Neutron Data Collection Research Articles

Cryotrapping peroxide in the active site of human mitochondrial manganese superoxide dismutase crystals for neutron diffraction.

Structurally identifying the enzymatic intermediates of redox proteins has been elusive due to difficulty in resolving the H atoms involved in catalysis and the susceptibility of ligand complexes to photoreduction from X-rays. Cryotrapping ligands for neutron protein crystallography combines two powerful tools that offer the advantage of directly identifying hydrogen positions in redox-enzyme intermediates without radiolytic perturbation of metal-containing active sites. However, translating cryogenic techniques from X-ray to neutron crystallography is not straightforward due to the large crystal volumes and long data-collection times. Here, methods have been developed to visualize the evasive peroxo complex of manganese superoxide dismutase (MnSOD) so that all atoms, including H atoms, could be visualized. The subsequent cryocooling and ligand-trapping methods resulted in neutron data collection to 2.30 Å resolution. The P6122 crystal form of MnSOD is challenging because it has some of the largest unit-cell dimensions (a = b = 77.8, c = 236.8 Å) ever studied using high-resolution cryo-neutron crystallography. The resulting neutron diffraction data permitted the visualization of a dioxygen species bound to the MnSOD active-site metal that was indicative of successful cryotrapping.

Crystallization of a potassium ion channel and X-ray and neutron data collection.

The mechanism by which potassium ions are transported through ion channels is currently being investigated by several groups using many different techniques. Clarification of the location of water molecules during transport is central to understanding how these integral membrane proteins function. Neutrons have a unique sensitivity to both hydrogen and potassium, rendering neutron crystallography capable of distinguishing waters from K+ ions. Here, the collection of a complete neutron data set from a potassium ion channel to a resolution of 3.55 Å using the Macromolecular Neutron Diffractometer (MaNDi) is reported. A room-temperature X-ray data set was also collected from the same crystal to a resolution of 2.50 Å. Upon further refinement, these results will help to further clarify the ion/water population within the selectivity filter of potassium ion channels.

Proton disorder in a short intramolecular hydrogen bond investigated by single-crystal neutron diffraction at 2.5 and 170 K.

The substituted acetylacetone 3-[2-(pyridin-4-yl)ethyl]pentane-2,4-dione, C12H15NO2, (1), with an ethylene bridge between the acetylacetone moiety and the heteroaromatic ring, represents an attractive linker for mixed-metal coordination polymers. In the crystal, (1) adopts an antiperiplanar conformation with respect to the C-C bond in the central ethylene group and almost coplanar acetylacetone and pyridyl groups. The ditopic molecule exists as the enol tautomer, with proton disorder in the short intramolecular hydrogen bond. Single-crystal neutron diffraction at 2.5 K indicated site occupancies of 0.602 (17) and 0.398 (17). The geometry of the acetylacetone moiety is in agreement with such a site preference of the bridging hydrogen: the O atom associated with the preferred H-atom site subtends the longer [1.305 (2) Å] and the more carbonyl-like O atom the shorter [1.288 (2) Å] C-O bond. Based on structure-factor calculations for the alternative H-atom sites, reflections particularly sensitive for proton distribution were identified and measured in a second neutron data collection at 170 K. At this temperature, 546 independent neutron intensities were used to refine positional and isotropic displacement parameters for a structure model in which parameters for C and O atoms were constrained to those obtained by single-crystal X-ray diffraction at the same temperature. The site occupancies for the disordered proton do not significantly differ from those at 2.5 K.

Preliminary neutron diffraction analysis of challenging human manganese superoxide dismutase crystals.

Superoxide dismutases (SODs) are enzymes that protect against oxidative stress by dismutation of superoxide into oxygen and hydrogen peroxide through cyclic reduction and oxidation of the active-site metal. The complete enzymatic mechanisms of SODs are unknown since data on the positions of hydrogen are limited. Here, methods are presented for large crystal growth and neutron data collection of human manganese SOD (MnSOD) using perdeuteration and the MaNDi beamline at Oak Ridge National Laboratory. The crystal from which the human MnSOD data set was obtained is the crystal with the largest unit-cell edge (240 Å) from which data have been collected via neutron diffraction to sufficient resolution (2.30 Å) where hydrogen positions can be observed.

Neutron and high-resolution room-temperature X-ray data collection from crystallized lytic polysaccharide monooxygenase.

Bacteria and fungi express lytic polysaccharide monooxgyenase (LPMO) enzymes that act in conjunction with canonical hydrolytic sugar-processing enzymes to rapidly convert polysaccharides such as chitin, cellulose and starch to single monosaccharide products. In order to gain a better understanding of the structure and oxidative mechanism of these enzymes, large crystals (1-3 mm(3)) of a chitin-processing LPMO from the Gram-positive soil bacterium Jonesia denitrificans were grown and screened for their ability to diffract neutrons. In addition to the collection of neutron diffraction data, which were processed to 2.1 Å resolution, a high-resolution room-temperature X-ray diffraction data set was collected and processed to 1.1 Å resolution in space group P212121. To our knowledge, this work marks the first successful neutron crystallographic experiment on an LPMO. Joint X-ray/neutron refinement of the resulting data will reveal new details of the structure and mechanism of this recently discovered class of enzymes.

Crystallization, neutron data collection, initial structure refinement and analysis of a xyloglucan heptamer bound to an engineered carbohydrate-binding module from xylanase.

Carbohydrate-binding modules (CBMs) are discrete parts of carbohydrate-hydrolyzing enzymes that bind specific types of carbohydrates. Ultra high-resolution X-ray crystallographic studies of CBMs have helped to decipher the basis for specificity in carbohydrate-protein interactions. However, additional studies are needed to better understand which structural determinants confer which carbohydrate-binding properties. To address these issues, neutron crystallographic studies were initiated on one experimentally engineered CBM derived from a xylanase, X-2 L110F, a protein that is able to bind several different plant carbohydrates such as xylan, β-glucan and xyloglucan. This protein evolved from a CBM present in xylanase Xyn10A of Rhodothermus marinus. The protein was complexed with a branched xyloglucan heptasaccharide. Large single crystals of hydrogenous protein (∼1.6 mm(3)) were grown at room temperature and subjected to H/D exchange. Both neutron and X-ray diffraction data sets were collected to 1.6 Å resolution. Joint neutron and X-ray refinement using phenix.refine showed significant density for residues involved in carbohydrate binding and revealed the details of a hydrogen-bonded water network around the binding site. This is the first report of a neutron structure of a CBM and will add to the understanding of protein-carbohydrate binding interactions.

Visualization of Pronation States in PLP Dependent Enzymes

Pyridoxal phosphate (PLP) dependent enzymes are essential proteins that catalyze a diverse number of reactions: transamination, racemization, phosphorylation, decarboxylation, aldol cleavage, elimination, and replacement. Despite completing a myriad of chemical reactions, these proteins have a unifying Schiff base catalytic intermediate but the overall mechanisms remain ambiguous. Based upon previous X‐ray structures and abundant literature of the different families of PLP dependent enzymes, one question must be answered: How do the different families of PLP dependent enzymes utilize different protonation profiles of pyridoxal phosphate that enable them to promote catalysis of a specific reaction? The objective of this research will be development of detailed model mechanisms for the different classes of PLP dependent enzymes, based on the protonation state of the coenzyme. Aspartate aminotransferase (AAT) and tryptophan synthase (TS) are two model PLP dependent enzymes that have shown to grow neutron diffraction quality crystals (1mm3). Neutron crystallography is an unparalleled tool to determine the atomic coordinates of the hydrogen/deuterium in macromolecules undetectable by other means. Initial neutron data collection and refinement of these proteins gives insight to the protonation state of PLP during catalysis.

Joint neutron/Xray cryocrystallography: developments for cryocooling experiments

Cryo-cooling of protein crystals is not often used in neutron crystallography. However cryo-temperatures are used to block the reaction processes at specific intermediate stages, and this has been widely used in X-ray studies (1); (2). In order to develop this area for joint neutron/X-ray applications, trypsin was chosen as a suitable system for which its interaction with the substrate succinyl-Ala-Ala-Pro-Arg-p-nitro-aniline could be studied (3). Here the neutron developments were carried out in parallel with complementary X-ray techniques, and also using in crystallo UV-visible and Raman spectroscopy. Various strategies for doing this have been tested. The installation of an N2-gas-cryostream system on the D19 single crystal diffractometer at the Institut Laue Langevin (ILL) and the development of a new carboloop mounting system, has opened new avenues to perform cryo-cooling experiments using a neutron source. Preliminary data collection carried out at the ILL and at the European Synchrotron Radiation Facility (ESRF), have confirmed the feasibility of the approach. A full description of the experimental procedures and results will be presented. As part of this a new carboloop mounting system has been developed that is suitable for both X-ray and neutron data collection. These mounts resolve the problems of activation and hydrogen incoherent scattering in neutron experiments We describe the use of these and their advantages over conventional X-ray mounting systems - including compatibility with standard magnetic goniometer heads and resistance to cryogenic temperatures.

3He spin filter based polarized neutron capability at the NIST Center for Neutron Research

A 3He neutron spin filter (NSF) program for polarized neutron scattering was launched in 2006 as part of the National Institute of Standards and Technology (NIST) Center for Neutron Research (NCNR) Expansion Initiative. The goal of the project was to enhance the NCNR polarized neutron measurement capabilities. Benefitting from more than a decade's development of spin-exchange optical pumping (SEOP) at NIST, we planned to employ SEOP based 3He neutron spin filters for the polarized neutron scattering community. These 3He NSF devices were planned for use on different classes of polarized neutron instrumentation at the NCNR, including triple-axis spectrometers (TAS), small-angle neutron scattering instruments (SANS), reflectometers, and wide-angle polarization analysis. Among them, the BT-7 thermal TAS, NG-3 SANS, and MAGIK reflectometer have already been in the user program for routine polarized beam experiments. Wide-angle polarization analysis on Multi-Axis Crystal Spectrometer (MACS) has been developed for user experiments. We describe briefly the SEOP systems dedicated for polarized beam experiments and polarizing neutron development for each instrument class. We summarize the current status and polarized neutronic performance for each instrument. We present a 3He NSF hardware and software interface to allow for synchronization of 3He polarization inversion (neutron spin flipping) and free-induction decay (FID) nuclear magnetic resonance (NMR) measurements with neutron data collection.

Cryogenic neutron protein crystallography: routine methods and potential benefits

The use of cryocooling in neutron diffraction has been hampered by several technical challenges, such as the need for specialized equipment and techniques. This article reports the recent development and deployment of equipment and strategies that allow routine neutron data collection on cryocooled crystals using off-the-shelf components. This system has several advantages compared to a closed displex cooling system, such as fast cooling coupled with easier crystal mounting and centering. The ability to routinely collect cryogenic neutron data for analysis will significantly broaden the range of scientific questions that can be examined by neutron protein crystallography. Cryogenic neutron data collection for macromolecules has recently become available at the new Biological Diffractometer BIODIFF at the FRM II and the Macromolecular Diffractometer (MaNDi) at the Spallation Neutron Source, Oak Ridge National Laboratory. To evaluate the benefits of a cryocooled neutron structure, a full neutron data set was collected on the BIODIFF instrument on a Toho-1 β-lactamase structure at 100 K.

Crystallization and preliminary neutron diffraction experiment of human farnesyl pyrophosphate synthase complexed with risedronate

Nitrogen-containing bisphosphonates (N-BPs), such as risedronate and zoledronate, are currently used as a clinical drug for bone-resorption diseases and are potent inhibitors of farnesyl pyrophosphate synthase (FPPS). X-ray crystallographic analyses of FPPS with N-BPs have revealed that N-BPs bind to FPPS with three magnesium ions and several water molecules. To understand the structural characteristics of N-BPs bound to FPPS, including H atoms and hydration by water, neutron diffraction studies were initiated using BIODIFF at the Heinz Maier-Leibnitz Zentrum (MLZ). FPPS-risedronate complex crystals of approximate dimensions 2.8 × 2.5 × 1.5 mm (∼3.5 mm(3)) were obtained by repeated macro-seeding. Monochromatic neutron diffraction data were collected to 2.4 Å resolution with 98.4% overall completeness. Here, the first successful neutron data collection from FPPS in complex with N-BPs is reported.

Neutron data collection at room temperature with tiny crystals of perdeuterated proteins

Neutron data collection at room temperature with tiny crystals of perdeuterated proteins

Rapid visualization of hydrogen positions in protein neutron crystallographic structures

Neutron crystallography is a powerful technique for experimental visualization of the positions of light atoms, including hydrogen and its isotope deuterium. In recent years, structural biologists have shown increasing interest in the technique as it uniquely complements X-ray crystallographic data by revealing the positions of D atoms in macromolecules. With this regained interest, access to macromolecular neutron crystallography beamlines is becoming a limiting step. In this report, it is shown that a rapid data-collection strategy can be a valuable alternative to longer data-collection times in appropriate cases. Comparison of perdeuterated rubredoxin structures refined against neutron data sets collected over hours and up to 5 d shows that rapid neutron data collection in just 14 h is sufficient to provide the positions of 269 D atoms without ambiguity.

Time-of-flight neutron diffraction study of bovine γ-chymotrypsin at the Protein Crystallography Station.

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 mm(3) 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.

A history of neutrons in biology: the development of neutron protein crystallography at BNL and LANL

The first neutron diffraction data were collected from crystals of myoglobin almost 42 years ago using a step-scan diffractometer with a single detector. Since then, major advances have been made in neutron sources, instrumentation and data collection and analysis, and in biochemistry. Fundamental discoveries about enzyme mechanisms, biological complex structures, protein hydration and H-atom positions have been and continue to be made using neutron diffraction. The promise of neutrons has not changed since the first crystal diffraction data were collected. Today, with the developments of beamlines at spallation neutron sources and the use of the Laue method for data collection, the field of neutrons in structural biology has renewed vitality.

Design of a novel Peltier-based cooling device and its use in neutron diffraction data collection of perdeuterated yeast pyrophosphatase

H atoms play a central role in enzymatic mechanisms, but H-atom positions cannot generally be determined by X-ray crystallography. Neutron crystallography, on the other hand, can be used to determine H-atom positions but it is experimentally very challenging. Yeast inorganic pyrophosphatase (PPase) is an essential enzyme that has been studied extensively by X-ray crystallography, yet the details of the catalytic mechanism remain incompletely understood. The temperature instability of PPase crystals has in the past prevented the collection of a neutron diffraction data set. This paper reports how the crystal growth has been optimized in temperature-controlled conditions. To stabilize the crystals during neutron data collection a Peltier cooling device that minimizes the temperature gradient along the capillary has been developed. This device allowed the collection of a full neutron diffraction data set.

High-resolution X-ray and neutron data collection on antifreeze protein

High-resolution X-ray and neutron data collection on antifreeze protein

Preface [Hot topic: Protein Crystallography in Drug Design (Guest Editor: Jonathan B. Cooper)

he value of structure analysis of proteins in rational drug design is undisputed. The ability to visualize the three dimensional structure of a receptor or enzyme provides a unique view of the residues that make up the protein's ligand binding or catalytic site. The analysis of complexes with ligands or inhibitors provides many clues on the roles that these residues play in the functioning of the molecule. The information derived from this 'picture' can be used to design the next generation of inhibitors with improved complementarity to the ligand-binding site. Whilst many other considerations such as solubility, lipophilicity, chemical stability and bioavailability also 'shape' the drug design process, crystallography can assist in designing compounds which minimize the problems of resistance that arise, for example, when drugs are used to treat infections by viruses that undergo rapid mutation. In this volume, Elspeth Garman (Oxford) and Graeme Laver (Canberra) describe the impact which structural studies have had on the design of influenza drugs. Despite the fact that influenza is a disease which affects millions of people, sometimes with fatal consequences, there has not, until recently, been any drug effective against all strains and vaccines may be relatively or totally ineffective. Screening of many thousands of compounds by pharmaceutical companies has resulted in only two compounds, amantadine and rimantidine, which target the M2 ion channel on the virus and these drugs have major disadvantages. Knowledge of the crystal structure of influenza virus neuraminidase, on the other hand, has allowed the rational design of four “plug-drugs” which bind to the active site of flu neuraminidase and stop replication of the virus. Two of these compounds, Relenza and Tamiflu, are now being used world wide and, although effective when used properly, suffer from problems of delivery. They need to be given very soon after infection to be effective, they only inhibit the influenza virus and none of the other respiratory agents which cause flu-like symptoms, and they are very expensive. The review by Leo Brady and Gus Cameron (Bristol) details the application of protein crystallography and other structural techniques to identifying novel drug targets from plasmodia, the causative agent of malaria. The current use of structure-based design in developing effective drugs for plasmodial targets is discussed. The review by Steve Wood and Simon Kolstoe (Southampton) summarises the known properties of amyloid fibres and proteins found associated with them. The role of X-ray crystallography in analyzing the amyloid proteins themselves and proteins that protect the fibres is discussed. In addition the analysis of complexes with compounds that inhibit fibre formation and compounds that inhibit the processing enzymes will be covered. In the next article, Chris Dealwis and Jonathan Wall (Knoxville) describe structural approaches to combating the pathogenicity of amyloid disease characterised by the deposition of monoclonal free immunoglobulin light chain proteins (LC) as amyloid fibrils within vital organs. Finally, in the review by Leighton Coates (Southampton) and Dean Myles (Grenoble), the manifold benefits of atomic resolution X-ray diffraction analysis of proteins are described. The ability to locate hydrogen atoms of interest and thus define the protonation states of many side chains including the active site groups is potentially of enormous benefit to the drug design process. In addition, the ability to improve definition of disordered regions of the molecule, to analyse ligands which bind with poor occupancy and to study anisotropic movements within the molecule provides further important data. Recent developments in neutron data collection mean that complementary information on proton positions can be provided for an increasing number of proteins. There are several hundred Xray crystal structures that have been refined to or beyond atomic resolution in the protein databank. A number of these structures are of proteins that are currently targets for drug discovery and these are surveyed to illustrate the benefits of atomic resolution X-ray analysis.

Crystallization of a large single crystal of a B-DNA decamer for a neutron diffraction experiment by the phase-diagram technique

Crystallization of a large single crystal of a B-DNA decamer, d(CCATTAATGG), for a neutron-diffraction experiment has been accomplished by an analysis of its solubility phase diagram and a large single crystal was successfully crystallized at around the minimum solubility point of the oligonucleotide: 30%(v/v) MPD, 100 mM MgCl(2) pD 6.6 using 0.4 ml D(2)O solutions of the DNA (sample concentration 1.5 mM). It is confirmed that the resulting crystal (dimensions: 1.7 x 1.3 x 0.6 mm) diffracts sufficiently well for neutron data collection.

Pentameric and decameric structures in solution of serum amyloid P component by X-ray and neutron scattering and molecular modelling analyses

Human serum amyloid P component (SAP) is a normal plasma glycoprotein and the precursor of amyloid P component which is a universal constituent of the abnormal tissue deposits in amyloidosis. X-ray and neutron scattering data showed that pentameric or decameric ring structures for SAP in solution are readily distinguished. Further neutron data collection showed that SAP pentamers were reproducibly obtained in the presence of Ca2+ at pH 5.5 or in the presence of methyl 4,6-O-(1-carboxyethylidene)-β-d-galactopyranoside (MOβDG) and Ca2+ at pH 6.0 to 8.0, while SAP decamers were obtained in the presence of EDTA between pH 5.5 and 8.0. SAP pentamers have a mean X-ray RG of 3.99(±0.11) nm and a mean neutron RG of 3.69(±0.12) nm in 100% 2H2O. SAP decamers have a mean X-ray RG of 4.23(±0.12) nm and a mean neutron RG of 4.09(±0.14) nm in 100% 2H2O. The absorption coefficients of SAP pentamers and decamers differ by 10%. If we infer that the two α-helical A-faces are in contact with each other in the SAP decamer, the lack of structural change of the decamer with pH may be explained by the absence of His residues from the A-face of the SAP pentamer, and the change in absorption coefficients is compatible with the presence of Trp residues at this A-face. The rigid ring structure of pentameric SAP provided a test of scattering curves calculated from crystal structures. The only structural unknown is the orientation of the five chemically homogeneous oligosaccharide chains relative to the protein, but extended oligosaccharide structures were found to account for its scattering curve. X-ray scattering curves were best calculated using a hydrated structure, while neutron scattering curves were best calculated using an unhydrated structure. The outcome of these analyses was used to model the structure of decameric SAP. The evaluation of 640 structures for two SAP pentamers brought face-to-face to form SAP decamers gave better curve fits for structures in which the two A-faces were in contact with each other, in which it is likely that the two pentamers were out of alignment by a rotation of 36° and the oligosaccharide chains were extended.