FEM: feature-enhanced map.

A method is presented that modifies a 2mFobs - DFmodel σA-weighted map such that the resulting map can strengthen a weak signal, if present, and can reduce model bias and noise. The method consists of first randomizing the starting map and filling in missing reflections using multiple methods. This is followed by restricting the map to regions with convincing density and the application of sharpening. The final map is then created by combining a series of histogram-equalized intermediate maps. In the test cases shown, the maps produced in this way are found to have increased interpretability and decreased model bias compared with the starting 2mFobs - DFmodel σA-weighted map.

X-ray crystallographic studies of family 11 xylanase Michaelis and product complexes: implications for the catalytic mechanism.

Xylanases catalyze the hydrolysis of plant hemicellulose xylan into oligosaccharides by cleaving the main-chain glycosidic linkages connecting xylose subunits. To study ligand binding and to understand how the pH constrains the activity of the enzyme, variants of the Trichoderma reesei xylanase were designed to either abolish its activity (E177Q) or to change its pH optimum (N44H). An E177Q-xylohexaose complex structure was obtained at 1.15 Šresolution which represents a pseudo-Michaelis complex and confirmed the conformational movement of the thumb region owing to ligand binding. Co-crystallization of N44H with xylohexaose resulted in a hydrolyzed xylotriose bound in the active site. Co-crystallization of the wild-type enzyme with xylopentaose trapped an aglycone xylotriose and a transglycosylated glycone product. Replacing amino acids near Glu177 decreased the xylanase activity but increased the relative activity at alkaline pH. The substrate distortion in the E177Q-xylohexaose structure expands the possible conformational itinerary of this xylose ring during the enzyme-catalyzed xylan-hydrolysis reaction.

Joint X-ray/neutron crystallographic study of HIV-1 protease with clinical inhibitor amprenavir: insights for drug design.

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.

Heterologous expression, purification, crystallization and preliminary X-ray analysis of Trichoderma reesei xylanase II and four variants.

Xylanase II from Trichoderma reesei catalyzes the hydrolysis of glycosidic bonds in xylan. Crystallographic studies of this commercially important enzyme have been initiated to investigate its reaction mechanism, substrate binding and dependence on basic pH conditions. The wild-type protein was heterologously expressed in an Escherichia coli host using the defined medium and four active-site amino acids were replaced to abolish its activity (E177Q and E86Q) or to change its pH optimum (N44D and N44H). Cation-exchange and size-exclusion chromatography were used to obtain >90% protein purity. The ligand-free proteins and variant complexes containing substrate (xylohexaose) or product (xylotriose) were crystallized in several different space groups and diffracted to high resolutions (from 1.07 to 1.55 Å).

Inhibition of D-xylose isomerase by polyols: atomic details by joint X-ray/neutron crystallography.

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 Ni(2+) cofactors and sorbitol inhibitor at pH 5.9 and a room-temperature X-ray structure of XI containing Mg(2+) 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 Ni(2+) ions occupying the catalytic metal site (M2) were found at two locations, while Mg(2+) 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.

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. 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.

Room-temperature ultrahigh-resolution time-of-flight neutron and X-ray diffraction studies of H/D-exchanged crambin.

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.

Neutron structure of human carbonic anhydrase II: a hydrogen-bonded water network "switch" is observed between pH 7.8 and 10.0.

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 show important differences in the protonation of key catalytic residues in the active site as well as a rearrangement of the H-bonded water network. For the first time, a completed H-bonded network stretching from the Zn-bound solvent to the proton shuttling residue, His64, has been directly observed.

High-resolution neutron crystallographic studies of the hydration of the coenzyme cob(II)alamin.

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.

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.

Exact direct-space asymmetric units for the 230 crystallographic space groups.

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.

Preliminary joint X-ray and neutron protein crystallographic studies of endoxylanase II from the fungus Trichoderma longibrachiatum.

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 Šresolution using a home source and to 1.80 Šresolution 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.

Neutron structure and mechanistic studies of diisopropyl fluorophosphatase (DFPase).

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.

Protonation states of histidine and other key residues in deoxy normal human adult hemoglobin by neutron protein crystallography.

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 ß chains, αHis20, αHis50, αHis58, αHis89, ßHis63, ßHis143 and ßHis146, have different protonation states. The protonation of distal His residues in the α(1)ß(1) 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.

Joint X-ray and neutron refinement with phenix.refine.

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.

Macromolecular neutron crystallography at the Protein Crystallography Station (PCS).

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.

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. 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.

Metal ion roles and the movement of hydrogen during 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 multistep reaction that involves 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. The 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.

Preliminary neutron and X-ray crystallographic studies of equine cyanomethemoglobin.

Room-temperature and 100 K X-ray and room-temperature neutron diffraction data have been measured from equine cyanomethemoglobin to 1.7 A resolution using a home source, to 1.6 A resolution on NE-CAT at the Advanced Photon Source and to 2.0 A 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.

Direct determination of protonation states of histidine residues in a 2 A neutron structure of deoxy-human normal adult hemoglobin and implications for the Bohr effect.

We have investigated the protonation states of histidine residues (potential Bohr groups) in the deoxy form (T state) of human hemoglobin by direct determination of hydrogen (deuterium) positions with the neutron protein crystallography technique. The reversible binding of protons is key to the allosteric regulation of human hemoglobin. The protonation states of 35 of the 38 His residues were directly determined from neutron scattering omit maps, with 3 of the remaining residues being disordered. Protonation states of 5 equivalent His residues--alpha His20, alpha His50, alpha His89, beta His143, and beta His146--differ between the symmetry-related globin subunits. The distal His residues, alpha His58 and beta His63, are protonated in the alpha 1 beta 1 heterodimer and are neutral in alpha 2 beta 2. Buried residue alpha His103 is found to be protonated in both subunits. These distal and buried residues have the potential to act as Bohr groups. The observed protonation states of His residues are compared to changes in their pK(a) values during the transition from the T to the R state and the results provide some new insights into our understanding of the molecular mechanism of the Bohr effect.