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1.  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.
doi:10.1021/jm400684f
PMCID: PMC3815997  PMID: 23772563
neutron diffraction; HIV-1 protease; enzyme-drug complex; amprenavir; drug design
2.  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
3.  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
4.  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.
doi:10.1021/bi201487b
PMCID: PMC3371383  PMID: 21988105
neutron diffraction; hydrogen bond; carbonic anhydrase; deuterium; proton transfer
5.  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
6.  Towards automated crystallographic structure refinement with phenix.refine  
phenix.refine is a program within the PHENIX package that supports crystallographic structure refinement against experimental data with a wide range of upper resolution limits using a large repertoire of model parameterizations. This paper presents an overview of the major phenix.refine features, with extensive literature references for readers interested in more detailed discussions of the methods.
phenix.refine is a program within the PHENIX package that supports crystallographic structure refinement against experimental data with a wide range of upper resolution limits using a large repertoire of model parameterizations. It has several automation features and is also highly flexible. Several hundred parameters enable extensive customizations for complex use cases. Multiple user-defined refinement strategies can be applied to specific parts of the model in a single refinement run. An intuitive graphical user interface is available to guide novice users and to assist advanced users in managing refinement projects. X-ray or neutron diffraction data can be used separately or jointly in refinement. phenix.refine is tightly integrated into the PHENIX suite, where it serves as a critical component in automated model building, final structure refinement, structure validation and deposition to the wwPDB. This paper presents an overview of the major phenix.refine features, with extensive literature references for readers interested in more detailed discussions of the methods.
doi:10.1107/S0907444912001308
PMCID: PMC3322595  PMID: 22505256
structure refinement; PHENIX; joint X-ray/neutron refinement; maximum likelihood; TLS; simulated annealing; subatomic resolution; real-space refinement; twinning; NCS
7.  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
8.  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.
doi:10.1107/S0907444910034013
PMCID: PMC2967418  PMID: 21041927
neutron crystallography; DFPase; enzymes; rational design; phosphotriesterases; mechanism
9.  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.
doi:10.1107/S0907444910025448
PMCID: PMC2967419  PMID: 21041929
human hemoglobin; deoxy form; protonation states; neutron protein crystallography; Bohr effect; histidine; deuterated water
10.  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.
doi:10.1107/S0907444910026582
PMCID: PMC2967420  PMID: 21041930
structure refinement; neutrons; joint X-ray and neutron refinement; PHENIX
11.  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.
doi:10.1107/S0907444910027198
PMCID: PMC2967422  PMID: 21041938
Protein Crystallography Station; neutron macromolecular crystallography; spallation neutron sources; deuteration; user support
12.  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.
doi:10.1107/S0907444910027915
PMCID: PMC2967424  PMID: 21041947
neutron diffraction; enzyme mechanisms; d-xylose isomerase
13.  Reintroducing Electrostatics into Macromolecular Crystallographic Refinement: Application to Neutron Crystallography and DNA Hydration 
Summary
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.
doi:10.1016/j.str.2011.01.015
PMCID: PMC3083928  PMID: 21481775
14.  Metal ion roles and the movement of hydrogen during the reaction catalyzed by d-xylose isomerase: a joint X-ray and neutron diffraction study 
SUMMARY
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.
doi:10.1016/j.str.2010.03.011
PMCID: PMC2887347  PMID: 20541506
15.  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
16.  Exact direct-space asymmetric units for the 230 crystallographic space groups 
A reference table of exact direct-space asymmetric units for the 230 crystallographic space groups is presented, based on a new geometric notation for asymmetric unit conditions.
It is well known that the direct-space asymmetric unit definitions found in the International Tables for Crystallography, Volume A, are inexact at the borders. Face- and edge-specific sub-conditions have to be added to remove parts redundant under symmetry. This paper introduces a concise geometric notation for asymmetric unit conditions. The notation is the foundation for a reference table of exact direct-space asymmetric unit definitions for the 230 crystallographic space-group types. The change-of-basis transformation law for the conditions is derived, which allows the information from the reference table to be used for any space-group setting. We also show how the vertices of an asymmetric unit can easily be computed from the information in the reference table.
doi:10.1107/S0108767311007008
PMCID: PMC3082335  PMID: 21487185
asymmetric unit; direct space; space groups
17.  Neutron Structure of Human Carbonic Anhydrase II: Implications for Proton Transfer† 
Biochemistry  2010;49(3):415-421.
Human carbonic anhydrase II (HCA II) catalyzes the reversible hydration of carbon dioxide to form bicarbonate and a proton. Despite many high-resolution X-ray crystal structures, mutagenesis, and kinetic data, the structural details of the active site, especially the proton transfer pathway, are unclear. A large HCA II crystal was prepared at pH 9.0 and subjected to vapor H–D exchange to replace labile hydrogens with deuteriums. Neutron diffraction studies were conducted at the Protein Crystallography Station at Los Alamos National Laboratory. The structure to 2.0 Å resolution reveals several interesting active site features: (1) the Zn-bound solvent appearing to be predominantly a D2O molecule, (2) the orientation and hydrogen bonding pattern of solvent molecules in the active site cavity, (3) the side chain of His64 being unprotonated (neutral) and predominantly in an inward conformation pointing toward the zinc, and (4) the phenolic side chain of Tyr7 appearing to be unprotonated. The implications of these details are discussed, and a proposed mechanism for proton transfer is presented.
doi:10.1021/bi901995n
PMCID: PMC2893723  PMID: 20025241
18.  Hydrogen location in stages of an enzyme-catalyzed reaction: time-of-flight neutron structure of d-xylose isomerase with bound d-xylulose†∞ 
Biochemistry  2008;47(29):7595-7597.
The time-of-flight neutron Laue technique has been used to determine the location of hydrogen atoms in the enzyme D-xylose isomerase (XI). The neutron structure of crystalline XI with bound product, D-xylulose, shows, unexpectedly, that O5 of D-xylulose is not protonated but is hydrogen-bonded to doubly protonated His54. Also, Lys289, which is neutral in native XI, is protonated (positively charged), while the catalytic water in native XI has become activated to a hydroxyl anion which is in close proximity to C1 and C2, the molecular site of isomerization of xylose. These findings impact our understanding of the reaction mechanism.
doi:10.1021/bi8005434
PMCID: PMC2488401  PMID: 18578508
20.  Generalized X-ray and neutron crystallographic analysis: more accurate and complete structures for biological macromolecules 
X-ray and neutron crystallographic data have been combined in a joint structure-refinement procedure that has been developed using recent advances in modern computational methodologies, including cross-validated maximum-likelihood target functions with gradient-based optimization and simulated annealing.
X-ray and neutron crystallographic techniques provide complementary information on the structure and function of biological macromolecules. X-ray and neutron (XN) crystallo­graphic data have been combined in a joint structure-refinement procedure that has been developed using recent advances in modern computational methodologies, including cross-validated maximum-likelihood target functions with gradient-based optimization and simulated annealing. The XN approach for complete (including hydrogen) macromolecular structure analysis provides more accurate and complete structures, as demonstrated for diisopropyl fluorophosphatase, photoactive yellow protein and human aldose reductase. Furthermore, this method has several practical advantages, including the easier determination of the orientation of water molecules, hydroxyl groups and some amino-acid side chains.
doi:10.1107/S0907444909011548
PMCID: PMC2685734  PMID: 19465771
joint X-ray and neutron crystallography; structure refinement
21.  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
22.  Protein structures by spallation neutron crystallography 
Journal of Synchrotron Radiation  2008;15(Pt 3):215-218.
The capabilities of the Protein Crystallography Station at Los Alamos Neutron Science Center for determining protein structures by spallation neutron crystallography are illustrated, and the methodological and technological advances that are emerging from the Macromolecular Neutron Crystallography consortium are described.
The Protein Crystallography Station 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. This capability also includes the Macromolecular Neutron Crystallography (MNC) consortium between Los Alamos (LANL) and Lawrence Berkeley National Laboratories for developing computational tools for neutron protein crystallography, a biological deuteration laboratory, the National Stable Isotope Production Facility, and an MNC drug design consortium between LANL and Case Western Reserve University.
doi:10.1107/S0909049508000824
PMCID: PMC2394804  PMID: 18421142
neutrons; proteins; macromolecular crystallography; deuteration; enzyme mechanisms; drug binding; hydration; joint XN structure refinement

Results 1-22 (22)