:

• The review provides essential guidance for selecting and preparing standard protein samples for serial crystallography.
• Optimized protocols contribute to enhanced reproducibility and reliable experimental performance at XFEL facilities.
• This work supports the broader scientific community in advancing structural biology through serial crystallography techniques.