Flow-aligned, single-shot fiber diffraction using a femtosecond X-ray free-electron laser

Area of Science:

• Structural biology
• Biophysics
• X-ray science

Background:

• Determining biological molecule structures without crystals is a major goal in X-ray free-electron laser (XFEL) science.
• Filament systems, with their one-dimensional translational symmetry, bridge the gap between single molecules and crystals for structural analysis.
• XFEL radiation offers potential for single macromolecular structure elucidation.

Purpose of the Study:

• To demonstrate flow alignment of biological filaments for XFEL-based structural studies.
• To determine the structures of specific amyloid fibrils and F-actin using this technique.
• To assess the potential of XFEL for analyzing ordered biological macromolecules.

Main Methods:

• Flow alignment of biological filaments (e.g., Escherichia coli pili, F-actin, amyloid fibrils) using microfluidics.
• Irradiation of aligned filaments with femtosecond X-ray pulses from an XFEL.
• Analysis of resulting diffraction patterns to determine molecular structure and order.

Main Results:

• Demonstrated successful flow alignment of as few as 100 filaments.
• Obtained diffraction patterns comparable to classical fiber diffraction studies.
• Determined F-actin can be flow-aligned with a disorientation of approximately 5 degrees.
• Elucidated that gelsolin amyloids consist of stacked β-strands perpendicular to the filament axis.
• Observed a spectrum of order, from fibrillar to crystalline, in individual α-synuclein amyloids.

Conclusions:

• Flow alignment coupled with XFEL radiation is a viable method for structural determination of biological filaments.
• This technique provides insights into the structural organization of amyloids and cytoskeletal proteins.
• XFEL-based fiber diffraction offers a powerful approach for studying macromolecular structures at high resolution.