Name: neutron crystallography data collection and processing for modelling hydrogen atoms in protein structures
Uploaded: 2020-12-01T15:00:00
Duration: 10 min 10 s
Description: Watch this Scientific Journal Video about Neutron Crystallography Data Collection and Processing for Modelling Hydrogen Atoms in Protein Structures at JoVE.com

Protein expression, purification and crystallization experiments were conducted at the Center for Structural Molecular Biology (CSMB), a U.S. Department of Energy Biological and Environmental Research User Facility at Oak Ridge National Laboratory. Neutron diffraction data was collected at BL-11B MaNDi at the Spallation Neutron Source (SNS) at ORNL which is sponsored by the Scientific User Facilities Division, Office of Basic Energy Sciences, U.S. Department of Energy. The authors thank Brendan Sullivan for assistance with data reduction. X-ray diffraction data was collected at the Molecular Education, Technology, and Research Innovation Center (METRIC) facilities at North Carolina State University, which is supported by the State of North Carolina. GCS acknowledges support in part from the National Research Foundation (NRF), South Africa and the Graduate Opportunities (GO!) program at ORNL. FM acknowledges support from USDA NIFA Hatch 211001.

The authors have nothing to disclose.

Neutron protein crystallography is a highly sensitive technique to probe protonation states and water molecule orientation in proteins. This information sheds light on protein catalytic mechanisms since changes in protonation and hydrogen bonding interactions are often central to enzyme chemistry10. Neutron protein crystallography, albeit an informative technique, has a number of factors that should be taken into consideration before planning to conduct a neutron diffraction experiment, namely:
<ol>
	<li>The requirement for large protein crystals for data collection.</li>
	<li>The scattering properties of hydrogen and other elements, such as metal ions.</li>
	<li>Limitations in the structure refinement and model building software when working with deuterated samples.</li>
</ol>
Neutron protein crystallography is a flux limited technique. In contrast to X-ray diffraction datasets, higher R-factors and lower completeness, redundancy and signal-to-noise ratios are expected for neutron datasets due to the technique inherent limitations (flux limited, quasi-Laue, longer wavelengths). Data collection of a single frame is typically 12 &#8211; 18 hours. Success of an experiment is highly dependent on sample size and quality with crystals of 0.1 mm3 often being the minimum requirement3. Neutron diffraction requires production of large amounts of protein to set up crystallization drops ranging from 10 to 800 &#956;L. The minimum volume for growing sufficiently large crystals can be estimated using a Volume Calculator given the crystal and sample parameters (https://neutrons.ornl.gov/imagine). Growth of large crystals has most prevalently been accomplished by vapor diffusion3. Hanging drop crystallization permits growth of crystals in large drops ranging from 10-25 &#956;L, while larger drops ranging up to ~ 50 &#956;L can be set up using commercially available sitting drop equipment14,54. Siliconized nine-well glass plates can be used to set up very large drops, with volumes up to 800 &#956;L. These glass plates are placed in &#8220;sandwich boxes&#8221; commercially available from Hampton Research. Further crystallization techniques include batch crystallization, in which the limit of the drop size is dictated by the vessel. Batch crystallization experiment set up can range from microliters to milliliters55. Crystallization can also be performed using the dialysis technique in which the protein is equilibrated with the precipitant via a dialysis membrane or by counter-diffusion along a precipitant concentration gradient or through a porous plug such as agarose56,57. Seeding offers another alternative to obtain crystals of the desired volume. Micro- and macroseeding have been successfully employed for large crystal growth, including large crystal of NcLPMO9D45. Some knowledge of the protein phase diagram, including the influence of temperature on solubility, aid in large crystal growth.
When planning a neutron diffraction experiment, optimization of the protein preparation to maximize signal-to-noise ratio during diffraction data collection is essential7. To circumvent density cancellation and high incoherent scattering caused by H atoms, neutron SLD maps can be improved by exchanging H atoms for its isotope D, which possesses a positive coherent scattering length and low incoherent scattering length. To accomplish this, vapor exchange of the hydrogenated protein crystal against deuterated crystallization buffer is performed. This ensures H/D exchange of solvent molecules and the labile, titratable protein H atoms23. Vapor exchange is performed by mounting the hydrogenated crystal in a quartz capillary with D2O-based,&#160;deuterated crystallization buffer &#8220;plugs&#8221; and it represents an effective, gentle technique that is most often applied14,23,35. The exchange can take several weeks and preferably requires the deuterated buffer to be frequently changed to ensure maximum H/D exchange. H/D exchange can also be performed by directly soaking the crystal in deuterated buffer. To avoid placing the crystal under stress due to D2O exposure, the soaking process should be performed gradually by incrementally increasing the D2O:H2O ratio58. In addition to this, crystallization of hydrogenated protein can also be performed in deuterated buffer for H/D exchange at labile H sites22,59. It should be noted, however, that D2O-based buffer has an effect on protein solubility requiring further adjustment of the known H2O-based conditions3,59. D2O-based buffers have also been observed to lead to smaller crystals in some cases59. Full exchange of titratable and carbon-bound H atoms to D can be achieved by expressing proteins in deuterated media to generate a perdeuterated sample20. The resulting neutron SLD maps of the perdeuterated sample will be significantly improved, no longer displaying the density cancellation of the hydrogenated sample counterpart. This is beneficial when characterizing H/D bound at non-exchangeable sites in a protein or cofactor. However, expression of perdeuterated protein is both high in cost and low in yield60. The Oak Ridge National Laboratory (ORNL) Center for Structural Molecular Biology (CSMB) offers a deuteration facility for users seeking to generate a perdeuterated sample (https://www.ornl.gov/facility/csmb). Perdeuterated expression is typically performed in a bioreactor on the 1 L scale yielding ~50 mg of purified protein61.
Following the collection of neutron diffraction data, refinement and interactive model building is performed. Refinement can be run using multiple software suites including phenix.refine, nCNS or SHELXL28,31,32,33. The Phenix suite is the most commonly utilized software for refinement of neutron diffraction data in conjunction with Coot which is used to manually build the model from the neutron SLD maps34. Although both Phenix and Coot allow for the processing of neutron diffraction data, they may lack certain features necessary to process the idiosyncrasies associated with neutron data and deuterated samples. For example, Coot does not contain geometry optimization for deuterated residues, which can lead to complications during model building since the &#8220;Real Space Refine&#8221; feature results in &#8220;exploding&#8221; residues (Supplementary Figure 26)62. This can be resolved by generating restraint files for all the deuterated residues. However, this is an intensive process and such libraries are not currently publicly available. When performing refinements in Phenix, exchangeable H/D sites will initially be set to 0.50 occupancy for H and D. As refinements are performed, the occupancy of H and D will be refined according to the neutron SLD maps. During interactive model building, difference density Fo-Fc maps are very informative in assessing H/D occupancies. Maps can be used to determine which sites possess high D occupancy, which is particularly informative at the active site where protonation states are catalytically relevant63. Ambiguous situations do arise, however, when the H:D occupancy is close to 0.70:0.30 which results in complete signal cancellation in neutron SLD maps64. It should also be taken into account that quasi-Laue neutron data sets often have completeness around 80%, which is lower than the routinely observed &#8805; 98% for X-ray diffraction data. When refining neutron diffraction data in Phenix, the missing observed amplitudes (Fo) are therefore calculated from the model to complete the reflection list, thus introducing model bias. To account for this potential bias &#8220;no_fill&#8221; maps should be examined during interactive model building as opposed to &#8220;filled&#8221; maps.
Users can choose to perform a joint X-ray/neutron data refinement of their structure, or a neutron-data only refinement. Visualizing neutron SLD maps, particularly at lower resolution, may initially be disconcerting especially for a hydrogenated protein in which H is still present at non-exchangeable sites despite H/D vapor exchange. This results in neutron density map cancellations, giving the impression of discontinuous maps65,66. Collecting a corresponding X-ray dataset advantageously complements these cancellations in a joint refinement (Figure 13A and Figure 13B). A joint-refinement strategy typically involves refining the protein backbone coordinates against the X-ray data, while the neutron diffraction data is used to refine the position and occupancy of the H/D atoms at exchangeable sites28. Since introduction of joint H/D occupancy at exchangeable sites increases the number of parameters being refined, a joint refinement with X-ray data also increases the data-to-parameter ratio. A joint refinement requires a corresponding X-ray dataset to be collected at the same temperature on the same crystal or a crystal grown under the same conditions. For neutron diffraction data collected at room temperature (300K), the corresponding X-ray dataset should be collected at room temperature using a low-dose data collection strategy to limit radiation damage. Perdeuterated samples, in contrast, provide improved and continuous neutron SLD maps since they do not possess the same magnitude of H/D signal cancellation. However, the neutron scattering length of certain elements including metals and sulfur make them poorly visible in neutron SLD maps, even if the protein has been perdeuterated (Figure 13C-F)18. If a metal needs to be characterized, it is best to utilize X-ray diffraction in a joint refinement or apply spectroscopic techniques to complement diffraction experiments. Neutron-only data refinements are often performed when the neutron dataset has high resolution or if a perdeuterated protein was used. In addition, neutron-only data refinement is particularly useful if a protein highly sensitive to radiation damage is being studied, since an X-ray derived structure may possess radiation-induced artefacts. If a neutron-data-only refinement is to be performed, it must be ascertained whether the corresponding neutron dataset has sufficient completeness and resolution.
ORNL offers two facilities for collection of neutron diffraction data: the IMAGINE beamline at the HFIR as well as the MaNDi beamline at the SNS36,67. While both instruments provide effective means for collecting a neutron diffraction dataset employing similar principles, each instrument has unique specifications that should be taken into account when applying for beam time. IMAGINE collects quasi-Laue data and is optimized for room temperature data collection on crystals with unit cells up to ~100 &#197;. MaNDi can be used for the collection of room temperature and cryo-temperature data employing TOF-Laue collection on crystals with unit cells up to ~ 300 &#197;. Prior to collecting a complete dataset, a test is performed on the crystal to evaluate the quality of the obtained diffraction pattern in which the crystal is exposed to the neutron beam for a single frame. If the crystal is of sufficient quality, a full neutron diffraction dataset will be collected, indexed, integrated, scaled and merged in a process analogous to X-ray data processing. IMAGINE makes use of Lauegen and Lscale and MaNDi utilizes the Mantid package and employs three-dimensional profile fitting48,50,51,68,69,70. Scientists who become users at either of these facilities will be provided with a dataset in MTZ or HKL format for further analysis.
Neutron diffraction is a non-destructive, highly sensitive technique for probing the protonation state and hydrogen bond interactions of biological macromolecules. It is particularly useful for photo-sensitive proteins and metalloproteins. Several considerations regarding the technique as well as the processing of the data must be taken into account&#160;before conducting an experiment, however the outcome yields results which may give valuable insight into the catalytic mechanism of the protein of interest. Neutron protein crystallography complements computational, structural, biochemical and spectroscopic studies, making it a valuable tool in the biologist&#8217;s toolbox of techniques used to characterize biological macromolecules.

Neutron macromolecular crystallography is a technique that provides a unique window into the structure and underlying chemistry of proteins. Conceptually similar to X-ray diffraction, neutron diffraction provides atomistic details of macromolecular structure, however, the interaction of neutrons with nuclei enables localization of light atoms, often difficult to detect with X-ray diffraction1. During X-ray diffraction, X-rays scatter from the electron cloud, making light atoms such as hydrogen (H) poorly visible in electron density maps that do not have near sub-&#197;ngstr&#246;m resolution2. In contrast, the scattering intensity of neutrons depends on complex interactions with the nucleus, with isotopes of the same element displaying different scattering lengths. Therefore, light atoms and their isotopes, such as hydrogen (1H) and deuterium (2H or D), have comparable visibility to the backbone carbon, nitrogen and oxygen atoms in neutron scattering length density (SLD) maps. Furthermore, since the magnitude of neutron scattering is independent of the number of electrons, scattering from light elements is not obscured by heavy elements when they are in close vicinity to each other, as is observed in X-ray scattering. The enhanced visibility of H and its isotope D when employing neutron diffraction provides valuable information about the protonation state of catalytically important residues, cofactors and ligands and aids the orientation of water molecules, revealing important information about catalytic mechanisms and protein chemistry3. Neutron diffraction also offers the advantage of being a non-destructive technique, particularly suited to biological samples sensitive to ionization such as proteins with metal centers or photosensitive redox cofactors2. The primary focus of this article is to provide an overview of the workflow to obtain a high-quality neutron protein crystal&#160;structure.&#160;We refer the interested reader to Podjarny et al.4, Blakeley5, Blakeley et al.6 and O&#8216;Dell et al.3 for an excellent overview of neutron protein diffraction and Ashkar et al.7 for further applications of neutron scattering.
Neutrons are primarily generated during nuclear reactions employing either of two processes: nuclear fission at reactor sources or spallation at accelerator-based sources8. Reactor sources provide a continuous neutron beam by employing nuclear fission of the 235U isotope while spallation neutron sources produce a pulsed neutron beam by bombarding a target, for example a liquid metal such as mercury, with protons9. Oak Ridge National Laboratory (ORNL) in Oak Ridge, Tennessee, hosts both a steady-state neutron source at the High Flux Isotope Reactor (HFIR) and a 60 Hz pulsed source&#160;at the Spallation Neutron Source (SNS). The IMAGINE beamline, located at the HFIR, is a neutron diffractometer optimized for biological macromolecules (Supplementary Figure 1)10. IMAGINE employs a neutron image plate detector to measure quasi-Laue data using a narrow bandpass in the range of 2.8 &#8211; 4.5 &#197; from single crystals with unit cell edges &#60;150 &#197;. The Macromolecular Neutron Diffractometer (MaNDi), located at the SNS, is a time-of-flight (TOF) Laue neutron diffractometer equipped with a spherical detector array frame (DAF) (Supplementary Figure 2)11. MaNDi measures data from single crystals with unit cell edges in the range of 10 &#8211; 300 &#197; by employing a tunable 2 &#197;-wavelength bandwidth between 2.0 &#8211; 6.0 &#197;12.
The process of generating neutrons is highly energy intensive, resulting in relatively weak neutron beam fluxes when contrasted to X-ray beam fluxes at synchrotron sources13. To ensure sufficient signal-to-noise ratios during data collection, it is necessary to grow crystals of suitable size and quality14. Typically, crystals with volumes &#62; 0.1 mm3 are needed to collect data with adequate statistics15. In addition to lower fluxes, inherent properties of the interaction between neutrons and the sample nuclei must be taken into consideration16. The scattering length of neutrons differs for isotopes of the same element, a property which can be advantageously exploited in small angle neutron scattering (SANS) to mask or highlight regions of a sample &#8211; a process known as contrast matching17. In diffraction experiments, the negative coherent neutron scattering length of H (-3.741 fm for 1H) can lead to cancellation of neutron scattering density map features since the coherent neutron scattering lengths of other biologically relevant atoms, including carbon (6.6511 fm for 12C), nitrogen (9.37 fm for 14N), oxygen (5.803 fm for 16O), phosphorus (5.13 fm for 31P) and sulfur (2.804 fm for 32S), are positive (Table 1)12,14. Furthermore, the large incoherent scattering length of H (25.274 fm), increases the background during data collection, hampering the quality of the dataset and compromising data resolution7. To circumvent these limitations introduced by H it is necessary, for neutron diffraction, to exchange H for its isotope deuterium, 2H(D), which has a positive coherent neutron scattering length (6.671 fm) and significantly lower incoherent scattering length (4.04 fm)19. This can be achieved by perdeuteration, a process in which the protein is expressed by organisms grown in fully deuterated media ensuring complete incorporation of D at H sites20. It is also possible to partially deuterate protein by replacing H with D solely at the exchangeable sites (titratable groups) while the non-exchangeable carbon-bound sites remain hydrogenated21. This can be achieved by growth of hydrogenated protein crystals in deuterated mother liquor22. Most commonly however, H/D exchange of hydrogenated proteins is performed by vapor exchange following growth of suitably large crystals in H2O-based buffer23. In such cases, crystals are mounted in a quartz capillary and vapor-equilibrated with a D20-based&#160;mother liquor.
The limited neutron fluxes at neutron sources result in longer data collection times, ranging from days to several weeks24. At ORNL, both IMAGINE and MaNDi employ a narrow wavelength bandpass in the 2&#8211;6 &#197; range to optimize data collection25. Data can be collected at room temperature&#160;or at cryo-temperature. Cryo-data collection can potentially&#160;improve data quality and opens up the possibility for freeze-trapping catalytic intermediates. Following neutron diffraction data collection, an X-ray dataset is typically collected on the same crystal at the same temperature or on a crystal grown under identical conditions26. Data collection at the same temperature allows structure refinement to be performed against both X-ray and neutron data, preventing any potential temperature-induced artefacts such as changes in the visibility and position of waters or the occupancies of residues with alternate conformations27. Joint X-ray neutron data refinement increases the data-to-parameter ratio and provides the advantage of allowing the protein backbone coordinates to be refined against the X-ray data, while the neutron diffraction data is used to refine the position of the H/D atoms28. This is particularly useful when using partially deuterated samples, where&#160;density cancellation due to H atoms at non-exchangeable sites on the protein is present. Although the number of X-ray structures far exceeds the number of neutron structures deposited in the Protein Data Bank (PDB), software packages initially designed for the refinement of X-ray data have been expanded to encompass neutron data as well3,29,30. Following data collection, models can be refined using refinement packages such as phenix.refine, CNSsolve (nCNS) or SHELXL28,31,32,33. During the refinement process, neutron scattering density maps can be visualized for manual fitting using COOT34. Following structure solution, the coordinates and the neutron and/or X-ray diffraction&#160;data files can be submitted to the PDB, who will validate and deposit the model, making it available for public access18,29,30.
Structural analysis of proteins is a multifaceted approach in which numerous techniques are used to probe their function and mechanism35. Neutron protein crystallography provides valuable chemical insights to expand on and complement findings from additional studies such as X-ray&#160;diffraction, spectroscopy, nuclear magnetic resonance (NMR) or micro crystal electron diffraction (microED)36. Neutron protein diffraction is uniquely positioned to provide insights into enzymatic mechanisms, since H atoms are central to their chemistry. The absence of radiation damage induced by neutrons make them a probe exceptionally suited to the study of metalloproteins37. We present here a representative example of the process of neutron protein diffraction from sample preparation to data collection, refinement and analysis (Figure 1). Crystals of sufficient size for neutron diffraction experiments have been grown of the metalloprotein Neurospora crassa LPMO9D (NcLPMO9D). NcLPMO9D is a copper-containing metalloprotein involved in the degradation of recalcitrant cellulose by oxygen atom insertion at the glycosidic bond38,39. The NcLPMO9D active site contains a mononuclear copper center within a characteristic &#8220;histidine-brace&#8221; composed of the N-terminal histidine and a second conserved histidine (Supplementary Figure 3)40. The N-terminal of fungal LPMOs is methylated but the post-transitional modification does not occur during recombinant expression in yeast. In the NcLPMO9D resting state, the copper center is present in a Cu2+ oxidation state and is activated by single electron reduction to Cu1+, allowing molecular oxygen to bind and be activated by rapidly being reduced to a superoxide species41,42. The overall NcLPMO9D reaction requires further addition of one electron and two protons to form the hydroxylated polysaccharide product43. The identity of the activated oxygen species responsible for hydrogen atom abstraction (HAA) from the polysaccharide substrate has not been identified and intensive structural and computational studies are currently ongoing44,45. Given the redox chemistry at the NcLPMO9D active site, mitigation of radiation damage is particularly pertinent. We illustrate here room temperature and cryo-temperature data collection on NcLPMO9D crystals to determine the NcLPMO9D&#160;structure in the resting state and in the&#160;activated reduced form, respectively46. Emphasis will be given to protein crystal mounting, beamline instrument setup for data collection, the preparation of the data and coordinate files and the refinement steps necessary to model an all-atom neutron structure.

<table>
	<tbody>
		<tr>
			<td>Absorbent Paper Points Size #30-#40, 60 mm length</td>
			<td>DiaDent/DiaVet</td>
			<td>218-292</td>
			<td></td>
		</tr>
		<tr>
			<td>Capillary wax</td>
			<td>Hampton</td>
			<td>HR4-328</td>
			<td></td>
		</tr>
		<tr>
			<td>CCP4</td>
			<td></td>
			<td>Version 7.0.077</td>
			<td></td>
		</tr>
		<tr>
			<td>Conical Centrifuge Tubes (15 mL)</td>
			<td>Corning</td>
			<td>CLS430790</td>
			<td></td>
		</tr>
		<tr>
			<td>Conical Centrifuge Tubes (50mL)</td>
			<td>Corning</td>
			<td>CLS430828</td>
			<td></td>
		</tr>
		<tr>
			<td>Coot</td>
			<td></td>
			<td>Version 0.8.9.2</td>
			<td></td>
		</tr>
		<tr>
			<td>CrystalCap ALS</td>
			<td>Hampton</td>
			<td>HR4-779</td>
			<td></td>
		</tr>
		<tr>
			<td>Curved-Tip Forceps</td>
			<td>Mitegen</td>
			<td>TW-CTF-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Deuterium chloride solution, 35 wt. % in D2O, &#8805;99 atom % D</td>
			<td>Sigma-Aldrich</td>
			<td>543047</td>
			<td></td>
		</tr>
		<tr>
			<td>Deuterium oxide 99.9 atom % D</td>
			<td>Sigma-Aldrich</td>
			<td>151882</td>
			<td></td>
		</tr>
		<tr>
			<td>Dual Thickness MicroLoops 1000 &#181;m</td>
			<td>Mitegen</td>
			<td>M5-L18SP-1000</td>
			<td></td>
		</tr>
		<tr>
			<td>FiveEasy pH meter F20-Std-Kit</td>
			<td>Mettler Toledo</td>
			<td>30266626</td>
			<td></td>
		</tr>
		<tr>
			<td>Foam Dewars Standard Vessel 800 ml</td>
			<td>Spearlab</td>
			<td>M-FD-800</td>
			<td></td>
		</tr>
		<tr>
			<td>Four Color Mounting Clay</td>
			<td>Hampton</td>
			<td>HR4-326</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES, BioUltra, for molecular biology, &#8805;99.5% (T),</td>
			<td>Sigma-Aldrich</td>
			<td>54457</td>
			<td></td>
		</tr>
		<tr>
			<td>High flux rotating anode X-ray diffractomemeter with EIGER 4M detector</td>
			<td>Rigaku, Oxford Cryostream and Dectris</td>
			<td>XtaLAB Synergy-R</td>
			<td>Home source X-ray diffractometer</td>
		</tr>
		<tr>
			<td>Magnetic Wand Straight</td>
			<td>Mitegen</td>
			<td>M-R-1013198</td>
			<td></td>
		</tr>
		<tr>
			<td>Microloader, tip for filling Femtotips and other glass microcapillaries (for research use only), 0.5 &#8211; 20 &#181;L, 100 mm, light gray, 192 pcs. (2 racks &#215; 96 pcs.)</td>
			<td>Eppendorf</td>
			<td>930001007</td>
			<td></td>
		</tr>
		<tr>
			<td>Microtubes volume 1.5 mL</td>
			<td>Eppendorf</td>
			<td>Z606340</td>
			<td></td>
		</tr>
		<tr>
			<td>Petri Dishes with Clear Lid 100 mm diameter</td>
			<td>Fischerbrand</td>
			<td>FB0875713</td>
			<td></td>
		</tr>
		<tr>
			<td>Phenix</td>
			<td></td>
			<td>Version 1.14-3260</td>
			<td></td>
		</tr>
		<tr>
			<td>Pin Tong 18 mm</td>
			<td>Mitegen</td>
			<td>M-R-1013196</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipette Volume 0.1-2.5 &#956;L</td>
			<td>Eppendorf Research</td>
			<td>Z683779</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipette Volume 100-1000 &#956;L</td>
			<td>Eppendorf Research</td>
			<td>Z683825</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipette Volume 10-100 &#956;L</td>
			<td>Eppendorf Research</td>
			<td>Z683809</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipette Volume 20-200 &#956;L</td>
			<td>Eppendorf Research</td>
			<td>Z683817</td>
			<td></td>
		</tr>
		<tr>
			<td>Poly(ethylene glycol) BioXtra, average mol wt 3,350, powder</td>
			<td>Sigma-Aldrich</td>
			<td>P4338</td>
			<td></td>
		</tr>
		<tr>
			<td>Quartz Capillary , 1.00 mm inner diameter, 80 mm length</td>
			<td>Hampton</td>
			<td>HR6-146</td>
			<td>Thin-walled capillary</td>
		</tr>
		<tr>
			<td>Research Stereomicroscope System</td>
			<td>Olympus</td>
			<td>SZX16</td>
			<td></td>
		</tr>
		<tr>
			<td>Reusable B3 (SSRL/SAM Style) Goniometer Bases</td>
			<td>Mitegen</td>
			<td>GB-B3-R</td>
			<td></td>
		</tr>
		<tr>
			<td>Round - Miniature Hollow Glass Tubing (VitroTubes) Clear Fused Quartz / 1.00 mm inner diameter, 100 mm length</td>
			<td>VitroCom</td>
			<td>CV1012</td>
			<td>Thick-walled capillary</td>
		</tr>
		<tr>
			<td>Sandwich Box with cover</td>
			<td>Hampton</td>
			<td>HR3-132</td>
			<td></td>
		</tr>
		<tr>
			<td>Siliconized 9 Well Glass Plate</td>
			<td>Hampton</td>
			<td>HR3-134</td>
			<td></td>
		</tr>
		<tr>
			<td>Sitting Drop Crystallization Plate (24 Big Well)</td>
			<td>Mitegen</td>
			<td>XQ-P-24S-A</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium deuteroxide solution, 40 wt. % in D2O, 99 atom % D</td>
			<td>Sigma-Aldrich</td>
			<td>176788</td>
			<td></td>
		</tr>
		<tr>
			<td>Thick Siliconized circle cover slides (22 mm x 0.96 mm)</td>
			<td>Hampton</td>
			<td>HR3-247</td>
			<td></td>
		</tr>
		<tr>
			<td>Universal Pipet Tips, 0.1 - 10 &#181;L</td>
			<td>VWR</td>
			<td>76322-528</td>
			<td></td>
		</tr>
		<tr>
			<td>Universal Pipet Tips, 1 - 100 &#181;L</td>
			<td>VWR</td>
			<td>76322-136</td>
			<td></td>
		</tr>
		<tr>
			<td>Universal Pipet Tips, 100 - 1000 &#181;L</td>
			<td>VWR</td>
			<td>76322-154</td>
			<td></td>
		</tr>
		<tr>
			<td>Universal Pipet Tips, 20 - 200 &#181;L</td>
			<td>VWR</td>
			<td>76322-150</td>
			<td></td>
		</tr>
		<tr>
			<td>Universal Pipet Tips, 1 - 20 &#181;L</td>
			<td>VWR</td>
			<td>76322-134</td>
			<td></td>
		</tr>
		<tr>
			<td>Wax pen</td>
			<td>Hampton</td>
			<td>HR4-342</td>
			<td></td>
		</tr>
	</tbody>
</table>

neutron crystallography data collection and processing for modelling hydrogen atoms in protein structures

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&#160;rapidly induces radiation damage of photosensitive&#160;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 (&#62; 0.1 mm3) has been grown. We demonstrate mounting of hydrogenated protein&#160;crystals in quartz capillaries for neutron diffraction data collection. Also presented is the vapor exchange process of the mounted crystals with D2O-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-of-flight 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 data-only 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.

Watch this Scientific Journal Video about Neutron Crystallography Data Collection and Processing for Modelling Hydrogen Atoms in Protein Structures at JoVE.com

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

Neutron protein crystallography is a structural technique that permits the localization of hydrogen atoms, thereby providing important mechanistic details of protein function. We present here the workflow for mounting a protein crystal, neutron diffraction data collection, structure refinement and analysis of the neutron scattering length density maps.

Neutron crystallography is a structural technique that allows determination of hydrogen atom positions within biological macromolecules, yielding ...

Research

JoVE Journal

Chemistry

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