Structure determination from single molecule X-ray scattering with three photons per image

Area of Science:

• Structural Biology
• Biophysics
• X-ray Scattering

Background:

• Femtosecond high-intensity free-electron laser (FEL) pulses offer a novel method for macromolecular structure determination.
• Current limitations include the need for nano-crystals or virus particles, with the ultimate goal of single biomolecule scattering.
• Key challenges are the low signal-to-noise ratio and random molecular orientation in scattering images.

Purpose of the Study:

• To develop a de novo correlation-based approach for macromolecular structure determination.
• To demonstrate the feasibility of structure determination using a minimal number of scattered photons.
• To overcome the limitations of current scattering experiments for single biomolecules.

Main Methods:

• A de novo correlation-based computational approach was developed.
• Synthetic scattering data from a small protein was utilized for analysis.
• The method focuses on analyzing coherently scattered photons to reconstruct molecular structure.

Main Results:

• Structure determination is achievable with as few as three coherently scattered photons per image.
• Near-atomic resolution of 3.3 Å was demonstrated using synthetic data.
• The approach successfully processed a large number of images (3.3 × 10^9) with a significant photon count (3.3 × 10^10).

Conclusions:

• The developed three-photon correlation approach significantly lowers the photon requirement for macromolecular structure determination.
• This method shows robustness against noise from incoherent scattering, provided minimal solvent molecules are present.
• The findings pave the way for advancing single biomolecule imaging and structural analysis using FELs.