Three-dimensional structure determination protocol for noncrystalline biomolecules using x-ray free-electron laser...

Area of Science:

• Structural biology
• Biophysics
• X-ray crystallography

Background:

• X-ray Free-Electron Laser (XFEL) sources offer intense X-ray pulses for structural determination of noncrystalline biomolecules.
• Current XFEL intensities are insufficient for single-molecule diffraction due to the low scattering cross-section of biomolecules.

Purpose of the Study:

• To propose an experimental protocol and analysis method for visualizing biomolecular structures using XFEL.
• To overcome the limitations of low X-ray intensity for noncrystalline biomolecular structure determination.

Main Methods:

• Utilizing coherent X-ray diffraction imaging and 3D reconstruction.
• Employing a thin vitreous ice plate with multiple biomolecules (hundreds/μm²) as the sample.
• Retrieving biomolecular images from diffraction patterns of the ice plate.

Main Results:

• The proposed method compensates for the low scattering cross-section of individual biomolecules.
• Realistic atomic simulations indicate feasibility for 3D structure determination of large biomolecules like the 50S ribosomal subunit.
• Achieved resolution of 0.8 nm is possible with a future X-ray beam of 10^16 photons/500×500 nm²/pulse.

Conclusions:

• The combined approach of XFEL, diffraction imaging, and 3D reconstruction offers a viable path for high-resolution biomolecular structure determination.
• This method significantly enhances the potential of XFEL in structural biology, particularly for challenging noncrystalline samples.
• Future advancements in XFEL intensity are crucial for realizing the full potential of this technique.