Neutron crystallography is a structural technique for determining hydrogen atom positions in biological macromolecules. Neutron structures reveal protonation states and water molecule orientations to elucidate reaction mechanisms and binding interactions. In contrast to x-ray diffraction, neutron diffraction has the advantage of being a non-destructive technique.
Proteins with photosensitive groups or metallosensors can be studied without suffering from radiation damage. To perform neutron protein crystallography, large fragile protein crystals are mounted in quartz capillaries for room temperature data collection. Crystal capillary mounting is not routinely performed, making demonstration of this technique instructive.
For crystal harvesting, open the sealed sandwich box containing the protein crystals in a nine-well large volume siliconized glass plate and use a micropipette to transfer 10 to 20 microliters from the crystallization reservoir solution onto a glass slide. Evaluate the crystals with a microscope. Use an appropriately sized microloop to harvest a crystal and place the crystal in the drop of reservoir solution.
For crystal mounting, take a two millimeter diameter 50 millimeter length quartz capillary, aspirate reservoir buffer with a pipette and fill one end of the capillary with reservoir buffer. Use a mounting loop to gently place the crystal into the reservoir buffer within the quartz capillary. Tap the tube to move the reservoir buffer and crystal down the capillary and use a long thin pipette to aspirate the solution from around the crystal.
Use a thin paper wick to carefully dry the crystal and dry the capillary walls and add 20 to 50 microliters of deuterated buffer solution to the end of the capillary. Use a heat wand to melt a portion of beeswax and gently insert the capillary in the melted beeswax until an airtight seal has formed. Replace the deuterated buffer with 20 to 50 microliters fresh deuterated buffer at days two, six, and 10 after crystal mounting and reseal the capillary with fresh melted beeswax after each vapor exchange as demonstrated.
After at least two weeks vapor exchange, use putty to secure the quartz capillary onto the IMAGINE neutron diffractometer goniometer sample stick that has been secured to the instrument sample stage and lower the sample and stick into the neutron beam and detector area. Open the data acquisition program on the Beamline control computer and click the setup tab to set up the data collection strategy and enter the experiment parameters including instrument name and the name of images to be collected. Click the collect tab and enter the data collection parameters including the exposure time and number of frames to be collected.
Within the optics graphic user interface, click 2.78 for the lambda minimum and 4.5 for the lambda maximum to set the quasi range for the data collection. Click start scan to initiate the data collection. After neutron data collection, collect a corresponding x-ray dataset on the same crystal at the same temperature.
Prior to cryo data collection, remove the reservoir solution from the sandwich box and fill the sandwich box with deuterated reservoir buffer solution, allow to equilibrate for one week and repeat three times. After three more rounds of reservoir solution exchange, harvest the crystal with a microloop and immerse the crystal in a cryoprotectant solution for two hours before mounting the crystal in a microloop attached to a cryo crystal mount. Plunge the mounted crystal and cryo mount into liquid nitrogen.
When crystal is frozen, mount the crystal on the macromolecular neutron diffractometer sample stage fitted with a cryo stream. Verify that the crystal is mounted and centered for data collection, complete the proposal information, and click the folder icon to load the data collection strategy table, then click submit to start the data collection. Diffracted neutrons will become visible in real time as they are detected by the MaNDi time-of-flight detectors.
For joint x-ray and neutron data refinement, first open CCP4 and select convert to modify extend MTZ program to match the R free data flags of the neutron data to those of the x-ray data. Import the neutron data reflection file in MTZ format. Select import free R data from another MTZ file and import the x-ray MTZ file.
Name the new matched MTZ file and click run. Next, open the Phoenix software package and under refinement, click ready set. Upload the protein coordinate file, select to add hydrogens to model if absent, and select HD at exchangeable sites, H elsewhere from the dropdown menu.
Select add deuterium to solvent molecules and click run to begin. For structure refinement, in the refinement tab, open the phoenix. refine program to set up the refinement using both the x-ray and neutron data.
In the configure tab, input the PDB file from the solved x-ray structure. Upload the MTZ file from the neutron data. Assign the MTZ file data as neutron data in neutron R free.
Upload the MTZ file from the x-ray data and assign it as x-ray data and x-ray R free. Under refinement settings, confirm that the standard refinement strategy is selected and increase the number of cycles to five. Select all parameters, advanced, and hydrogens.
Change the hydrogen refinement model to individual and turn off the force riding ADP, then search for nuclear. Select use the nuclear distances from X-HD. Click run to initiate the refinement.
For model building in Phoenix, click open in Coot. In Coot, visualize the x-ray electron density and neutron scattering length density maps. Select the display manager and delete the neutron 2FOFC neutron scattering length density map.
Select open MTZ and select open MTZ and open the neutron data mtz file. For both the amplitudes and phases options, select no_fill_neutron data from the dropdown menus to open the unfilled neutron scattering length density maps. Perform visual inspection of the residues to determine whether the model fits the data and analyze the difference density map peaks of hydrogen-deuterium exchangeable sites to determine the correct orientation and occupancy.
Reorient the water molecules according to the neutron SLD maps and hydrogen bond interactions. Adjust the protonation state and orientation of the protein residue hydrogen-deuterium exchangeable sites according to the neutron SLD maps. Perform further rounds of interactive model building and refinement to obtain a complete structure.
Hydrogenated protein crystals grown in water-based buffer measure approximately 1, 000 by 900 microns. The crystals can be mounted in quartz capillaries for vapor exchange with deuterium oxide-based buffer for three weeks before neutron diffraction data collection. Neutron diffraction data was collected for several days at 2.30 angstrom resolution and an x-ray diffraction dataset was collected on the same crystal.
Peaks in FO minus FC neutron scattering length density maps provide valuable information on the orientation of residues such asparagine and positive peaks in FO minus FC neutron scattering length density omit maps are also very informative in determining the protonation states of residues with titratable groups such as histidine. Map overlays of electron and neutron scattering length density maps for water molecules indicate that while hydrogen bond interactions can be inferred from x-ray data, neutrons provide clear information about the positions of these hydrogen bonds. Neutron scattering length density omit maps can be used to determine hydrogen-deuterium side chain functional group orientations.
Neutron scattering length density maps in which non-exchangeable hydrogen atoms are bonded to carbon appear incomplete when compared to their electron density map counterparts due to density cancellation. It is therefore preferable to perform a joint refinement of a sample with both x-ray and neutron data in which the x-ray data can be used to determine the position of the protein backbone. Neutron protein crystallography requires large crystals.
Care should be taken during crystal handling and mounting to avoid damaging the crystals which can easily crack, compromising data quality. Neutron protein crystallography provides insight into the protein reaction mechanism, potentially revealing catalytically relevant residues or water molecules whose role can be further probed by kinetics, mutagenesis, or spectroscopy.
