Multi-wavelength anomalous diffraction de novo phasing using a two-colour X-ray free-electron laser with wide...

Area of Science:

• Structural Biology
• Biophysics
• Crystallography

Background:

• Serial femtosecond crystallography (SFX) at X-ray free-electron lasers (XFELs) enables macromolecular structure determination.
• De novo structure determination using SFX is challenging due to data inaccuracies and radiation damage.
• Accurate diffraction intensities for phasing require extensive data, large sample volumes, and significant beamtime.

Purpose of the Study:

• To investigate the utility of simultaneous two-color X-ray free-electron laser pulses for multiple wavelength anomalous dispersion (MAD) phasing.
• To assess the accuracy of phase angle determination using two-color MAD compared to single-color phasing in SFX.

Main Methods:

• Collecting serial femtosecond crystallography data using simultaneous two-color X-ray free-electron laser pulses.
• Applying multiple wavelength anomalous dispersion (MAD) phasing principles to the collected data.
• Comparing phase angle accuracy between two-color and single-color MAD phasing strategies.

Main Results:

• Two-color XFEL pulses can be effectively utilized for MAD phasing in SFX.
• Phase angle determination accuracy is significantly improved with two-color MAD phasing compared to single-color methods.
• The enhanced accuracy facilitates de novo structure determination of macromolecules.

Conclusions:

• Two-color MAD phasing is a viable and more accurate approach for SFX.
• This technique is expected to advance the structure determination of difficult-to-phase proteins at XFELs.
• It offers a pathway to more efficient and accurate macromolecular structure determination.