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1.  Preliminary neutron and ultrahigh-resolution X-ray diffraction studies of the aspartic proteinase endothiapepsin cocrystallized with a gem-diol inhibitor 
Endothiapepsin has been cocrystallized with the gem-diol inhibitor PD-135,040 in a low solvent-content (39%) unit cell, which is unprecedented for this enzyme—inhibitor complex and enables ultrahigh-resolution (1.0 Å) X-ray diffraction data to be collected. This atomic resolution X-ray data set will be used to deduce the protonation states of the catalytic aspartate residues. A room-temperature neutron data set has also been collected for joint refinement with a room-temperature X-ray data set in order to locate the H/D atoms at the active site.
PMCID: PMC2344097  PMID: 18084100
2.  A preliminary neutron diffraction study of γ-­chymotrypsin 
A 2.0 Å resolution neutron diffraction data set has been collected from a D2O-soaked γ-chymotrypsin crystal at low pH on the Institute Laue–Langevin LADI-III beamline.
The crystal preparation and preliminary neutron diffraction analysis of γ-­chymotrypsin are presented. Large hydrogenated crystals of γ-chymotrypsin were exchanged into deuterated buffer via vapor diffusion in a capillary and neutron Laue diffraction data were collected from the resulting crystal to 2.0 Å resolution on the LADI-III diffractometer at the Institut Laue–Langevin (ILL) at room temperature. The neutron structure of a well studied protein such as γ-­chymotrypsin, which is also amenable to ultrahigh-resolution X-ray crystallo­graphy, represents the first step in developing a model system for the study of H atoms in protein crystals.
PMCID: PMC2650460  PMID: 19255494
γ-chymotrypsin; neutron diffraction
3.  Room-temperature ultrahigh-resolution time-of-flight neutron and X-ray diffraction studies of H/D-exchanged crambin 
The 0.85 Å room-temperature ultrahigh-resolution structure of H/D-exchanged crambin is reported. Preliminary 1.1 Å resolution neutron diffraction data have been collected at the neutron Protein Crystallography Station at LANSCE.
The room-temperature (RT) X-ray structure of H/D-exchanged crambin is reported at 0.85 Å resolution. As one of the very few proteins refined with anisotropic atomic displacement parameters at two temperatures, the dynamics of atoms in the RT and 100 K structures are compared. Neutron diffraction data from an H/D-exchanged crambin crystal collected at the Protein Crystallo­graphy Station (PCS) showed diffraction beyond 1.1 Å resolution. This is the highest resolution neutron diffraction reported to date for a protein crystal and will reveal important details of the anisotropic motions of H and D atoms in protein structures.
PMCID: PMC3274385  PMID: 22297981
crambin; neutron diffraction; ultrahigh resolution; H/D exchange
4.  Inorganic pyrophosphatase crystals from Thermococcus thioreducens for X-ray and neutron diffraction 
Inorganic pyrophosphatase from T. thioreducans has been crystallized and the crystals were deemed to be suitable for both X-ray and neutron diffraction at room temperature.
Inorganic pyrophosphatase (IPPase) from the archaeon Thermococcus thioreducens was cloned, overexpressed in Escherichia coli, purified and crystallized in restricted geometry, resulting in large crystal volumes exceeding 5 mm3. IPPase is thermally stable and is able to resist denaturation at temperatures above 348 K. Owing to the high temperature tolerance of the enzyme, the protein was amenable to room-temperature manipulation at the level of protein preparation, crystallization and X-ray and neutron diffraction analyses. A complete synchrotron X-ray diffraction data set to 1.85 Å resolution was collected at room temperature from a single crystal of IPPase (monoclinic space group C2, unit-cell parameters a = 106.11, b = 95.46, c = 113.68 Å, α = γ = 90.0, β = 98.12°). As large-volume crystals of IPPase can be obtained, preliminary neutron diffraction tests were undertaken. Consequently, Laue diffraction images were obtained, with reflections observed to 2.1 Å resolution with I/σ(I) greater than 2.5. The preliminary crystallographic results reported here set in place future structure–function and mechanism studies of IPPase.
PMCID: PMC3509969  PMID: 23192028
inorganic pyrophosphatase; Thermococcus thioreducens; neutron diffraction
5.  X-ray structure of perdeuterated diisopropyl fluorophosphatase (DFPase): perdeuteration of proteins for neutron diffraction 
The crystal structure of perdeuterated diisopropyl fluorophosphatase is reported and compared with the hydrogenated structure. Diffraction guidelines for neutron crystallography experiments are summarized.
The signal-to-noise ratio is one of the limiting factors in neutron macromolecular crystallography. Protein perdeuteration, which replaces all H atoms with deuterium, is a method of improving the signal-to-noise ratio of neutron crystallography experiments by reducing the incoherent scattering of the hydrogen isotope. Detailed analyses of perdeuterated and hydrogenated structures are necessary in order to evaluate the utility of perdeuterated crystals for neutron diffraction studies. The room-temperature X-ray structure of perdeuterated diisopropyl fluorophosphatase (DFPase) is reported at 2.1 Å resolution. Comparison with an independently refined hydrogenated room-temperature structure of DFPase revealed no major systematic differences, although the crystals of perdeuterated DFPase did not diffract neutrons. The lack of diffraction is examined with respect to data-collection and crystallo­graphic parameters. The diffraction characteristics of successful neutron structure determinations are presented as a guideline for future neutron diffraction studies of macromolecules. X-ray diffraction to beyond 2.0 Å resolution appears to be a strong predictor of successful neutron structures.
PMCID: PMC2852326  PMID: 20383004
diisopropyl fluorophosphatase; perdeuteration
6.  Time-of-flight neutron diffraction study of bovine γ-­chymotrypsin at the Protein Crystallography Station 
In order to begin an exact determination of hydrogen positions in proteins, a neutron diffraction study of bovine gamma-chymotrypsin has been conducted. This paper details the data collection of the protein at pD (pH*) 7.1.
The overarching goal of this research project is to determine, for a subset of proteins, exact hydrogen positions using neutron diffraction, thereby improving H-atom placement in proteins so that they may be better used in various computational methods that are critically dependent upon said placement. In order to be considered applicable for neutron diffraction studies, the protein of choice must be amenable to ultrahigh-resolution X-ray crystallography, be able to form large crystals (1 mm3 or greater) and have a modestly sized unit cell (no dimension longer than 100 Å). As such, γ-chymotrypsin is a perfect candidate for neutron diffraction. To understand and probe the role of specific active-site residues and hydrogen-bonding patterns in γ-chymotrypsin, neutron diffraction studies were initiated at the Protein Crystallography Station (PCS) at Los Alamos Neutron Science Center (LANSCE). A large single crystal was subjected to H/D exchange prior to data collection. Time-of-flight neutron diffraction data were collected to 2.0 Å resolution at the PCS with ∼85% completeness. Here, the first time-of-flight neutron data collection from γ-­chymotrypsin is reported.
PMCID: PMC3087647  PMID: 21543868
neutron diffraction; γ-chymotrypsin
7.  Large crystal growth by thermal control allows combined X-ray and neutron crystallographic studies to elucidate the protonation states in Aspergillus flavus urate oxidase 
Journal of the Royal Society Interface  2009;6(Suppl 5):S599-S610.
Urate oxidase (Uox) catalyses the oxidation of urate to allantoin and is used to reduce toxic urate accumulation during chemotherapy. X-ray structures of Uox with various inhibitors have been determined and yet the detailed catalytic mechanism remains unclear. Neutron crystallography can provide complementary information to that from X-ray studies and allows direct determination of the protonation states of the active-site residues and substrate analogues, provided that large, well-ordered deuterated crystals can be grown. Here, we describe a method and apparatus used to grow large crystals of Uox (Aspergillus flavus) with its substrate analogues 8-azaxanthine and 9-methyl urate, and with the natural substrate urate, in the presence and absence of cyanide. High-resolution X-ray (1.05–1.20 Å) and neutron diffraction data (1.9–2.5 Å) have been collected for the Uox complexes at the European Synchrotron Radiation Facility and the Institut Laue-Langevin, respectively. In addition, room temperature X-ray data were also collected in preparation for joint X-ray and neutron refinement. Preliminary results indicate no major structural differences between crystals grown in H2O and D2O even though the crystallization process is affected. Moreover, initial nuclear scattering density maps reveal the proton positions clearly, eventually providing important information towards unravelling the mechanism of catalysis.
PMCID: PMC2843968  PMID: 19586953
urate oxidase; neutron and X-ray crystallography; crystal growth; phase diagram; H–D exchange; protonation states
8.  A preliminary neutron diffraction study of rasburicase, a recombinant urate oxidase enzyme, complexed with 8-azaxanthin 
Neutron diffraction data of hydrogenated recombinant urate oxidase enzyme (Rasburicase), complexed with a purine-type inhibitor 8-azaxanthin, was collected to 2.1 Å resolution from a crystal grown in D2O by careful control and optimization of crystallization conditions via knowledge of the phase diagram. Deuterium atoms were clearly seen in the neutron-scattering density map.
Crystallization and preliminary neutron diffraction measurements of rasburicase, a recombinant urate oxidase enzyme expressed by a genetically modified Saccharomyces cerevisiae strain, complexed with a purine-type inhibitor (8-­azaxanthin) are reported. Neutron Laue diffraction data were collected to 2.1 Å resolution using the LADI instrument from a crystal (grown in D2O) with volume 1.8 mm3. The aim of this neutron diffraction study is to determine the protonation states of the inhibitor and residues within the active site. This will lead to improved comprehension of the enzymatic mechanism of this important enzyme, which is used as a protein drug to reduce toxic uric acid accumulation during chemotherapy. This paper illustrates the high quality of the neutron diffraction data collected, which are suitable for high-resolution structural analysis. In comparison with other neutron protein crystallography studies to date in which a hydrogenated protein has been used, the volume of the crystal was relatively small and yet the data still extend to high resolution. Furthermore, urate oxidase has one of the largest primitive unit-cell volumes (space group I222, unit-cell parameters a = 80, b = 96, c = 106 Å) and molecular weights (135 kDa for the homotetramer) so far successfully studied with neutrons.
PMCID: PMC2197182  PMID: 16511330
urate oxidase; heavy water; phase diagram; neutron Laue diffraction
9.  Crystallization of porcine pancreatic elastase and a preliminary neutron diffraction experiment 
To investigate the structural characteristics of a covalent inhibitor bound to porcine pancreatic elastase (PPE), including H atoms and hydration by water, a crystal of porcine pancreatic elastase with its inhibitor was grown to a size of 1.6 mm3 for neutron diffraction study. The crystal diffracted to 2.3 Å resolution with sufficient quality for further structure determination owing to the similar atomic scattering properties of deuterium and carbon.
Porcine pancreatic elastase (PPE) resembles the attractive drug target leukocyte elastase, which has been implicated in a number of inflammatory disorders. In order to investigate the structural characteristics of a covalent inhibitor bound to PPE, including H atoms and the hydration by water, a single crystal of PPE for neutron diffraction study was grown in D2O containing 0.2 M sodium sulfate (pD 5.0) using the sitting-drop vapour-diffusion method. The crystal was grown to a size of 1.6 mm3 by repeated macroseeding. Neutron diffraction data were collected at room temperature using a BIX-3 diffractometer at the JRR-3 research reactor of the Japan Atomic Energy Agency (JAEA). The data set was integrated and scaled to 2.3 Å resolution in space group P212121, with unit-cell parameters a = 51.2, b = 57.8, c = 75.6 Å.
PMCID: PMC2330211  PMID: 17401204
porcine pancreatic elastase; neutron diffraction
10.  Hirshfeld atom refinement 
IUCrJ  2014;1(Pt 5):361-379.
The new automated iterative Hirshfeld atom refinement method is explained and validated through comparison of structural models of Gly–l-Ala obtained from synchrotron X-ray and neutron diffraction data at 12, 50, 150 and 295 K. Structural parameters involving hydrogen atoms are determined with comparable precision from both experiments and agree mostly to within two combined standard uncertainties.
Hirshfeld atom refinement (HAR) is a method which determines structural parameters from single-crystal X-ray diffraction data by using an aspherical atom partitioning of tailor-made ab initio quantum mechanical molecular electron densities without any further approximation. Here the original HAR method is extended by implementing an iterative procedure of successive cycles of electron density calculations, Hirshfeld atom scattering factor calculations and structural least-squares refinements, repeated until convergence. The importance of this iterative procedure is illustrated via the example of crystalline ammonia. The new HAR method is then applied to X-ray diffraction data of the dipeptide Gly–l-Ala measured at 12, 50, 100, 150, 220 and 295 K, using Hartree–Fock and BLYP density functional theory electron densities and three different basis sets. All positions and anisotropic displacement parameters (ADPs) are freely refined without constraints or restraints – even those for hydrogen atoms. The results are systematically compared with those from neutron diffraction experiments at the temperatures 12, 50, 150 and 295 K. Although non-hydrogen-atom ADPs differ by up to three combined standard uncertainties (csu’s), all other structural parameters agree within less than 2 csu’s. Using our best calculations (BLYP/cc-pVTZ, recommended for organic molecules), the accuracy of determining bond lengths involving hydrogen atoms from HAR is better than 0.009 Å for temperatures of 150 K or below; for hydrogen-atom ADPs it is better than 0.006 Å2 as judged from the mean absolute X-ray minus neutron differences. These results are among the best ever obtained. Remarkably, the precision of determining bond lengths and ADPs for the hydrogen atoms from the HAR procedure is comparable with that from the neutron measurements – an outcome which is obtained with a routinely achievable resolution of the X-ray data of 0.65 Å.
PMCID: PMC4174878  PMID: 25295177
aspherical atom partitioning; quantum mechanical molecular electron densities; X-ray structure refinement; hydrogen atom modelling; anisotropic displacement parameters
11.  Crystallization and preliminary neutron diffraction studies of ADP-ribose pyrophosphatase-I from Thermus thermophilus HB8 
ADP-ribose pyrophosphatase-I, a Nudix enzyme, from T. thermophilus was crystallized for neutron diffraction. Neutron and X-ray diffraction data sets were collected to 2.1 and 1.5 Å resolution, respectively.
ADP-ribose pyrophosphatase-I from Thermus thermophilus HB8 (TtADPRase-I) prevents the intracellular accumulation of ADP-ribose by hydrolyzing it to AMP and ribose 5′-phosphate. To understand the catalytic mechanism of TtADPRase-I, it is necessary to investigate the role of glutamates and metal ions as well as the coordination of water molecules located at the active site. A macroseeding method was developed in order to obtain a large TtADPRase-I crystal which was suitable for a neutron diffraction study to provide structural information. Neutron and X-ray diffraction experiments were performed at room temperature using the same crystal. The crystal diffracted to 2.1 and 1.5 Å resolution in the neutron and X-ray diffraction experiments, respectively. The crystal belonged to the primitive space group P3221, with unit-cell parameters a = b = 50.7, c = 119 Å.
PMCID: PMC3253833  PMID: 22232170
ADP-ribose pyrophosphatase; Ndx4; neutron diffraction; Thermus thermophilus
12.  Crystallization and preliminary neutron diffraction studies of HIV-1 protease cocrystallized with inhibitor KNI-272 
In order to determine the protonation states of the residues within the active site of an HIV-1 protease–inhibitor complex, a crystal of HIV-1 protease complexed with inhibitor (KNI-272) was grown to a size of 1.4 mm3 for neutron diffraction study. The crystal diffracted to 2.3 Å resolution with sufficient quality for further structure determination.
This paper reports the crystallization and preliminary neutron diffraction measurements of HIV-1 protease, a potential target for anti-HIV therapy, complexed with an inhibitor (KNI-272). The aim of this neutron diffraction study is to obtain structural information about the H atoms and to determine the protonation states of the residues within the active site. The crystal was grown to a size of 1.4 mm3 by repeated macroseeding and a slow-cooling method using a two-liquid system. Neutron diffraction data were collected at room temperature using a BIX-4 diffractometer at the JRR-3 research reactor of the Japan Atomic Energy Agency (JAEA). The data set was integrated and scaled to 2.3 Å resolution in space group P21212, with unit-cell parameters a = 59.5, b = 87.4, c = 46.8 Å.
PMCID: PMC2581681  PMID: 18997326
HIV-1 protease; inhibitors; neutron diffraction
13.  Preliminary neutron and X-ray crystallographic studies of equine cyanomethemoglobin 
Equine cyanomethemoglobin has been crystallized and X-ray and neutron diffraction data have been measured. Joint X-ray–neutron refinement is under way; the structural results should help to elucidate the differences between the hemoglobin R and T states.
Room-temperature and 100 K X-ray and room-temperature neutron diffraction data have been measured from equine cyanomethemoglobin to 1.7 Å resolution using a home source, to 1.6 Å resolution on NE-CAT at the Advanced Photon Source and to 2.0 Å resolution on the PCS at Los Alamos Neutron Science Center, respectively. The cyanomethemoglobin is in the R state and preliminary room-temperature electron and neutron scattering density maps clearly show the protonation states of potential Bohr groups. Interestingly, a water molecule that is in the vicinity of the heme group and coordinated to the distal histidine appears to be expelled from this site in the low-temperature structure.
PMCID: PMC2852348  PMID: 20383026
equine hemoglobin; time-of-flight neutron diffraction; R state; joint XN refinement; protonation
14.  On the temperature dependence of H-U iso in the riding hydrogen model 
The temperature dependence of hydrogen U iso and parent U eq in the riding hydrogen model is investigated by neutron diffraction, aspherical-atom refinements and QM/MM and MO/MO cluster calculations. Fixed values of 1.2 or 1.5 appear to be underestimated, especially at temperatures below 100 K.
The temperature dependence of H-U iso in N-acetyl-l-4-hydroxyproline monohydrate is investigated. Imposing a constant temperature-independent multiplier of 1.2 or 1.5 for the riding hydrogen model is found to be inaccurate, and severely underestimates H-U iso below 100 K. Neutron diffraction data at temperatures of 9, 150, 200 and 250 K provide benchmark results for this study. X-ray diffraction data to high resolution, collected at temperatures of 9, 30, 50, 75, 100, 150, 200 and 250 K (synchrotron and home source), reproduce neutron results only when evaluated by aspherical-atom refinement models, since these take into account bonding and lone-pair electron density; both invariom and Hirshfeld-atom refinement models enable a more precise determination of the magnitude of H-atom displacements than independent-atom model refinements. Experimental efforts are complemented by computing displacement parameters following the TLS+ONIOM approach. A satisfactory agreement between all approaches is found.
PMCID: PMC4075069
riding hydrogen model; QM/MM computations; neutron diffraction; invariom refinement; Hirshfeld-atom refinement; synchrotron radiation
15.  Preliminary joint X-ray and neutron protein crystallographic studies of endoxylanase II from the fungus Trichoderma longibrachiatum  
A fungal family 11 endoxylanase has been crystallized at pH 8.5 and room-temperature X-ray and neutron diffraction data have been collected. Joint X-ray/neutron refinement is under way; the structural results will aid in rational engineering of the enzyme.
Room-temperature X-ray and neutron diffraction data were measured from a family 11 endoxylanase holoenzyme (XynII) originating from the filamentous fungus Trichoderma longibrachiatum to 1.55 Å resolution using a home source and to 1.80 Å resolution using the Protein Crystallography Station at LANSCE. Crystals of XynII, which is an important enzyme for biofuel production, were grown at pH 8.5 in order to examine the effect of basic conditions on the protonation-state distribution in the active site and throughout the protein molecule and to provide insights for rational engineering of catalytically improved XynII for industrial applications.
PMCID: PMC3034629  PMID: 21301107
biofuels; glycosidic enzymes; endoxylanases; joint X-ray/neutron crystallography; catalytic mechanism; protonation
16.  X-CHIP: an integrated platform for high-throughput protein crystallization and on-the-chip X-ray diffraction data collection 
The X-CHIP (X-ray Crystallography High-throughput Integrated Platform) is a novel microchip that has been developed to combine multiple steps of the crystallographic pipeline from crystallization to diffraction data collection on a single device to streamline the entire process.
The X-CHIP (X-ray Crystallization High-throughput Integrated Platform) is a novel microchip that has been developed to combine multiple steps of the crystallographic pipeline from crystallization to diffraction data collection on a single device to streamline the entire process. The system has been designed for crystallization condition screening, visual crystal inspection, initial X-ray screening and data collection in a high-throughput fashion. X-ray diffraction data acquisition can be performed directly on-the-chip at room temperature using an in situ approach. The capabilities of the chip eliminate the necessity for manual crystal handling and cryoprotection of crystal samples, while allowing data collection from multiple crystals in the same drop. This technology would be especially beneficial for projects with large volumes of data, such as protein-complex studies and fragment-based screening. The platform employs hydrophilic and hydrophobic concentric ring surfaces on a miniature plate transparent to visible light and X-rays to create a well defined and stable microbatch crystallization environment. The results of crystallization and data-collection experiments demonstrate that high-quality well diffracting crystals can be grown and high-resolution diffraction data sets can be collected using this technology. Furthermore, the quality of a single-wavelength anomalous dispersion data set collected with the X-CHIP at room temperature was sufficient to generate interpretable electron-density maps. This technology is highly resource-efficient owing to the use of nanolitre-scale drop volumes. It does not require any modification for most in-house and synchrotron beamline systems and offers a promising opportunity for full automation of the X-ray structure-determination process.
PMCID: PMC3107051  PMID: 21636893
protein crystallization devices; in situ X-ray analysis; crystallization; crystal visual inspection; diffraction data collection
17.  Room-temperature macromolecular serial crystallography using synchrotron radiation 
IUCrJ  2014;1(Pt 4):204-212.
The room-temperature structure of lysozyme is determined using 40000 individual diffraction patterns from micro-crystals flowing in liquid suspension across a synchrotron microfocus beamline.
A new approach for collecting data from many hundreds of thousands of microcrystals using X-ray pulses from a free-electron laser has recently been developed. Referred to as serial crystallography, diffraction patterns are recorded at a constant rate as a suspension of protein crystals flows across the path of an X-ray beam. Events that by chance contain single-crystal diffraction patterns are retained, then indexed and merged to form a three-dimensional set of reflection intensities for structure determination. This approach relies upon several innovations: an intense X-ray beam; a fast detector system; a means to rapidly flow a suspension of crystals across the X-ray beam; and the computational infrastructure to process the large volume of data. Originally conceived for radiation-damage-free measurements with ultrafast X-ray pulses, the same methods can be employed with synchrotron radiation. As in powder diffraction, the averaging of thousands of observations per Bragg peak may improve the ratio of signal to noise of low-dose exposures. Here, it is shown that this paradigm can be implemented for room-temperature data collection using synchrotron radiation and exposure times of less than 3 ms. Using lysozyme microcrystals as a model system, over 40 000 single-crystal diffraction patterns were obtained and merged to produce a structural model that could be refined to 2.1 Å resolution. The resulting electron density is in excellent agreement with that obtained using standard X-ray data collection techniques. With further improvements the method is well suited for even shorter exposures at future and upgraded synchrotron radiation facilities that may deliver beams with 1000 times higher brightness than they currently produce.
PMCID: PMC4107920  PMID: 25075341
serial crystallography; room-temperature protein crystallography; radiation damage; CrystFEL; microfocus beamline
18.  High-resolution neutron crystallographic studies of the hydration of the coenzyme cob(II)alamin 
High-resolution crystallographic studies of the hydration of the coenzyme cob(II)alamin have provided hydrogen-bond parameters of unprecedented accuracy for a biomacromolecule.
The hydration of the coenzyme cob(II)alamin has been studied using high-resolution monochromatic neutron crystallographic data collected at room temperature to a resolution of 0.92 Å on the original D19 diffractometer with a prototype 4° × 64° detector at the high-flux reactor neutron source run by the Institute Laue–Langevin. The resulting structure provides hydrogen-bonding parameters for the hydration of biomacromolecules to unprecedented accuracy. These experimental parameters will be used to define more accurate force fields for biomacromolecular structure refinement. The presence of a hydrophobic bowl motif surrounded by flexible side chains with terminal functional groups may be significant for the efficient scavenging of ligands. The feasibility of extending the resolution of this structure to ultrahigh resolution was investigated by collecting time-of-flight neutron crystallographic data during commissioning of the TOPAZ diffracto­meter with a prototype array of 14 modular 2° × 21° detectors at the Spallation Neutron Source run by Oak Ridge National Laboratory.
PMCID: PMC3107055  PMID: 21636899
cob(II)alamin; neutron crystallography; hydration; hydrogen bonding; high resolution; D19; TOPAZ
19.  Perdeuteration, purification, crystallization and preliminary neutron diffraction of an ocean pout type III antifreeze protein 
Perdeuterated type III antifreeze protein has been expressed, purified and crystallized. Preliminary neutron data collection showed diffraction to 1.85 Å resolution from a 0.13 mm3 crystal.
The highly homologous type III antifreeze protein (AFP) subfamily share the capability to inhibit ice growth at subzero temperatures. Extensive studies by X-­ray crystallography have been conducted, mostly on AFPs from polar fishes. Although interactions between a defined flat ice-binding surface and a particular lattice plane of an ice crystal have now been identified, the fine structural features underlying the antifreeze mechanism still remain unclear owing to the intrinsic difficulty in identifying H atoms using X-ray diffraction data alone. Here, successful perdeuteration (i.e. complete deuteration) for neutron crystallo­graphic studies of the North Atlantic ocean pout (Macrozoarces americanus) AFP in Escherichia coli high-density cell cultures is reported. The perdeuterated protein (AFP D) was expressed in inclusion bodies, refolded in deuterated buffer and purified by cation-exchange chromatography. Well shaped perdeuterated AFP D crystals have been grown in D2O by the sitting-drop method. Preliminary neutron Laue diffraction at 293 K using LADI-III at ILL showed that with a few exposures of 24 h a very low background and clear small spots up to a resolution of 1.85 Å were obtained using a ‘radically small’ perdeuterated AFP D crystal of dimensions 0.70 × 0.55 × 0.35 mm, corresponding to a volume of 0.13 mm3.
PMCID: PMC2664773  PMID: 19342793
type III antifreeze proteins; neutron diffraction; perdeuteration
20.  Engineering an improved crystal contact across a solvent-mediated interface of human fibroblast growth factor 1 
A solvent-mediated crystal contact in fibroblast growth factor-1 was subjected to mutagenesis to improve crystal growth. The results indicate that improved growth was achieved upon elimination of the solvent-mediated interface and introduction of direct crystal contacts.
Large-volume protein crystals are a prerequisite for neutron diffraction studies and their production represents a bottleneck in obtaining neutron structures. Many protein crystals that permit the collection of high-resolution X-ray diffraction data are inappropriate for neutron diffraction owing to a plate-type morphology that limits the crystal volume. Human fibroblast growth factor 1 crystallizes in a plate morphology that yields atomic resolution X-ray diffraction data but has insufficient volume for neutron diffraction. The thin physical dimension has been identified as corresponding to the b cell edge and the X-ray structure identified a solvent-mediated crystal contact adjacent to position Glu81 that was hypothesized to limit efficient crystal growth in this dimension. In this report, a series of mutations at this crystal contact designed to both reduce side-chain entropy and replace the solvent-mediated interface with direct side-chain contacts are reported. The results suggest that improved crystal growth is achieved upon the introduction of direct crystal contacts, while little improvement is observed with side-chain entropy-reducing mutations alone.
PMCID: PMC2777043  PMID: 19923735
protein crystallization; side-chain entropy; neutron diffraction; protein engineering; crystal growth
21.  Experimental Electron Density and Neutron Diffraction Studies on the Polymorphs of Sulfathiazole 
Crystal Growth & Design  2014;14(3):1227-1239.
High resolution X-ray diffraction data on forms I–IV of sulfathiazole and neutron diffraction data on forms II–IV have been collected at 100 K and analyzed using the Atoms in Molecules topological approach. The molecular thermal motion as judged by the anisotropic displacement parameters (adp’s) is very similar in all four forms. The adp of the thiazole sulfur atom had the greatest amplitude perpendicular to the five-membered ring, and analysis of the temperature dependence of the adps indicates that this is due to genuine thermal motion rather than a concealed disorder. A minor disorder (∼1–2%) is evident for forms I and II, but a statistical analysis reveals no deleterious effect on the derived multipole populations. The topological analysis reveals an intramolecular S–O···S interaction, which is consistently present in all experimental topologies. Analysis of the gas-phase conformation of the molecule indicates two low-energy theoretical conformers, one of which possesses the same intramolecular S–O···S interaction observed in the experimental studies and the other an S–O···H–N intermolecular interaction. These two interactions appear responsible for “locking” the molecular conformation. The lattice energies of the various polymorphs computed from the experimental multipole populations are highly dependent on the exact refinement model. They are similar in magnitude to theoretically derived lattice energies, but the relatively high estimated errors mean that this method is insufficiently accurate to allow a definitive stability order for the sulfathiazole polymorphs at 0 K to be determined.
High resolution X-ray diffraction data on sulfathiazole (forms I−IV) and neutron diffraction data have been used to analyze the polymorphic electron density using Quantum Theory of Atoms in Molecules. Two low-energy theoretical conformers are found in the gas phase, one of which possesses an S−O···S interaction (a) and the other an S−O···H−N (b) intermolecular interaction. These interactions appear responsible for “locking” the molecular conformation.
PMCID: PMC3963452  PMID: 24672285
22.  Room-temperature X-ray diffraction studies of cisplatin and carboplatin binding to His15 of HEWL after prolonged chemical exposure 
Acta Crystallographica Section F  2012;68(Pt 11):1300-1306.
Binding of cisplatin to His15 in hen egg-white lysozyme in aqueous media is observed after prolonged chemical exposure for 15 months, in contrast to the lack of binding that was observed after 4 d in a previous study. Binding of carboplatin is seen in greater detail in the case of room-temperature data collection compared with cryo data collection.
The anticancer complexes cisplatin and carboplatin are known to bind to both the Nδ and the N∊ atoms of His15 of hen egg-white lysozyme (HEWL) in the presence of dimethyl sulfoxide (DMSO). However, neither binds in aqueous media after 4 d of crystallization and crystal growth, suggesting that DMSO facilitates cisplatin/carboplatin binding to the N atoms of His15 by an unknown mechanism. Crystals of HEWL cocrystallized with cisplatin in both aqueous and DMSO media, of HEWL cocrystallized with carboplatin in DMSO medium and of HEWL cocrystallized with cisplatin and N-acetylglucosamine (NAG) in DMSO medium were stored for between seven and 15 months. X-ray diffraction studies of these crystals were carried out on a Bruker APEX II home-source diffractometer at room temperature. Room-temperature X-ray diffraction data collection removed the need for cryoprotectants to be used, ruling out any effect that the cryoprotectants might have had on binding to the protein. Both cisplatin and carboplatin still bind to both the Nδ and N∊ atoms of His15 in DMSO media as expected, but more detail for the cyclobutanedicarboxylate (CBDC) moiety of carboplatin was observed at the N∊ binding site. However, two molecules of cisplatin were now observed to be bound to His15 in aqueous conditions. The platinum peak positions were identified using anomalous difference electron-density maps as a cross-check with F o − F c OMIT electron-density maps. The occupancies of each binding site were calculated using SHELXTL. These results show that over time cisplatin binds to both N atoms of His15 of HEWL in aqueous media, whereas this binding is speeded up in the presence of DMSO. The implication of cisplatin binding to proteins after a prolonged period of time is an important consideration for the length of treatment in patients who are given cisplatin.
PMCID: PMC3515368  PMID: 23143236
cisplatin; carboplatin; histidine; aqueous media; DMSO media; data collection at room temperature; data collection with ω versus ϕ scans with capillaries
23.  Preliminary time-of-flight neutron diffraction study on diisopropyl fluorophosphatase (DFPase) from Loligo vulgaris  
Diisopropyl fluorophosphatase (DFPase) effectively hydrolyzes a number of organophosphorus nerve agents, including sarin, cyclohexylsarin, soman and tabun. Neutron diffraction data have been collected from DFPase crystals to 2.2 Å resolution in an effort to gain further insight into the mechanism of this enzyme.
The enzyme diisopropyl fluorophosphatase (DFPase) from Loligo vulgaris is capable of decontaminating a wide variety of toxic organophosphorus nerve agents. DFPase is structurally related to a number of enzymes, such as the medically important paraoxonase (PON). In order to investigate the reaction mechanism of this phosphotriesterase and to elucidate the protonation state of the active-site residues, large-sized crystals of DFPase have been prepared for neutron diffraction studies. Available H atoms have been exchanged through vapour diffusion against D2O-containing mother liquor in the capillary. A neutron data set has been collected to 2.2 Å resolution on a relatively small (0.43 mm3) crystal at the spallation source in Los Alamos. The sample size and asymmetric unit requirements for the feasibility of neutron diffraction studies are summarized.
PMCID: PMC2330113  PMID: 17183172
neutron diffraction; DFPase; time-of-flight; phosphotriesterase
24.  Synthesis, capillary crystallization and preliminary joint X-ray and neutron crystallographic study of Z-­DNA without polyamine at low pH 
Joint X-ray and neutron crystallographic data have been collected from the oligonucleotide d(CGCGCG) crystallized without polyamine and at low pH in order to study hydration in the protein-binding major groove of Z-DNA.
In order to crystallographically study the hydration of the major groove (convex surface) of Z-DNA, the oligonucleotide d(CGCGCG) has been synthesized. Single crystals were grown by vapor diffusion using the hanging-drop and sitting-drop methods for X-ray studies and by batch crystallization and evaporation within silicon tubes for neutron studies. Hexagonal crystals were obtained without the use of duplex-stabilizing polyamines and at an acid pH. X-­ray data collected at room temperature (1.5 Å resolution; unit-cell parameters a = 17.90, b = 30.59, c = 44.61 Å) and at 100 K (1 Å resolution; a = 17.99, b = 30.98, c = 44.07 Å) and neutron data collected at room temperature (1.6 Å resolution; a = 18.00, b = 31.16, c = 44.88 Å) indicate that the DNA is in the Z-form packing in space group P212121.
PMCID: PMC2219980  PMID: 16682774
d(CGCGCG); Z-DNA; hydration
25.  Crystallization and preliminary X-ray diffraction analysis of a Lys49-phospholipase A2 complexed with caffeic acid, a molecule with inhibitory properties against snake venoms 
Piratoxin I, a noncatalytic and myotoxic Lys49-phospholipase A2 from B. pirajai venom, was cocrystallized with the inhibitor caffeic acid and a data set was collected to a resolution of 1.65 Å. The electron-density map unambiguously indicated that three inhibitor molecules interact with the C-­terminus of the protein.
Phospholipases A2 (PLA2s) are one of the main components of bothropic venoms; in addition to their phospholipid hydrolysis action, they are involved in a wide spectrum of pharmacological activities, including neurotoxicity, myo­toxicity and cardiotoxicity. Caffeic acid is an inhibitor that is present in several plants and is employed for the treatment of ophidian envenomations in the folk medicine of many developing countries; as bothropic snake bites are not efficiently neutralized by conventional serum therapy, it may be useful as an antivenom. In this work, the cocrystallization and preliminary X-ray diffraction analysis of the Lys49-PLA2 piratoxin I from Bothrops pirajai venom in the presence of the inhibitor caffeic acid (CA) are reported. The crystals diffracted X-rays to 1.65 Å resolution and the structure was solved by molecular-replacement techniques. The electron-density map unambiguously indicated the presence of three CA molecules that interact with the C-terminus of the protein. This is the first time a ligand has been observed bound to this region and is in agreement with various experiments previously reported in the literature.
PMCID: PMC3034620  PMID: 21301098
Lys49-phospholipases A2; caffeic acid; snake venoms; piratoxin I

Results 1-25 (973384)