Neutron diffraction techniques permit direct determination of the hydrogen (H) and deuterium (D) positions in crystal structures of biological macromolecules at resolutions of ∼1.5 and 2.5 Å, respectively. In addition, neutron diffraction data can be collected from a single crystal at room temperature without radiation damage issues. By locating the positions of H/D-atoms, protonation states and water molecule orientations can be determined, leading to a more complete understanding of many biological processes and drug-binding. In the last ca. 5 years, new beamlines have come online at reactor neutron sources, such as BIODIFF at Heinz Maier-Leibnitz Zentrum and IMAGINE at Oak Ridge National Laboratory (ORNL), and at spallation neutron sources, such as MaNDi at ORNL and iBIX at the Japan Proton Accelerator Research Complex. In addition, significant improvements have been made to existing beamlines, such as LADI-III at the Institut Laue-Langevin. The new and improved instrumentations are allowing sub-mm3 crystals to be regularly used for data collection and permitting the study of larger systems (unit-cell edges >100 Å). Owing to this increase in capacity and capability, many more studies have been performed and for a wider range of macromolecules, including enzymes, signalling proteins, transport proteins, sugar-binding proteins, fluorescent proteins, hormones and oligonucleotides; of the 126 structures deposited in the Protein Data Bank, more than half have been released since 2013 (65/126, 52%). Although the overall number is still relatively small, there are a growing number of examples for which neutron macromolecular crystallography has provided the answers to questions that otherwise remained elusive.

X-rays and neutrons: hydrogen atoms and radiation damage

More than one hundred and twenty thousand macromolecular structures have now been determined using X-ray diffraction, contributing significantly to the comprehension of a vast array of ever-more complex biological systems and processes. The great majority (∼98%) of these structures, however, provide us with no, or very little, information relating to hydrogen (H), owing to the fact that, with only one electron, H-atoms scatter X-rays very weakly, while protons (H+) do not scatter X-rays at all. As such, details of H-bonding and protonation are most often inferred from the positions of the non-H-atoms. To directly locate the positions of some H-atoms in electron density maps, X-ray diffraction data must extend to atomic resolution (dmin ≤ 1.2 Å) or better [1,2]. However, more mobile H-atoms (atomic displacement parameters approximately >10 Å2) are generally not located (e.g. water molecule H-atoms) even when ultra-high-resolution X-ray data (dmin ≤ 0.8 Å) are available [37].

Scatter plot of the molecular mass of the biological macromolecules deposited in the PDB, which were refined using neutron diffraction data versus the deposition year.

Figure 1.
Scatter plot of the molecular mass of the biological macromolecules deposited in the PDB, which were refined using neutron diffraction data versus the deposition year.

As can be seen, in recent years much larger systems are being successfully studied, and in general, the number of structures being deposited each year is increasing.

Figure 1.
Scatter plot of the molecular mass of the biological macromolecules deposited in the PDB, which were refined using neutron diffraction data versus the deposition year.

As can be seen, in recent years much larger systems are being successfully studied, and in general, the number of structures being deposited each year is increasing.

The basic principles of neutron scattering are similar to those of X-ray scattering in many respects [8], but the key difference is that for neutrons the scattering centers are the atomic nuclei rather than the electrons. The total scattering by a nucleus is the sum of the coherent and incoherent scattering terms; the coherent scattering gives rise to Bragg reflections, while the incoherent scattering produces a uniform background radiation. The coherent scattering lengths show no correlation to the number of electrons, but rather depend on the nuclear forces, which can even vary between different isotopes of the same element. Positive values of coherent scattering length b reflect a 180° change in phase between the incident and the scattered neutron waves, while negative values of b are associated with a resonance level that produces an extra 180° phase shift. Hydrogen, and in particular its isotope deuterium (2H, D), are more readily located using neutrons than X-rays [9,10] because their respective neutron coherent scattering lengths are comparable to those of the other atoms of a macromolecule (Table 1).

Table 1
Neutron coherent scattering lengths and incoherent scattering cross sections, and X-ray scattering lengths, for the common elements of a macromolecule
Isotope Atomic number Neutron incoherent cross section (barns, 1 barn = 10−24 cm2Neutron coherent scattering length, b (10−12 cm) X-ray scattering lengths (10−12 cm) 
sin θ = 0 sin θ/λ = 0.5 Å−1 
180.27 −0.374 0.28 0.02 
2H (D) 2.05 0.667 0.28 0.02 
120.00 0.665 1.69 0.48 
140.50 0.937 1.97 0.53 
160.00 0.580 2.25 0.62 
24Mg 12 0.00 0.549 3.38 1.35 
3216 0.00 0.280 4.50 1.90 
3919 0.25 0.379 5.30 2.20 
55Mn 25 0.40 −0.375 7.00 3.10 
56Fe 26 0.00 1.010 7.30 3.30 
Isotope Atomic number Neutron incoherent cross section (barns, 1 barn = 10−24 cm2Neutron coherent scattering length, b (10−12 cm) X-ray scattering lengths (10−12 cm) 
sin θ = 0 sin θ/λ = 0.5 Å−1 
180.27 −0.374 0.28 0.02 
2H (D) 2.05 0.667 0.28 0.02 
120.00 0.665 1.69 0.48 
140.50 0.937 1.97 0.53 
160.00 0.580 2.25 0.62 
24Mg 12 0.00 0.549 3.38 1.35 
3216 0.00 0.280 4.50 1.90 
3919 0.25 0.379 5.30 2.20 
55Mn 25 0.40 −0.375 7.00 3.10 
56Fe 26 0.00 1.010 7.30 3.30 

An important difference between X-rays and neutrons is that while X-rays are ionizing radiation that cause damage to the crystals, mostly through the generation of free radicals, neutrons are neutral particles that (at the energies/wavelengths used for neutron macromolecular crystallography) are a nondestructive probe. X-ray radiation damage can lead to significant changes in the structure, and so, diffraction data have been routinely collected at 100 K in order to minimize the damage. However, while X-ray radiation damage at 100 K can be largely controlled via the use of multiple crystals, limited doses or crystal translation at microfocus beams, it nevertheless still takes place and remains a limiting factor for structure solution [11]. Moreover, it is a widely agreed objective to seek structural results as near to physiological temperatures as possible, and so recently, serial crystallography experiments at room temperature (RT) — in which diffraction patterns are recorded one at a time, each from a fresh crystal delivered to the beam in a random and unknown orientation — have been performed at synchrotron radiation and X-ray free electron (XFEL) sources. It has been shown that by using the very short X-ray pulses (i.e. a few tens of fs) from an XFEL allows ‘diffraction-before-destruction’ at RT with no significant radiation damage [12]. Although an exciting development, a consequence of this approach is that a very large number of microcrystals (i.e. many thousands) are required to collect a complete dataset. In the case of neutron diffraction, data can be collected from a single crystal (albeit much larger) at RT, without radiation damage issues.

H/D exchange and perdeuteration

In neutron scattering studies, experiments are often performed which exploit the benefits H/D isotopic replacement can provide. This is the case for macromolecular crystallography, where it is advantageous to replace H-atoms by D-atoms because deuterium has both a lower incoherent scattering cross section and a larger coherent scattering length than hydrogen (Table 1). Since H-atoms constitute around half of the atoms of a biological macromolecule, the signal-to-noise ratio of neutron diffraction data collected from non-exchanged H-crystals tends to be limited due to the large incoherent scattering signal arising from the H-atoms. H/D isotopic replacement lowers the incoherent background, and as the coherent scattering length of deuterium (+0.667 × 10−12 cm) is both positive and approximately twice that of hydrogen (−0.374 × 10−12 cm), it also enhances the coherent scattering signal, thereby improving the signal-to-noise ratio.

Neutron studies have been most commonly performed using H/D-exchanged crystals [1369]; 96 of the 126 structures (76%) deposited in the Protein Data Bank (PDB) arise from data collected from H/D-exchanged crystals. H/D exchange can be achieved by vapor exchange or by soaking in, or growing from, D2O solutions, and allows exchange of solvent-accessible H-atoms attached to oxygen (O), nitrogen (N) or sulfur (S), but not those H-atoms attached to carbon (C). Neutron data collected from H/D-exchanged crystals allow D-atoms attached to O, N or S to be readily visualized at a resolution of ∼2.5 Å [13,17,64] or better. To readily locate H-atoms, data must extend to a resolution of ∼1.5 Å [1416] or better [38], since at lower resolutions cancellation effects (between positive and negative neutron scatterers) limit visualization of H-atoms attached to C-atoms (i.e. CHn groups).

Becoming more prevalent are studies performed using perdeuterated samples [7090] (30/126 structures deposited in the PDB overall, 24%; 20/65 structures deposited in the PDB since 2013, 31%), produced via bacterial expression on deuterated media [91,92]. As perdeuteration provides (near to) complete deuteration (i.e. all H replaced by D), neutron diffraction data from perdeuterated crystals have improved signal-to-noise ratios, allowing shorter data collection times [76,85] and/or providing data to higher resolution [77]. Since the historical bottleneck for successful neutron crystallographic studies has been the need for sufficiently large crystal volumes, it is important that perdeuteration permits data collection from much smaller crystal volumes (cf. H/D-exchanged samples) [72,74,84], making larger unit-cell systems more accessible to study [87]. Furthermore, neutron map cancellation effects are avoided, allowing all D-atoms to be readily visualized (at ∼2.5 Å resolution), including those attached to carbon atoms (i.e. CDn groups) [70,82]. Neutron facilities throughout the world have now developed dedicated laboratories for the partial or complete deuteration of biological macromolecules, such as the Deuteration Laboratory at the Institut Laue-Langevin (ILL), the Bio-Deuteration Laboratory at Oak Ridge National Laboratory (ORNL), the National Deuteration Facility at the Australian Nuclear Science and Technology Organisation (ANSTO) and the Deuteration and Macromolecular Crystallization platform (DEMAX) at the European Spallation Source (ESS).

Applications of neutron macromolecular crystallography

Since neutron diffraction techniques permit direct determination of the positions of H- and D-atoms, the protonation states of amino acid residues can be determined (and if present, those of ligands, inhibitors etc.), along with the orientations of individual water molecules, allowing H-bonding networks to be visualized. In general, these details are lacking using X-rays, and yet are required for a more complete understanding of many biological processes. As such, neutron macromolecular crystallography has found particular application in the study of enzymes (82/126, 65% of all structures deposited in the PDB), mainly focused on improving understanding of a catalytic mechanism or inhibitor/drug-binding interactions. Table 2 provides details of the 126 structures of macromolecules and their complexes deposited in the PDB that were refined using neutron diffraction data (either alone or in a joint X-ray/neutron strategy), including data collection details and crystallographic parameters for each.

Table 2
Macromolecules and their complexes deposited in the PDB that were refined using neutron diffraction data (either alone or in a joint X-ray/neutron strategy), including data collection details and crystallographic parameters for each
graphic
 
graphic
 

* Denotes macromolecules, ligands or substrates listed that are perdeuterated.

The structures are ordered in terms of the ratio of the crystal volume to the asymmetric unit volume, from lowest to highest. Highlighted in purple is a study of perdeuterated fatty acid binding protein in complex with oleic acid [84], which has the lowest ratio (15 × 1014) for the crystal volume (0.05 mm3) to the asymmetric unit volume (34 000 Å3), and the smallest crystal volume (0.05 mm3) for any deposited model. Highlighted in orange are two studies of inorganic pyrophosphatase [41,113], which at 125 kDa is the largest system to be studied. Highlighted in blue is a study of the Compound II intermediate of ascorbate peroxidase at 100 K [62], which has the lowest ratio (22 × 1014) for the crystal volume (0.14 mm3) to the asymmetric unit volume (63 000 Å 3), and the smallest crystal volume (0.14 mm3) for any study performed with a H/D-exchanged crystal. Highlighted in green is a study of perdeuterated rubredoxin [76], which at 14 h has the fastest data collection reported and highlighted in yellow is another study of perdeuterated rubredoxin [77], which at 1.05 Å resolution is the highest resolution structure deposited thus far. Highlighted in pink is a study of crambin [38], which at 1.1 Å resolution is the highest resolution structure reported for any study performed with a H/D-exchanged crystal. Those studies highlighted in turquoise [17,45,81,85,89] are, along with the study of ascorbate peroxidase [62] highlighted in blue, the studies that have been performed at cryo-temperatures.

Abbreviations: APV, amprenavir; DRV, darunavir; FAD, flavin-adenine dinucleotide; IPP, isopentenyl pyrophosphate; NAG, N-acetyl-D-glucosamine; RIS, risedronate; SAH, S-adenosylhomocysteine; SRH, S-ribosylhomocysteine.

Studies have been most commonly performed for hydrolases (EC 3: 48/126, 38% of all structures deposited), including serine proteases (EC 3.4.21: e.g. trypsin [38,68], thrombin [43], elastase [25] and protease I [44]), glycoside hydrolases (EC 3.2.1: e.g. lysozyme [89,90], xylanase II [56] and inverting cellulase [54]), aspartic endopeptidases (EC 3.4.23: e.g. HIV-1 protease [26,78,83,88] and endothiapepsin [20]) as well as other hydrolases such as β-lactamase [33,75,79,81,85,86], diisopropyl fluorophosphatase [23], 5′-methylthioadenosine nucleosidase [61], inorganic pyrophosphatase [41], RAS GTPase [52] and ribonuclease A [27,60].

A variety of structures of oxidoreductases (EC 1: 14/126, 11% of all structures deposited) have been determined, including heme peroxidases (EC 1.11.1: e.g. ascorbate peroxidase [62] and cytochrome c peroxidase [45]) and others such as aldose reductase [72], cholesterol oxidase [66], dihydrofolate reductase [50], lytic polysaccharide monooxygenases [65,67], phycocyanobilin:ferredoxin oxidoreductase [55] and urate oxidase [22,49]. A few transferase (EC 2) structures have been determined, such as those for aspartate aminotransferase [87], farnesyl pyrophosphate synthase [57] and protein kinase G1β [47]. Several structures of the isomerase (EC 5) xylose isomerase (XI, EC 5.3.1.5) have been deposited [29,34,40,46,48], including a variety of sugar complexes (e.g. with d-glucose, d-sorbitol or l-arabinose) with different divalent metal cations (e.g. Ni2+, Cd2+) bound, and structures of holo-XI and apo-XI (i.e. with no metals present) at different pH values. Currently, XI is the most extensively studied enzyme using neutron crystallography (10/126, 8% of all structures). Many structures of the lyase (EC 4) carbonic anhydrase II (CA-II, EC 4.2.1.1) have been deposited [30,37,39,53,58], including apo-CA-II, CA-II/sulfonamide complexes and holo-CA-II structures at different pH values. As yet, no structure of a ligase (EC 6) determined using neutron diffraction data has been deposited in the PDB.

Many structures of transport proteins have been determined, including electron transport proteins involved in redox processes, such as amicyanin [31] and rubredoxin [10,15,16,76,77], oxygen transport proteins such as myoglobin [14,70] and hemoglobin [32,59], a fatty acid-binding protein in complex with oleic acid [84] and transthyretin [42,80], which carries the thyroid hormone, thyroxine and retinol-binding protein bound to retinol.

Other macromolecules for which structures have been deposited in the PDB using neutron diffraction data include an engineered photo-switching chromogenic protein, dathail [63], wild-type and mutant forms of the signalling protein, photoactive yellow protein [24,69], the peptide hormone insulin at different pH values [21,28], saccharide-free and α-1,2-mannobiose-bound forms of concanavalin A [1,13,17,64], perdeuterated and selectively protonated (Leu/Val CH3) forms of the ice-structuring protein, type III antifreeze protein [74,82], the hydrophobic plant protein crambin [38], a carbohydrate-binding module (CBM) derived from CBM4-2 of the Xyn10 xylanase [51] and oligonucleotides in the B- and Z-forms of DNA [18,19,35].

For summaries of the results of neutron macromolecular crystallography studies over the years, the readers should refer to the reviews by Blakeley [93], Schoenborn [94], Niimura and Podjarny [95], Golden and Vrielink [96], O'Dell et al. [97], Chen et al. [98], Fisher et al. [99] and Oksanen et al. [100].

Neutron cryo-crystallography

Although the majority of neutron diffraction studies have been performed at RT (120/126, 95% of structures deposited in the PDB), a small number have been performed at cryo-temperatures, including those of ascorbate peroxidase at 100 K [62], β-lactamase at 100 K in the ligand-free form [85] and in complex with fully deuterated boronic acid (BZB) [81], concanavalin A at 15 K [17], cytochrome c peroxidase at 100 K [45] and T4 phage lysozyme at 80 K [89]. Data collection at cryo-temperatures can be of interest for many reasons. Since the reduction of dynamic disorder lowers atomic displacement parameters, this can lead to improvements in the nuclear scattering density definition, as has been illustrated by the study of saccharide-free concanavalin A at 15 K [17], in which the visibility of the water molecules (such as those in the saccharide-binding site) was enhanced, and more recently by the study of the β-lactamase/BZB complex at 100 K [81], in which the visibility of the BZB ligand was improved. Moreover, in the neutron cryo-crystallography studies performed thus far, smaller crystals have been used for data collection (cf. RT) to achieve (in most cases) similar resolution data. Indeed, in the study of β-lactamase at 100 K in the ligand-free form [85], a significant gain in the resolution limit was observed (1.7 Å at 100 K; cf. 2.1 Å at RT). The ability to use smaller crystal volumes at cryo-temperatures may allow studies to be performed that would otherwise be unfeasible.

Neutron cryo-crystallography opens up new categories of experiment, such as the study of cryo-trapped enzyme reaction intermediates. This was demonstrated in recent studies of two heme peroxidases, cytochrome c peroxidase [45] and ascorbate peroxidase [62], for which the ferryl heme intermediate states, known as Compound I (CI) and Compound II (CII), were successfully cryo-trapped within the crystals and neutron diffraction data collected at 100 K. The exact nature of the CI and CII intermediates has been the subject of a long-standing controversy and conflicting interpretation of indirect evidence, but was directly revealed from the neutron diffraction data at 100 K with CI shown to be the Fe(IV)=O form, and CII the Fe(IV)–OH form, while in both intermediates the active-site histidine was observed in the doubly protonated form. These observations indicate that the widely assumed reaction mechanism, particularly the role of the distal histidine, requires detailed revision.

Instrumentation for neutron macromolecular crystallography

At reactor neutron sources, the use of cylindrical neutron-sensitive image-plate detectors that completely surround the sample and provide a large coverage of reciprocal space (>2π steradian) has been incorporated into the design of instrumentation, such as the LADI-III instrument [101] at the ILL's High Flux Reactor (HFR), the IMAGINE instrument [102] at the High Flux Isotope Reactor (HFIR) of ORNL and the BIODIFF instrument [103] at the Forschungsreaktor München II (FRM II) of Heinz Maier-Leibnitz Zentrum (MLZ). The LADI-III and IMAGINE instruments utilize quasi-Laue methods for data collection, while BIODIFF is a monochromatic instrument, in many ways similar to the BIX-3 [104] and BIX-4 [105] instruments, which were used for neutron macromolecular crystallography studies in Japan from ca. 2002 and 2004, respectively, until they were closed down in 2011 due to the Fukushima Daiichi nuclear incident. Table 3 provides further details of diffractometers in operation at reactor neutron sources.

Table 3
Instruments for macromolecular crystallography at reactor neutron sources
 Instrument name 
 LADI-III IMAGINE BIODIFF D19 
Facility, source ILL, ILL HFR ORNL, HFIR MLZ, FRM II ILL, ILL HFR 
Reactor power 58 MW 84 MW 20 MW 58 MW 
Data collection method Quasi-Laue Quasi-Laue Monochromatic Monochromatic 
Detector type Image plate Image plate Image plate 3He PSD 
Sample environment (i) Ambient
(ii) Oxford Cryosystems Cobra: 80–500 K 
(i) Ambient
(ii) Closed-cycle refrigerator: 4–450 K 
(i) Ambient
(ii) Oxford Cryosystems 700 Plus: 90–500 K
(iii) Closed-cycle refrigerator 3.5–325 K 
(i) Ambient
(ii) Oxford Cryosystems 700 Plus: 90–500 K 
Data collection statistics for each instrument since 2013 
Structures deposited in PDB 12 13 13 
Mean (median) crystal volume 1.0 mm3 (0.48 mm31.1 mm3 (0.7 mm32.5 mm3 (2.4 mm35.4 mm3 (4.5 mm3
Smallest crystal volume [PDB code] 0.05 mm3 [5CE4] 0.35 mm3 [5TKI] 0.7 mm3 [4CVJ] 2.00 mm3 [4AR4] 
Mean asymmetric unit volume 77 000 Å3 62 000 Å3 69 000 Å3 45 000 Å3 
Largest asymmetric unit volume [PDB code] 223 000 Å3 [5VJZ] 119 000 Å3 [5KWF] 115 000 Å3 [5CG5] 123 000 Å3 [4QDW] 
Mean crystal volume/asymmetric unit volume ratio 148 × 1014 233 × 1014 400 × 1014 2334 × 1014 
Lowest ratio for crystal volume/asymmetric unit volume [PDB code] 15 × 1014 [5CE4] 34 × 1014 [5KWF] 65 × 1014 [4CVJ] 548 × 1014 [4PVN] 
Mean data collection time 17d 17d 13d 10d 
Fastest data collection [PDB code] 5d [4PVM] 3d [4K9F] 7d [4C3Q/4BD1] 3d [4AR4] 
Mean resolution of structures 2.1 Å 2.2 Å 1.9 Å 1.7 Å 
Highest resolution structure [PDB code] 1.90 Å [5CE4] 1.75 Å [4K9F] 1.42 Å [5MNX/5MON] 1.05 Å [4AR3] 
 Instrument name 
 LADI-III IMAGINE BIODIFF D19 
Facility, source ILL, ILL HFR ORNL, HFIR MLZ, FRM II ILL, ILL HFR 
Reactor power 58 MW 84 MW 20 MW 58 MW 
Data collection method Quasi-Laue Quasi-Laue Monochromatic Monochromatic 
Detector type Image plate Image plate Image plate 3He PSD 
Sample environment (i) Ambient
(ii) Oxford Cryosystems Cobra: 80–500 K 
(i) Ambient
(ii) Closed-cycle refrigerator: 4–450 K 
(i) Ambient
(ii) Oxford Cryosystems 700 Plus: 90–500 K
(iii) Closed-cycle refrigerator 3.5–325 K 
(i) Ambient
(ii) Oxford Cryosystems 700 Plus: 90–500 K 
Data collection statistics for each instrument since 2013 
Structures deposited in PDB 12 13 13 
Mean (median) crystal volume 1.0 mm3 (0.48 mm31.1 mm3 (0.7 mm32.5 mm3 (2.4 mm35.4 mm3 (4.5 mm3
Smallest crystal volume [PDB code] 0.05 mm3 [5CE4] 0.35 mm3 [5TKI] 0.7 mm3 [4CVJ] 2.00 mm3 [4AR4] 
Mean asymmetric unit volume 77 000 Å3 62 000 Å3 69 000 Å3 45 000 Å3 
Largest asymmetric unit volume [PDB code] 223 000 Å3 [5VJZ] 119 000 Å3 [5KWF] 115 000 Å3 [5CG5] 123 000 Å3 [4QDW] 
Mean crystal volume/asymmetric unit volume ratio 148 × 1014 233 × 1014 400 × 1014 2334 × 1014 
Lowest ratio for crystal volume/asymmetric unit volume [PDB code] 15 × 1014 [5CE4] 34 × 1014 [5KWF] 65 × 1014 [4CVJ] 548 × 1014 [4PVN] 
Mean data collection time 17d 17d 13d 10d 
Fastest data collection [PDB code] 5d [4PVM] 3d [4K9F] 7d [4C3Q/4BD1] 3d [4AR4] 
Mean resolution of structures 2.1 Å 2.2 Å 1.9 Å 1.7 Å 
Highest resolution structure [PDB code] 1.90 Å [5CE4] 1.75 Å [4K9F] 1.42 Å [5MNX/5MON] 1.05 Å [4AR3] 

The table provides details of instrument specifications and data collection statistics for the structures deposited in the PDB since 2013.

At spallation neutron sources, neutrons produced by proton pulses are ‘time-stamped’ such that by recording time-of-flight (TOF) information, the corresponding energy and wavelength of each neutron can be calculated. TOF techniques in combination with large position-sensitive detectors (PSDs) allow wavelength-resolved Laue patterns to be collected using all the available neutrons. In 2002, the first instrument dedicated to neutron macromolecular crystallography at a spallation neutron source was built at the Los Alamos Neutron Science Center (LANSCE). The instrument PCS [98] user program was funded by the Department of Energy for 13 years from 2002 until 2014, at which time it was sadly decommissioned. In the last few years, two new TOF Laue diffractometers dedicated to neutron macromolecular crystallography have become operational: MaNDi [106] at the Spallation Neutron Source (SNS) at ORNL and iBIX [107] at the Materials and Life Science Experimental Facility (MLF) of the Japan Proton Accelerator Research Complex (J-PARC). Table 4 provides further details of diffractometers in operation at spallation neutron sources.

Table 4
Instruments for macromolecular crystallography at spallation neutron sources
 Instrument name 
 MaNDi iBIX 
Facility, source ORNL, SNS J-PARC, MLF 
Reactor power 2 MW Hg target, 60/30 Hz 1 MW Hg target, 25 Hz 
Data collection method TOF Laue TOF Laue 
Detector type Anger cameras λ-shifting fiber 
Sample environment (i) Ambient
(ii) Oxford Cryosystems Cobra: 80–500 K 
(i) Ambient
(ii) Gas-flow-type cooling system, He: down to ∼20 K; N2: down to ∼90 K 
Data collection statistics for each instrument since 2013 
Structures deposited in PDB 
Mean (median) crystal volume 3.8 mm3 (2.9 mm34.5 mm3 (4.7 mm3
Smallest crystal volume [PDB code] 0.4 mm3 [5A93] 2.7 mm3 [4QCD] 
Mean asymmetric unit volume 105 000 Å3 70 000 Å3 
Largest asymmetric unit volume [PDB code] 285 000 Å3 [5TY5] 115 000 Å3 [5CG6] 
Mean crystal volume/asymmetric unit volume ratio 475 × 1014 844 × 1014 
Lowest ratio for crystal volume/asymmetric unit volume [PDB code] 53 × 1014 [5A93] 287 × 1014 [5CG6] 
Mean data collection time 8d 12d 
Fastest data collection [PDB code] 2d [5A93] 9d [5CG6, 4QCD] 
Mean resolution of structures 2.2 Å 1.8 Å 
Highest-resolution structure [PDB code] 2.0 Å [4S2F] 1.5 Å [3X2O, 3X2P] 
 Instrument name 
 MaNDi iBIX 
Facility, source ORNL, SNS J-PARC, MLF 
Reactor power 2 MW Hg target, 60/30 Hz 1 MW Hg target, 25 Hz 
Data collection method TOF Laue TOF Laue 
Detector type Anger cameras λ-shifting fiber 
Sample environment (i) Ambient
(ii) Oxford Cryosystems Cobra: 80–500 K 
(i) Ambient
(ii) Gas-flow-type cooling system, He: down to ∼20 K; N2: down to ∼90 K 
Data collection statistics for each instrument since 2013 
Structures deposited in PDB 
Mean (median) crystal volume 3.8 mm3 (2.9 mm34.5 mm3 (4.7 mm3
Smallest crystal volume [PDB code] 0.4 mm3 [5A93] 2.7 mm3 [4QCD] 
Mean asymmetric unit volume 105 000 Å3 70 000 Å3 
Largest asymmetric unit volume [PDB code] 285 000 Å3 [5TY5] 115 000 Å3 [5CG6] 
Mean crystal volume/asymmetric unit volume ratio 475 × 1014 844 × 1014 
Lowest ratio for crystal volume/asymmetric unit volume [PDB code] 53 × 1014 [5A93] 287 × 1014 [5CG6] 
Mean data collection time 8d 12d 
Fastest data collection [PDB code] 2d [5A93] 9d [5CG6, 4QCD] 
Mean resolution of structures 2.2 Å 1.8 Å 
Highest-resolution structure [PDB code] 2.0 Å [4S2F] 1.5 Å [3X2O, 3X2P] 

The table provides details of instrument specifications and data collection statistics for the structures deposited in the PDB since 2013.

Large crystal growth

The historical bottleneck for neutron macromolecular crystallography studies has been the need for large crystal volumes — the actual crystal volume required depending on the sample unit-cell volume. Over the years, improvements made to instrumentation combined with the use of perdeuteration have allowed ever-smaller crystal volumes to be used for data collection. Nevertheless, larger crystals will almost always be beneficial since higher-resolution data can generally be collected and data collection times can be reduced. For example, in recent neutron diffraction studies of the C-terminal domain of galectin-3 (galectin-3C), the diffraction resolution was improved from 1.9 to 1.7 Å by using a crystal of 1.8 mm3 rather than a crystal of 0.35 mm3. Moreover, the data collection time could be reduced from 21 to 6 days [108], of particular significance given the high demand for neutron beamtime. Clearly, growth of large crystals is a challenge; however, to be in a position to commence neutron diffraction studies, the macromolecule will have already been crystallized and the X-ray structure determined, such that the initial crystallization conditions can be used as a guide for growing the larger crystals needed with neutrons. Methods for growing large crystals via use of the crystallization phase diagram (CPD) are explained in detail in Neutron Protein Crystallography [95], but briefly the key is to apply knowledge of the crystallization phase diagram of the macromolecule. A CPD consists of three regions — (i) the undersaturated region, a solution phase in which crystals dissolve; (ii) the metastable region, in which nucleation of new crystals does not occur but macromolecules crystallize on the seed crystal; and (iii) the spontaneous nucleation region, in which both crystal growth and nucleation occur. Determination of the CPD thus provides the location and boundaries of these regions. By placing a seed crystal within the metastable region, and maintaining it there via adjustment of various parameters of the crystallization (e.g. protein concentration and temperature), larger crystals can be grown [22]. Devices which allow manipulation of the crystallization parameters in order to promote large crystal growth are now being developed [109]. Other methods for large crystal growth, such as the use of counter-diffusion in restricted geometry and Ostwald ripening, have been successfully used to prepare large crystals for neutron studies [41,110].

Computational tools for structure refinement

Given the lack of radiation damage with neutrons, the crystal used for neutron diffraction data collection can then be used to collect X-ray diffraction data at the same temperature. This is particularly useful since structural refinement tools have been developed which allow structural refinement in a joint refinement strategy with both X-ray and neutron diffraction data. With the addition of H- and D-atoms, a macromolecular structure has approximately twice as many atoms, therefore, attempting to refine such structures at medium resolution (e.g. dmin from 2.0 to 2.5 Å), specifically those with large unit cells, can be problematic as the data-to-parameter ratio is low. By combining X-ray and neutron diffraction data collected from the same crystal, the data-to-parameter ratio is increased, while the influence of systematic errors can be reduced. Joint X-ray/neutron refinement strategies make it possible to allow refinement of all the atoms within the structure, resulting in more accurate structures. The PHENIX [111] and nCNS [112] softwares are both able to refine structures in a joint X-ray/neutron strategy (as well as against neutron data alone) and have been used extensively since their development. Of the 126 structures deposited, more than half (66/126, 52%) employed joint X-ray/neutron refinement strategies.

Extending the limits of neutron macromolecular crystallography

All the structures of macromolecules and their complexes deposited in the PDB, which were refined using neutron diffraction data (either alone or in a joint X-ray/neutron strategy), are listed in Table 2; ordered by increasing crystal volume to asymmetric unit volume ratio, those with the lowest ratios can be considered the most challenging. Currently, the lowest ratio for the crystal volume to asymmetric unit volume of any of the studies is 15 × 1014 for the study of perdeuterated fatty acid-binding protein (FABP) in complex with oleic acid (crystal volume = 0.05 mm3, asymmetric unit volume = 34 000 Å3) from which the structure was determined to a resolution of 1.9 Å (PDB code: 4CE4) using LADI-III [84]. The FABP crystal used for data collection also has the smallest crystal volume (0.05 mm3) from any of the studies reported thus far. The lowest ratio for the crystal volume to the asymmetric unit volume for any study performed with a H/D-exchanged crystal is 22 × 1014 for the study of the CII enzyme intermediate of ascorbate peroxidase (APX) at 100 K (crystal volume = 0.14 mm3, asymmetric unit volume = 63 000 Å3), which was determined to a resolution of 2.31 Å using LADI-III (PDB code: 5JPR) [62]. The APX crystal (0.14 mm3) used for data collection has the smallest crystal volume for any study performed with a H/D-exchanged crystal. The largest structure reported thus far is that of a 125 kDa inorganic pyrophosphatase (I-PPase). This was first determined to a resolution of 2.5 Å (PDB code: 3Q3L) using LADI-IIII with data collected from a large 5 mm3 H/D-exchanged crystal of I-PPase [41]. Very recently, however, a new higher-resolution structure of I-PPase, to a resolution of 2.3 Å, has been deposited in the PDB (PDB code: 5TY5) using MaNDi at SNS. This structure was determined using a large (>6 mm3) H/D-exchanged crystal that was grown in microgravity on board the International Space Station [113]. The fastest data collection reported thus far (14 h) is for a study of perdeuterated rubredoxin [76] determined to a resolution of 1.75 Å (PDB code: 3RZT) using LADI-IIII, while the highest resolution structure reported, at a resolution of 1.05 Å, is for another study of perdeuterated rubredoxin (PDB code: 4AR3) with data collected using D19 at the ILL [77]. The highest resolution structure reported for any study performed with a H/D-exchanged crystal is for a study of crambin (PDB code: 4FC1). Data were collected to a resolution of 1.1 Å using the PCS at LANSCE [38].

In addition to the structures reported in Table 2, preliminary diffraction data have recently been presented for manganese superoxide dismutase (MnSOD) [114] and the photosystem II subunit PsbO [115]. With a cell edge of ∼242 Å, MnSOD represents a challenging system and yet TOF neutron diffraction data have been collected from a 0.26 mm3 perdeuterated crystal at 293 K to a resolution of 2.30 Å using MaNDi at SNS. The ratio of the crystal volume (0.26 mm3) to the asymmetric unit volume (115 000 Å3) for the MnSOD study is, at 23 × 1014, one of the lowest ratios reported thus far. In the case of PsbO, which has a cell edge of ∼195 Å, TOF neutron diffraction data were collected from a 0.25 mm3 H/D-exchanged crystal at 293 K to a resolution of 2.30 Å using MaNDi at SNS. The ratio of the crystal volume (0.25 mm3) to the asymmetric unit volume (44 000 Å3) for the PsbO study is 57 × 1014, which is one of the lowest ratios reported thus far for an experiment performed with a H/D-exchanged crystal.

Conclusions

Owing to the recent enhancements in capability and capacity for neutron macromolecular crystallography, notably the improvements in data collection facilities and the use of perdeuteration of samples, there has been a marked increase in the number of neutron structures deposited in the PDB. The limits of the field continue to expand with new examples illustrating further reductions in crystal volume or data collection time, and increases in resolution or unit-cell volume achievable (Figure 1). Moreover, the feasibility of data collection from cryo-trapped enzyme reaction intermediates has been demonstrated [45,62], allowing a greater array of studies to be performed. This trend is set to continue through further improvements planned to existing instrumentation, and the construction of new instrumentation, such as the LADI-B instrument, currently being proposed to be built at the ILL as part of their Endurance Programme, and the TOF Laue diffractometer NMX (https://europeanspallationsource.se/instruments/nmx), which is planned to be one of the first 10 instruments to come online at the ESS [99].

Summary
  • Neutron diffraction techniques provide direct determination of the hydrogen (H) and deuterium (D) positions in crystal structures of biological macromolecules at resolutions of ∼1.5 and 2.5 Å, respectively, leading to a more complete understanding of many biological processes and drug-binding.

  • Advances in instrumentation and sample preparation now allow sub-mm3 crystals to be used for data collection and permit the study of larger systems (unit-cell edges >100 Å). As such, many more studies have become feasible as indicated by the increase in the number of structures that have been deposited in the Protein Data Bank in the last 5 years.

  • The application of neutron macromolecular crystallography to address challenging questions in Structural Biology can be expected to expand with the introduction of new beamlines at existing neutron sources such as the Institut Laue-Langevin, and those currently under construction, such as the European Spallation Source.

Abbreviations

     
  • APX

    ascorbate peroxidase

  •  
  • BZB

    deuterated boronic acid

  •  
  • C

    carbon

  •  
  • CA-II

    carbonic anhydrase II

  •  
  • CBM

    carbohydrate-binding module

  •  
  • CI

    compound I

  •  
  • CII

    compound II

  •  
  • CPD

    crystallization phase diagram

  •  
  • D

    deuterium

  •  
  • FABP

    fatty acid-binding protein

  •  
  • FRM II

    Forschungsreaktor München II

  •  
  • H

    hydrogen

  •  
  • HFIR

    high flux isotope reactor

  •  
  • HFR

    high flux reactor

  •  
  • I-PPase

    inorganic pyrophosphatase

  •  
  • J-PARC

    Japan Proton Accelerator Research Complex

  •  
  • LANSCE

    Los Alamos Neutron Science Center

  •  
  • MLZ

    Maier-Leibnitz Zentrum

  •  
  • MnSOD

    manganese superoxide dismutase

  •  
  • N

    nitrogen

  •  
  • O

    oxygen

  •  
  • PSDs

    position-sensitive detectors

  •  
  • RT

    room temperature

  •  
  • S

    sulfur

  •  
  • SNS

    Spallation Neutron Source

  •  
  • TOF

    time of flight

  •  
  • XFEL

    X-ray free electron

  •  
  • XI

    xylose isomerase

Competing Interests

The Authors declare that there are no competing interests associated with the manuscript.

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