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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.
doi:10.1107/S1744309107061283
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.
doi:10.1107/S1744309109006630
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.
doi:10.1107/S1744309111051499
PMCID: PMC3274385  PMID: 22297981
crambin; neutron diffraction; ultrahigh resolution; H/D exchange
4.  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.
doi:10.1107/S1744309111044551
PMCID: PMC3253833  PMID: 22232170
ADP-ribose pyrophosphatase; Ndx4; neutron diffraction; Thermus thermophilus
5.  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.
doi:10.1107/S1744309110007840
PMCID: PMC2852348  PMID: 20383026
equine hemoglobin; time-of-flight neutron diffraction; R state; joint XN refinement; protonation
6.  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.
doi:10.1107/S174430911005075X
PMCID: PMC3034629  PMID: 21301107
biofuels; glycosidic enzymes; endoxylanases; joint X-ray/neutron crystallography; catalytic mechanism; protonation
7.  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.
doi:10.1107/S1744309110004318
PMCID: PMC2852326  PMID: 20383004
diisopropyl fluorophosphatase; perdeuteration
8.  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.
doi:10.1107/S1744309111009341
PMCID: PMC3087647  PMID: 21543868
neutron diffraction; γ-chymotrypsin
9.  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.
doi:10.1107/S1744309109036987
PMCID: PMC2777043  PMID: 19923735
protein crystallization; side-chain entropy; neutron diffraction; protein engineering; crystal growth
10.  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.
doi:10.1107/S090744491101496X
PMCID: PMC3107055  PMID: 21636899
cob(II)alamin; neutron crystallography; hydration; hydrogen bonding; high resolution; D19; TOPAZ
11.  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.
doi:10.1107/S1744309106006439
PMCID: PMC2197182  PMID: 16511330
urate oxidase; heavy water; phase diagram; neutron Laue diffraction
12.  Crystallization and preliminary X-ray diffraction analysis of a complex between the electron-transfer partners hexameric Cu-containing nitrite reductase and pseudoazurin 
The hexameric Cu-containing nitrite reductase and its electron-donor protein pseudoazurin have been cocrystallized using the hanging-drop vapour-diffusion method. X-ray diffraction data were collected to 3.3 Å resolution using a synchrotron-radiation source.
The complex between Cu-containing nitrite reductase (HdNIR) and its electron-donor protein pseudoazurin (HdPAz) from Hyphomicrobium denitrificans has been crystallized. The crystals were obtained from a mixture of the two proteins using the hanging-drop vapour-diffusion method in the presence of polyethylene glycol (PEG) and 2-methyl-2,4-pentanediol (MPD) as precipitants. SDS–PAGE analysis demonstrated that the crystals contained both proteins. The X-ray diffraction experiment was carried out at SPring-8 and diffraction data were collected to 3.3 Å resolution. The crystals were tetragonal (space group P41212), with unit-cell parameters a = b = 130.39, c = 505.55 Å. Preliminary analysis indicated that there was one HdNIR and at least two HdPAz molecules in the asymmetric unit of the crystal.
doi:10.1107/S1744309108040219
PMCID: PMC2635864  PMID: 19193999
Cu-containing nitrite reductases; pseudoazurins; electron-transfer complexes
13.  Crystallization and preliminary X-ray diffraction analysis of Salmonella typhimurium uridine phosphorylase complexed with 5-fluorouracil 
Uridine phosphorylase from S. typhimurium was expressed and purified and cocrystallized with the drug 5-fluorouracil. The crystals diffracted X-rays to 2.2 Å resolution using synchrotron radiation.
Uridine phosphorylase (UPh; EC 2.4.2.3) catalyzes the phosphorolytic cleavage of the N-glycosidic bond of uridine to form ribose 1-phosphate and uracil. This enzyme also activates pyrimidine-containing drugs, including 5-fluorouracil (5-FU). In order to better understand the mechanism of the enzyme–drug interaction, the complex of Salmonella typhimurium UPh with 5-FU was cocrystallized using the hanging-drop vapour-diffusion method at 294 K. X-ray diffraction data were collected to 2.2 Å resolution. Analysis of these data revealed that the crystal belonged to space group C2, with unit-cell parameters a = 158.26, b = 93.04, c = 149.87 Å, α = γ = 90, β = 90.65°. The solvent content was 45.85% assuming the presence of six hexameric molecules of the complex in the unit cell.
doi:10.1107/S1744309109016133
PMCID: PMC2688420  PMID: 19478441
uridine phosphorylase; 5-fluorouracil; Salmonella typhimurium
14.  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.
doi:10.1107/S1744309108029679
PMCID: PMC2581681  PMID: 18997326
HIV-1 protease; inhibitors; neutron diffraction
15.  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.
doi:10.1107/S174430910601236X
PMCID: PMC2219980  PMID: 16682774
d(CGCGCG); Z-DNA; hydration
16.  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.
doi:10.1107/S1744309110051407
PMCID: PMC3034620  PMID: 21301098
Lys49-phospholipases A2; caffeic acid; snake venoms; piratoxin I
17.  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.
doi:10.1107/S1744309109008574
PMCID: PMC2664773  PMID: 19342793
type III antifreeze proteins; neutron diffraction; perdeuteration
18.  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.
doi:10.1098/rsif.2009.0162.focus
PMCID: PMC2843968  PMID: 19586953
urate oxidase; neutron and X-ray crystallography; crystal growth; phase diagram; H–D exchange; protonation states
19.  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.
doi:10.1107/S1744309106052924
PMCID: PMC2330113  PMID: 17183172
neutron diffraction; DFPase; time-of-flight; phosphotriesterase
20.  Diffraction Techniques in Structural Biology 
A detailed understanding of chemical and biological function and the mechanisms underlying the activities ultimately requires atomic-resolution structural data. Diffraction-based techniques such as single-crystal X-ray crystallography, electron microscopy and neutron diffraction are well established and have paved the road to the stunning successes of modern-day structural biology. The major advances achieved in the last 20 years in all aspects of structural research, including sample preparation, crystallization, the construction of synchrotron and spallation sources, phasing approaches and high-speed computing and visualization, now provide specialists and non-specialists alike with a steady flow of molecular images of unprecedented detail. The present chapter combines a general overview of diffraction methods with a step-by-step description of the process of a single-crystal X-ray structure determination experiment, from chemical synthesis or expression to phasing and refinement, analysis and quality control. For novices it may serve as a stepping-stone to more in-depth treatises of the individual topics. Readers relying on structural information for interpreting functional data may find it a useful consumer guide.
doi:10.1002/0471142700.nc0713s41
PMCID: PMC2917260  PMID: 20517991
21.  Preliminary time-of-flight neutron diffraction study on diisopropyl fluorophosphatase (DFPase) from Loligo vulgaris 
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 2.2Å 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.
doi:10.1107/S1744309106052924
PMCID: PMC2330113  PMID: 17183172
22.  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.
doi:10.1021/cg401757z
PMCID: PMC3963452  PMID: 24672285
23.  Complicated water orientations in the minor groove of the B-DNA decamer d(CCATTAATGG)2 observed by neutron diffraction measurements 
Nucleic Acids Research  2005;33(9):3017-3024.
It has long been suspected that the structure and function of a DNA duplex can be strongly dependent on its degree of hydration. By neutron diffraction experiments, we have succeeded in determining most of the hydrogen (H) and deuterium (D) atomic positions in the decameric d(CCATTAATGG)2 duplex. Moreover, the D positions in 27 D2O molecules have been determined. In particular, the complex water network in the minor groove has been observed in detail. By a combined structural analysis using 2.0 Å resolution X-ray and 3.0 Å resolution neutron data, it is clear that the spine of hydration is built up, not only by a simple hexagonal hydration pattern (as reported in earlier X-ray studies), but also by many other water bridges hydrogen-bonded to the DNA strands. The complexity of the hydration pattern in the minor groove is derived from an extraordinary variety of orientations displayed by the water molecules.
doi:10.1093/nar/gki616
PMCID: PMC1140084  PMID: 15914673
24.  Inhibition of d-xylose isomerase by polyols: atomic details by joint X-ray/neutron crystallography 
A joint X-ray/neutron structure of d-xylose isomerase in complex with the inhibitor sorbitol was determined at room temperature at an acidic pH of 5.9. Protonation of the O5 O atom of the sugar was directly observed in the nuclear density maps. Under acidic conditions sorbitol gains a water-mediated interaction with the enzyme active site, which may explain the increased potency of the inhibitor at low pH.
d-Xylose isomerase (XI) converts the aldo-sugars xylose and glucose to their keto analogs xylulose and fructose, but is strongly inhibited by the polyols xylitol and sorbitol, especially at acidic pH. In order to understand the atomic details of polyol binding to the XI active site, a 2.0 Å resolution room-temperature joint X-ray/neutron structure of XI in complex with Ni2+ cofactors and sorbitol inhibitor at pH 5.9 and a room-temperature X-ray structure of XI containing Mg2+ ions and xylitol at the physiological pH of 7.7 were obtained. The protonation of oxygen O5 of the inhibitor, which was found to be deprotonated and negatively charged in previous structures of XI complexed with linear glucose and xylulose, was directly observed. The Ni2+ ions occupying the catalytic metal site (M2) were found at two locations, while Mg2+ in M2 is very mobile and has a high B factor. Under acidic conditions sorbitol gains a water-mediated interaction that connects its O1 hydroxyl to Asp257. This contact is not found in structures at basic pH. The new interaction that is formed may improve the binding of the inhibitor, providing an explanation for the increased affinity of the polyols for XI at low pH.
doi:10.1107/S0907444912024808
PMCID: PMC3489103  PMID: 22948921
d-xylose isomerase; joint X-ray/neutron crystallography; protonation; hydration; metalloenzymes
25.  Crystallization and preliminary X-ray analysis of phage Mu activator protein C in a complex with promoter DNA 
The isolation and preliminary X-ray analysis of crystals of phage Mu activator protein C bound to promoter DNA are reported.
Bacteriophage Mu C protein is an activator of the four Mu late promoters that drive the expression of genes encoding DNA-modification as well as phage head and tail morphogenesis proteins. This report describes the purification and cocrystallization of wild-type and selenomethionine-substituted C protein with a synthetic late promoter Psym, together with preliminary X-ray diffraction data analysis using SAD phasing. The selenomethionine peak data set was collected from a single crystal which diffracted to 3.1 Å resolution and belonged to space group P41 or P43, with unit-cell parameters a = 68.9, c = 187.6 Å and two complexes per asymmetric unit. The structure will reveal the amino acid–DNA interactions and any conformational changes associated with DNA binding.
doi:10.1107/S1744309107025286
PMCID: PMC2335125  PMID: 17620727
activator protein C; bacteriophage Mu; transcription factor

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