Advances in long-wavelength native phasing at X-ray free-electron lasers.

Area of Science:

• Structural Biology
• Crystallography
• X-ray Science

Background:

• Serial femtosecond crystallography (SFX) enables protein structure determination using X-ray free-electron lasers (XFELs).
• Native single-wavelength anomalous diffraction (native-SAD) phasing is crucial for de novo protein structure determination but can be data-intensive.

Purpose of the Study:

• To quantify improvements in native-SAD phasing at XFELs using longer X-ray wavelengths.
• To demonstrate reduced data requirements for de novo protein structure determination via optimized native-SAD.

Main Methods:

• Utilized long-wavelength XFEL pulses for native-SAD phasing of SFX data.
• Employed sensitive anomalous data-quality indicators and model proteins.
• Optimized experimental geometry and applied advanced post-refinement and partiality correction.

Main Results:

• Achieved up to a tenfold reduction in the number of indexed images needed for native-SAD compared to shorter wavelengths.
• Demonstrated significantly lowered sample consumption and beam-time requirements.
• Enhanced data quality and anomalous signal for challenging protein structures.

Conclusions:

• Longer X-ray wavelengths substantially enhance native-SAD phasing efficiency at XFELs.
• Optimized native-SAD protocols lower barriers for de novo protein structure determination.
• These advancements benefit various SFX experiments requiring precise weak signal measurements, including time-resolved studies.