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

Abstract

Neutron crystallography is a structural technique that allows determination of hydrogen atom positions within biological macromolecules, yielding mechanistically important information about protonation and hydration states while not inducing radiation damage. X-ray diffraction, in contrast, provides only limited information on the position of light atoms and the X-ray beam rapidly induces radiation damage of photosensitive cofactors and metal centers. Presented here is the workflow employed for the IMAGINE and MaNDi beamlines at Oak Ridge National Laboratory (ORNL) to obtain a neutron diffraction structure once a protein crystal of suitable size (> 0.1 mm 3 ) has been grown. We demonstrate mounting of hydrogenated protein crystals in quartz capillaries for neutron diffraction data collection. Also presented is the vapor exchange process of the mounted crystals with D 2 O-containing buffer to ensure replacement of hydrogen atoms at exchangeable sites with deuterium. The incorporation of deuterium reduces the background arising from the incoherent scattering of hydrogen atoms and prevents density cancellation caused by their negative coherent scattering length.Sample alignment and room temperature data collection strategies are illustrated using quasi-Laue data collection at IMAGINE at the High Flux Isotope Reactor (HFIR). Furthermore, crystal mounting and rapid freezing in liquid nitrogen for cryo-data collection to trap labile reaction intermediates is demonstrated at the MaNDi time-offlight instrument at the Spallation Neutron Source (SNS). Preparation of the model coordinate and diffraction data files and visualization of the neutron scattering length density (SLD) maps will also be addressed. Structure refinement against neutron dataonly or against joint X-ray/neutron data to obtain an all-atom structure of the protein of interest will finally be discussed. The process of determining a neutron structure will be demonstrated using crystals of the lytic polysaccharide monooxygenase Neurospora crassa LPMO9D, a copper-containing metalloprotein involved in the degradation of recalcitrant polysaccharides via oxidative cleavage of the glycosidic bond.

Development of an FeII Complex Exhibiting Intermolecular Proton Shifting Coupled Spin Transition

“…The availability of neutron data considerably enhances the precision of studying hydrogen-involved structural changes by improving the accuracy of determining atomic positions and thermal parameters. [29] Single-crystal neutron diffraction measurements of 1 were conducted at 300 and 210 K. Figure 2 shows the obtained structures, and Table 1 lists the hydrogen bond parameters. Notably, the bond lengths of and HB-2 did not significantly change with temperature.…”

Development of an FeII Complex Exhibiting Intermolecular Proton Shifting Coupled Spin Transition

“…The availability of neutron data considerably enhances the precision of studying hydrogen-involved structural changes by improving the accuracy of determining atomic positions and thermal parameters. [29] Single-crystal neutron diffraction measurements of 1 were conducted at 300 and 210 K. Figure 2 shows the obtained structures, and Table 1 lists the hydrogen bond parameters. Notably, the bond lengths of HB-1 and HB-2 did not significantly change with temperature.…”

Mapping H+ in the Nanoscale (A2C4)2-Ag8 Fluorophore

“…33 To directly identify these hydrogens, isotope exchange with D 2 O was used because deuterium scatters neutrons more efficiently than hydrogen. 34 Neutron scattering length density (NSLD) maps show that the former exocyclic amines are fully protonated/deuterated and thus capable of hydrogen bonding (Figure 1C). The combination of both silver and hydrogen bonds supports a metal-mediated sub-duplex within the overall (A 2 C 4 ) 2 -Ag 8 complex.…”

“…On the basis of inferred hydrogen positions in the X-ray diffraction data, the measured N–H/O bond distances of 1.9–2.5 Å are consistent with hydrogen bonds . To directly identify these hydrogens, isotope exchange with D 2 O was used because deuterium scatters neutrons more efficiently than hydrogen . Neutron scattering length density (NSLD) maps show that the former exocyclic amines are fully protonated/deuterated and thus capable of hydrogen bonding (Figure C).…”