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1.  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
2.  Effect of lignin content on changes occurring in poplar cellulose ultrastructure during dilute acid pretreatment 
Biotechnology for Biofuels  2014;7(1):150.
Obtaining a better understanding of the complex mechanisms occurring during lignocellulosic deconstruction is critical to the continued growth of renewable biofuel production. A key step in bioethanol production is thermochemical pretreatment to reduce plant cell wall recalcitrance for downstream processes. Previous studies of dilute acid pretreatment (DAP) have shown significant changes in cellulose ultrastructure that occur during pretreatment, but there is still a substantial knowledge gap with respect to the influence of lignin on these cellulose ultrastructural changes. This study was designed to assess how the presence of lignin influences DAP-induced changes in cellulose ultrastructure, which might ultimately have large implications with respect to enzymatic deconstruction efforts.
Native, untreated hybrid poplar (Populus trichocarpa x Populus deltoids) samples and a partially delignified poplar sample (facilitated by acidic sodium chlorite pulping) were separately pretreated with dilute sulfuric acid (0.10 M) at 160°C for 15 minutes and 35 minutes, respectively . Following extensive characterization, the partially delignified biomass displayed more significant changes in cellulose ultrastructure following DAP than the native untreated biomass. With respect to the native untreated poplar, delignified poplar after DAP (in which approximately 40% lignin removal occurred) experienced: increased cellulose accessibility indicated by increased Simons’ stain (orange dye) adsorption from 21.8 to 72.5 mg/g, decreased cellulose weight-average degree of polymerization (DPw) from 3087 to 294 units, and increased cellulose crystallite size from 2.9 to 4.2 nm. These changes following DAP ultimately increased enzymatic sugar yield from 10 to 80%.
Overall, the results indicate a strong influence of lignin content on cellulose ultrastructural changes occurring during DAP. With the reduction of lignin content during DAP, the enlargement of cellulose microfibril dimensions and crystallite size becomes more apparent. Further, this enlargement of cellulose microfibril dimensions is attributed to specific processes, including the co-crystallization of crystalline cellulose driven by irreversible inter-chain hydrogen bonding (similar to hornification) and/or cellulose annealing that converts amorphous cellulose to paracrystalline and crystalline cellulose. Essentially, lignin acts as a barrier to prevent cellulose crystallinity increase and cellulose fibril coalescence during DAP.
PMCID: PMC4205766  PMID: 25342973
Cellulose ultrastructure; Lignin content; Dilute acid pretreatment; Delignification; Enzymatic sugar release; Biomass recalcitrance
3.  Joint X-ray/neutron crystallographic study of HIV-1 protease with clinical inhibitor amprenavir – insights for drug design 
Journal of medicinal chemistry  2013;56(13):10.1021/jm400684f.
HIV-1 protease is an important target for the development of antiviral inhibitors to treat AIDS. A room-temperature joint X-ray/neutron structure of the protease in complex with clinical drug amprenavir has been determined at 2.0 Å resolution. The structure provides direct determination of hydrogen atom positions in the enzyme active site. Analysis of the enzyme-drug interactions suggests that some hydrogen bonds may be weaker than deduced from the non-hydrogen interatomic distances. This information may be valuable for the design of improved protease inhibitors.
PMCID: PMC3815997  PMID: 23772563
neutron diffraction; HIV-1 protease; enzyme-drug complex; amprenavir; drug design
4.  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
5.  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.
PMCID: PMC3489103  PMID: 22948921
d-xylose isomerase; joint X-ray/neutron crystallography; protonation; hydration; metalloenzymes
6.  Low- and room-temperature X-ray structures of protein kinase A ternary complexes shed new light on its activity 
ATP bound in the active site of protein kinase A is readily hydrolysed to ADP and free phosphate by X-ray irradiation at room temperature. The phosphate ion observed in the active site causes a dramatic conformational change of the bound peptide inhibitor.
Post-translational protein phosphorylation by protein kinase A (PKA) is a ubiquitous signalling mechanism which regulates many cellular processes. A low-temperature X-ray structure of the ternary complex of the PKA catalytic subunit (PKAc) with ATP and a 20-residue peptidic inhibitor (IP20) at the physiological Mg2+ concentration of ∼0.5 mM (LT PKA–MgATP–IP20) revealed a single metal ion in the active site. The lack of a second metal in LT PKA–MgATP–IP20 renders the β- and γ-phosphoryl groups of ATP very flexible, with high thermal B factors. Thus, the second metal is crucial for tight positioning of the terminal phosphoryl group for transfer to a substrate, as demonstrated by comparison of the former structure with that of the LT PKA–Mg2ATP–IP20 complex obtained at high Mg2+ concentration. In addition to its kinase activity, PKAc is also able to slowly catalyze the hydrolysis of ATP using a water molecule as a substrate. It was found that ATP can be readily and completely hydrolyzed to ADP and a free phosphate ion in the crystals of the ternary complex PKA–Mg2ATP–IP20 by X-ray irradiation at room temperature. The cleavage of ATP may be aided by X-ray-generated free hydroxyl radicals, a very reactive chemical species, which move rapidly through the crystal at room temperature. The phosphate anion is clearly visible in the electron-density maps; it remains in the active site but slides about 2 Å from its position in ATP towards Ala21 of IP20, which mimics the phosphorylation site. The phosphate thus pushes the peptidic inhibitor away from the product ADP, while resulting in dramatic conformational changes of the terminal residues 24 and 25 of IP20. X-ray structures of PKAc in complex with the non­hydrolysable ATP analogue AMP-PNP at both room and low temperature demonstrated no temperature effects on the conformation and position of IP20.
PMCID: PMC3388813  PMID: 22751671
protein kinase A; ATPases; ATP hydrolysis
7.  Insights into the Phosphoryl Transfer Catalyzed by cAMP-Dependent Protein Kinase: An X-ray Crystallographic Study of Complexes with Various Metals and Peptide Substrate SP20 
Biochemistry  2013;52(21):3721-3727.
X-ray structures of several ternary substrate and product complexes of the catalytic subunit of cAMP-dependent protein kinase (PKAc) have been determined with different bound metal ions. In the PKAc complexes, Mg2+, Ca2+, Sr2+, and Ba2+ metal ions could bind to the active site and facilitate the phosphoryl transfer reaction. ATP and a substrate peptide (SP20) were modified, and the reaction products ADP and the phosphorylated peptide were found trapped in the enzyme active site. Finally, we determined the structure of a pseudo-Michaelis complex containing Mg2+, nonhydrolyzable AMP-PCP (β,γ-methyleneadenosine 5′-triphosphate) and SP20. The product structures together with the pseudo-Michaelis complex provide snapshots of different stages of the phosphorylation reaction. Comparison of these structures reveals conformational, coordination, and hydrogen bonding changes that might occur during the reaction and shed new light on its mechanism, roles of metals, and active site residues.
PMCID: PMC3666212  PMID: 23672593
8.  Reversible swelling of the cell wall of poplar biomass by ionic liquid at room temperature 
Bioresource technology  2010;102(6):4518-4523.
Time-resolved autofluorescence, Raman microspectroscopy, and scanning microprobe X-ray diffraction were combined in order to characterize lignocellulosic biomass from poplar trees and how it changes during treatment with the ionic liquid 1-n-ethyl-3-methylimidazolium acetate (EMIMAC) at room temperature. The EMIMAC penetrates the cell wall from the lumen, swelling the cell wall by about a factor of two towards the empty lumen. However, the middle lamella remains unchanged, preventing the cell wall from swelling outwards. During this swelling, most of the cellulose microfibrils are solubi-lized but chain migration is restricted and a small percentage of microfibrils persist. When the EMIMAC is expelled, the cellulose recrystallizes as microfibrils of cellulose I. There is little change in the relative chemical composition of the cell wall after treatment. The action of EMIMAC on the poplar cell wall at room temperature would therefore appear to be a reversible swelling and a reversible decrystallization of the cell wall.
PMCID: PMC3487160  PMID: 21247757
Pretreatment; Lignocellulosic biomass; Cellulose; Wood; Ionic liquid
9.  Ionic liquid induced changes in cellulose structure associated with enhanced biomass hydrolysis 
Biomacromolecules  2011;12(8):3091-3098.
The effects of varying ionic liquid pretreatment parameters on various sources of lignocellulosic biomass have been studied using X-ray powder diffraction, X-ray fiber diffraction and compositional analysis. Comparative enzymatic hydrolysis and sugar analysis were used to relate the observed changes in cellulose structure to biomass digestibility. In this study the factor most clearly associated with enhanced biomass hydrolysis is the conversion of cellulose fibers from the cellulose I to the cellulose II crystal phase.
PMCID: PMC3177539  PMID: 21740062
10.  A coarse-grained model for synergistic action of multiple enzymes on cellulose 
Degradation of cellulose to glucose requires the cooperative action of three classes of enzymes, collectively known as cellulases. Endoglucanases randomly bind to cellulose surfaces and generate new chain ends by hydrolyzing β-1,4-D-glycosidic bonds. Exoglucanases bind to free chain ends and hydrolyze glycosidic bonds in a processive manner releasing cellobiose units. Then, β-glucosidases hydrolyze soluble cellobiose to glucose. Optimal synergistic action of these enzymes is essential for efficient digestion of cellulose. Experiments show that as hydrolysis proceeds and the cellulose substrate becomes more heterogeneous, the overall degradation slows down. As catalysis occurs on the surface of crystalline cellulose, several factors affect the overall hydrolysis. Therefore, spatial models of cellulose degradation must capture effects such as enzyme crowding and surface heterogeneity, which have been shown to lead to a reduction in hydrolysis rates.
We present a coarse-grained stochastic model for capturing the key events associated with the enzymatic degradation of cellulose at the mesoscopic level. This functional model accounts for the mobility and action of a single cellulase enzyme as well as the synergy of multiple endo- and exo-cellulases on a cellulose surface. The quantitative description of cellulose degradation is calculated on a spatial model by including free and bound states of both endo- and exo-cellulases with explicit reactive surface terms (e.g., hydrogen bond breaking, covalent bond cleavages) and corresponding reaction rates. The dynamical evolution of the system is simulated by including physical interactions between cellulases and cellulose.
Our coarse-grained model reproduces the qualitative behavior of endoglucanases and exoglucanases by accounting for the spatial heterogeneity of the cellulose surface as well as other spatial factors such as enzyme crowding. Importantly, it captures the endo-exo synergism of cellulase enzyme cocktails. This model constitutes a critical step towards testing hypotheses and understanding approaches for maximizing synergy and substrate properties with a goal of cost effective enzymatic hydrolysis.
PMCID: PMC3475064  PMID: 22853643
Cellulose degradation; Synergy; Exo-cellulase; Endo-cellulase; Agent-based model; Spatial heterogeneity
11.  Water in Crystalline Fibers of Dihydrate β-Chitin Results in Unexpected Absence of Intramolecular Hydrogen Bonding 
PLoS ONE  2012;7(6):e39376.
The complete crystal structure (including hydrogen) of dihydrate β-chitin, a homopolymer of N-acetylglucosamine hydrate, was determined using high-resolution X-ray and neutron fiber diffraction data collected from bathophilous tubeworm Lamellibrachia satsuma. Two water molecules per N-acetylglucosamine residue are clearly localized in the structure and these participate in most of the hydrogen bonds. The conformation of the labile acetamide groups and hydroxymethyl groups are similar to those found in anhydrous β-chitin, but more relaxed. Unexpectedly, the intrachain O3-H…O5 hydrogen bond typically observed for crystalline β,1–4 glycans is absent, providing important insights into its relative importance and its relationship to solvation.
PMCID: PMC3378566  PMID: 22724007
12.  Time-resolved X-ray diffraction microprobe studies of the conversion of cellulose I to ethylenediamine-cellulose I 
Cellulose (London, England)  2010;17(4):735-745.
Structural changes during the treatment of films of highly crystalline microfibers of Cladophora cellulose with ethylenediamine (EDA) have been studied by time-resolved X-ray microprobe diffraction methods. As EDA penetrates the sample and converts cellulose I to EDA-cellulose I, the measured profile widths of reflections reveal changes in the shapes and average dimensions of cellulose I and EDA-cellulose I crystals. The (200) direction of cellulose I is most resistant to EDA penetration, with EDA penetrating most effectively at the hydrophilic edges of the hydrogen bonded sheets of cellulose chains. Most of the cellulose chains in the initial crystals of cellulose I are incorporated into crystals of EDA-cellulose I. The size of the emerging EDA-cellulose I crystals is limited to about half of their size in cellulose I, most likely due to strains introduced by the penetration of EDA molecules. There is no evidence of any gradual structural transition from cellulose I to EDA-cellulose I involving a continuously changing intermediate phase. Rather, the results point to a rapid transition to EDA-cellulose I in regions of the microfibrils that have been penetrated by EDA.
PMCID: PMC3371382  PMID: 22693365
X-ray crystallography; Time-resolved; Cellulose; Ethylenediamine; Scanning microprobe
13.  Neutron structure of human carbonic anhydrase II: A hydrogen bonded water network “switch” is observed between pH 7.8 and 10.0 
Biochemistry  2011;50(44):9421-9423.
The neutron structure of wild type human carbonic anhydrase II at pH 7.8 has been determined to 2.0 Å resolution. Detailed analysis and comparison to the previously determined structure at pH 10.0 shows important differences in protonation of key catalytic residues in the active site as well as a rearrangement of the hydrogen bonded water network. For the first time, a completed hydrogen bonded network stretching from the Zn-bound solvent to the proton shuttling residue His64 has been directly observed.
PMCID: PMC3371383  PMID: 21988105
neutron diffraction; hydrogen bond; carbonic anhydrase; deuterium; proton transfer
14.  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
15.  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
16.  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
17.  Neutron structure and mechanistic studies of diisopropyl fluorophosphatase (DFPase) 
The structure and mechanism of diisopropyl fluorophosphatase (DFPase) have been studied using a variety of methods, including isotopic labelling, X-ray crystallography and neutron crystallography. The neutron structure of DFPase, mechanistic studies and subsequent rational design efforts are described.
Diisopropyl fluorophosphatase (DFPase) is a calcium-dependent phosphotriesterase that acts on a variety of highly toxic organophosphorus compounds that act as inhibitors of acetylcholinesterase. The mechanism of DFPase has been probed using a variety of methods, including isotopic labelling, which demonstrated the presence of a phosphoenzyme intermediate in the reaction mechanism. In order to further elucidate the mechanism of DFPase and to ascertain the protonation states of the residues and solvent molecules in the active site, the neutron structure of DFPase was solved at 2.2 Å resolution. The proposed nucleophile Asp229 is deprotonated, while the active-site solvent molecule W33 was identified as water and not hydroxide. These data support a mechanism involving direct nucleophilic attack by Asp229 on the substrate and rule out a mechanism involving metal-assisted water activation. These data also allowed for the re-engineering of DFPase through rational design to bind and productively orient the more toxic S P stereoisomers of the nerve agents sarin and cyclosarin, creating a modified enzyme with enhanced overall activity and significantly increased detoxification properties.
PMCID: PMC2967418  PMID: 21041927
neutron crystallography; DFPase; enzymes; rational design; phosphotriesterases; mechanism
18.  Protonation states of histidine and other key residues in deoxy normal human adult hemoglobin by neutron protein crystallography 
Using neutron diffraction analysis, the protonation states of 35 of 38 histidine residues were determined for the deoxy form of normal human adult hemoglobin. Distal and buried histidines may contribute to the increased affinity of the deoxy state for hydrogen ions and its decreased affinity for oxygen compared with the oxygenated form.
The protonation states of the histidine residues key to the function of deoxy (T-state) human hemoglobin have been investigated using neutron protein crystallography. These residues can reversibly bind protons, thereby regulating the oxygen affinity of hemoglobin. By examining the OMIT F o − F c and 2F o − F c neutron scattering maps, the protonation states of 35 of the 38 His residues were directly determined. The remaining three residues were found to be disordered. Surprisingly, seven pairs of His residues from equivalent α or β chains, αHis20, αHis50, αHis58, αHis89, βHis63, βHis143 and βHis146, have different protonation states. The proton­ation of distal His residues in the α1β1 heterodimer and the protonation of αHis103 in both subunits demonstrates that these residues may participate in buffering hydrogen ions and may influence the oxygen binding. The observed protonation states of His residues are compared with their ΔpK a between the deoxy and oxy states. Examination of inter-subunit interfaces provided evidence for interactions that are essential for the stability of the deoxy tertiary structure.
PMCID: PMC2967419  PMID: 21041929
human hemoglobin; deoxy form; protonation states; neutron protein crystallography; Bohr effect; histidine; deuterated water
19.  Joint X-ray and neutron refinement with phenix.refine  
The implementation of crystallographic structure-refinement procedures that include both X-ray and neutron data (separate or jointly) in the PHENIX system is described.
Approximately 85% of the structures deposited in the Protein Data Bank have been solved using X-ray crystallography, making it the leading method for three-dimensional structure determination of macromolecules. One of the limitations of the method is that the typical data quality (resolution) does not allow the direct determination of H-atom positions. Most hydrogen positions can be inferred from the positions of other atoms and therefore can be readily included into the structure model as a priori knowledge. However, this may not be the case in biologically active sites of macromolecules, where the presence and position of hydrogen is crucial to the enzymatic mechanism. This makes the application of neutron crystallo­graphy in biology particularly important, as H atoms can be clearly located in experimental neutron scattering density maps. Without exception, when a neutron structure is determined the corresponding X-ray structure is also known, making it possible to derive the complete structure using both data sets. Here, the implementation of crystallographic structure-refinement procedures that include both X-ray and neutron data (separate or jointly) in the PHENIX system is described.
PMCID: PMC2967420  PMID: 21041930
structure refinement; neutrons; joint X-ray and neutron refinement; PHENIX
20.  Macromolecular neutron crystallography at the Protein Crystallography Station (PCS) 
The Protein Crystallography Station user facility at Los Alamos National Laboratory not only offers open access to a high-performance neutron beamline, but also actively supports and develops new methods in protein expression, deuteration, purification, robotic crystallization and the synthesis of substrates with stable isotopes and provides assistance with data-reduction and structure-refinement software and comprehensive neutron structure analysis.
The Protein Crystallography Station (PCS) at Los Alamos Neutron Science Center is a high-performance beamline that forms the core of a capability for neutron macromolecular structure and function determination. Neutron diffraction is a powerful technique for locating H atoms and can therefore provide unique information about how biological macro­molecules function and interact with each other and smaller molecules. Users of the PCS have access to neutron beam time, deuteration facilities, the expression of proteins and the synthesis of substrates with stable isotopes and also support for data reduction and structure analysis. The beamline exploits the pulsed nature of spallation neutrons and a large electronic detector in order to collect wavelength-resolved Laue patterns using all available neutrons in the white beam. The PCS user facility is described and highlights from the user program are presented.
PMCID: PMC2967422  PMID: 21041938
Protein Crystallography Station; neutron macromolecular crystallography; spallation neutron sources; deuteration; user support
21.  Hemoglobin redux: combining neutron and X-ray diffraction with mass spectrometry to analyse the quaternary state of oxidized hemoglobins 
X-ray and neutron diffraction studies of cyanomethemoglobin are being used to evaluate the structural waters within the dimer–dimer interface involved in quaternary-state transitions.
Improvements in neutron diffraction instrumentation are affording the opportunity to re-examine the structures of vertebrate hemoglobins and to interrogate proton and solvent position changes between the different quaternary states of the protein. For hemoglobins of unknown primary sequence, structural studies of cyanomethemoglobin (CNmetHb) are being used to help to resolve sequence ambiguity in the mass spectra. These studies have also provided additional structural evidence for the involvement of oxidized hemoglobin in the process of erythrocyte senescence. X-ray crystal studies of Tibetan snow leopard CNmetHb have shown that this protein crystallizes in the B state, a structure with a more open dyad, which possibly has relevance to RBC band 3 protein binding and erythrocyte senescence. R-state equine CNmetHb crystal studies elaborate the solvent differences in the switch and hinge region compared with a human deoxyhemoglobin T-­state neutron structure. Lastly, comparison of histidine protonation between the T and R state should enumerate the Bohr-effect protons.
PMCID: PMC2967423  PMID: 21041946
equine hemoglobin; snow leopard hemoglobin; mass spectrometry; protein sequencing; time-of-flight neutron diffraction; R state; T state; B state; joint XN refinement; protonation state
22.  Using neutron protein crystallography to understand enzyme mechanisms 
A description is given of the results of neutron diffraction studies of the structures of four different metal-ion complexes of deuterated d-xylose isomerase.
A description is given of the results of neutron diffraction studies of the structures of four different metal-ion complexes of deuterated d-xylose isomerase. These represent four stages in the progression of the biochemical catalytic action of this enzyme. Analyses of the structural changes observed between the various three-dimensional structures lead to some insight into the mechanism of action of this enzyme.
PMCID: PMC2967424  PMID: 21041947
neutron diffraction; enzyme mechanisms; d-xylose isomerase
23.  Reintroducing Electrostatics into Macromolecular Crystallographic Refinement: Application to Neutron Crystallography and DNA Hydration 
Most current crystallographic structure refinements augment the diffraction data with a priori information consisting of bond, angle, dihedral, planarity restraints and atomic repulsion based on the Pauli exclusion principle. Yet, electrostatics and van der Waals attraction are physical forces that provide additional a priori information. Here we assess the inclusion of electrostatics for the force field used for all-atom (including hydrogen) joint neutron/X-ray refinement. Two DNA and a protein crystal structure were refined against joint neutron/X-ray diffraction data sets using force fields without electrostatics or with electrostatics. Hydrogen bond orientation/geometry favors the inclusion of electrostatics. Refinement of Z-DNA with electrostatics leads to a hypothesis for the entropic stabilization of Z-DNA that may partly explain the thermodynamics of converting the B form of DNA to its Z form. Thus, inclusion of electrostatics assists joint neutron/X-ray refinements, especially for placing and orienting hydrogen atoms.
PMCID: PMC3083928  PMID: 21481775
24.  Metal ion roles and the movement of hydrogen during the reaction catalyzed by d-xylose isomerase: a joint X-ray and neutron diffraction study 
Conversion of aldo to keto sugars by the metalloenzyme d-xylose isomerase (XI) is a multi-step reaction involving hydrogen transfer. We have determined the structure of this enzyme by neutron diffraction in order to locate H atoms (or their isotope D). Two studies are presented, one of XI containing cadmium and cyclic d-glucose (before sugar ring opening has occurred), and the other containing nickel and linear d-glucose (after ring opening has occurred but before isomerization). Previously we reported the neutron structures of ligand-free enzyme and enzyme with bound product. Data show that His54 is doubly protonated on the ring N in all four structures. Lys289 is neutral before ring opening, and gains a proton after this, the catalytic metal-bound water is deprotonated to hydroxyl during isomerization and O5 is deprotonated. These results lead to new suggestions as to how changes might take place over the course of the reaction.
PMCID: PMC2887347  PMID: 20541506
25.  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

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