Neutron Crystallography Data Collection and Processing for Modelling Hydrogen Atoms in Protein Structures.

Area of Science:

• Structural Biology
• Biophysics
• Biochemistry

Background:

• Neutron crystallography excels at locating hydrogen atoms in biological macromolecules, crucial for understanding protonation and hydration states.
• Unlike X-ray diffraction, neutron crystallography avoids radiation damage to sensitive biological samples.
• X-ray diffraction offers limited information on light atoms and can damage photosensitive cofactors.

Purpose of the Study:

• To present the workflow for obtaining neutron diffraction structures at the IMAGINE and MaNDi beamlines at Oak Ridge National Laboratory (ORNL).
• To demonstrate sample preparation techniques, including mounting and deuterium exchange, for neutron diffraction.
• To illustrate data collection strategies and structure refinement methods for neutron crystallography.

Main Methods:

• Mounting hydrogenated protein crystals in quartz capillaries for neutron diffraction.
• Vapor exchange with D2O-containing buffer to replace hydrogen with deuterium, reducing background noise.
• Quasi-Laue data collection at IMAGINE (HFIR) and cryo-data collection at MaNDi (SNS).
• Preparation of model coordinate and diffraction data files, and visualization of neutron scattering length density (SLD) maps.
• Structure refinement using neutron-only or joint X-ray/neutron data.

Main Results:

• Successful demonstration of mounting and deuterium exchange for protein crystals.
• Illustration of room temperature and cryo-data collection strategies for neutron diffraction.
• Detailed workflow for obtaining all-atom structures using neutron crystallography.
• Application of the method to determine the neutron structure of *Neurospora crassa* LPMO9D.

Conclusions:

• Neutron crystallography provides a powerful method for determining hydrogen atom positions in biological macromolecules without causing radiation damage.
• The presented workflow enables comprehensive structural analysis, including labile intermediates, using neutron diffraction.
• This technique is vital for understanding the mechanisms of metalloproteins like LPMO9D involved in polysaccharide degradation.