The IMAGINE instrument: first neutron protein structure and new capabilities for neutron macromolecular crystallography.
01 Oct 2013-Acta Crystallographica Section D-biological Crystallography (International Union of Crystallography)-Vol. 69, Iss: 10, pp 2157-2160
TL;DR: The first high-resolution neutron protein structure of perdeuterated rubredoxin from Pyrococcus furiosus (PfRd) determined using the new IMAGINE macromolecular neutron crystallography instrument at the Oak Ridge National Laboratory is reported.
Abstract: The first high-resolution neutron protein structure of perdeuterated rubredoxin from Pyrococcus furiosus (PfRd) determined using the new IMAGINE macromolecular neutron crystallography instrument at the Oak Ridge National Laboratory is reported. Neutron diffraction data extending to 1.65 A resolution were collected from a relatively small 0.7 mm3 PfRd crystal using 2.5 d (60 h) of beam time. The refined structure contains 371 out of 391, or 95%, of the D atoms of the protein and 58 solvent molecules. The IMAGINE instrument is designed to provide neutron data at or near atomic resolution (1.5 A) from crystals with volume <1.0 mm3 and with unit-cell edges <100 A. Beamline features include novel elliptical focusing mirrors that deliver neutrons into a 2.0 × 3.2 mm focal spot at the sample position with full-width vertical and horizontal divergences of 0.5 and 0.6°, respectively. Variable short- and long-wavelength cutoff optics provide automated exchange between multiple-wavelength configurations (λmin = 2.0, 2.8, 3.3 A to λmax = 3.0, 4.0, 4.5, ∼20 A). These optics produce a more than 20-fold increase in the flux density at the sample and should help to enable more routine collection of high-resolution data from submillimetre-cubed crystals. Notably, the crystal used to collect these PfRd data was 5–10 times smaller than those previously reported.
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TL;DR: Neutron crystallography and sub-atomic X-ray crystallography complement each other in defining hydrogen positions in macromolecules and much effort is still required to become a mainstream activity.
Abstract: The International Year of Crystallography saw the number of macromolecular structures deposited in the Protein Data Bank cross the 100000 mark, with more than 90000 of these provided by X-ray crystallography. The number of X-ray structures determined to sub-atomic resolution (i.e. ≤1 A) has passed 600 and this is likely to continue to grow rapidly with diffraction-limited synchrotron radiation sources such as MAX-IV (Sweden) and Sirius (Brazil) under construction. A dozen X-ray structures have been deposited to ultra-high resolution (i.e. ≤0.7 A), for which precise electron density can be exploited to obtain charge density and provide information on the bonding character of catalytic or electron transfer sites. Although the development of neutron macromolecular crystallography over the years has been far less pronounced, and its application much less widespread, the availability of new and improved instrumentation, combined with dedicated deuteration facilities, are beginning to transform the field. Of the 83 macromolecular structures deposited with neutron diffraction data, more than half (49/83, 59%) were released since 2010. Sub-mm3 crystals are now regularly being used for data collection, structures have been determined to atomic resolution for a few small proteins, and much larger unit-cell systems (cell edges >100 A) are being successfully studied. While some details relating to H-atom positions are tractable with X-ray crystallography at sub-atomic resolution, the mobility of certain H atoms precludes them from being located. In addition, highly polarized H atoms and protons (H+) remain invisible with X-rays. Moreover, the majority of X-ray structures are determined from cryo-cooled crystals at 100 K, and, although radiation damage can be strongly controlled, especially since the advent of shutterless fast detectors, and by using limited doses and crystal translation at micro-focus beams, radiation damage can still take place. Neutron crystallography therefore remains the only approach where diffraction data can be collected at room temperature without radiation damage issues and the only approach to locate mobile or highly polarized H atoms and protons. Here a review of the current status of sub-atomic X-ray and neutron macromolecular crystallography is given and future prospects for combined approaches are outlined. New results from two metalloproteins, copper nitrite reductase and cytochrome c′, are also included, which illustrate the type of information that can be obtained from sub-atomic-resolution (∼0.8 A) X-ray structures, while also highlighting the need for complementary neutron studies that can provide details of H atoms not provided by X-ray crystallography.
98 citations
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TL;DR: Methods to produce isotopically-substituted proteins and to grow large crystals are provided in the context of neutron structures reported in the literature along with technique-specific strategies including joint X-ray/neutron structure refinement.
Abstract: Neutron protein crystallography is a powerful tool for investigating protein chemistry because it directly locates hydrogen atom positions in a protein structure. The visibility of hydrogen and deuterium atoms arises from the strong interaction of neutrons with the nuclei of these isotopes. Positions can be unambiguously assigned from diffraction at resolutions typical of protein crystals. Neutrons have the additional benefit to structural biology of not inducing radiation damage in protein crystals. The same crystal could be measured multiple times for parametric studies. Here, we review the basic principles of neutron protein crystallography. The information that can be gained from a neutron structure is presented in balance with practical considerations. Methods to produce isotopically-substituted proteins and to grow large crystals are provided in the context of neutron structures reported in the literature. Available instruments for data collection and software for data processing and structure refinement are described along with technique-specific strategies including joint X-ray/neutron structure refinement. Examples are given to illustrate, ultimately, the unique scientific value of neutron protein crystal structures.
68 citations
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TL;DR: High-resolution X-ray crystal structures of an enzyme from Neurospora crassa in the resting state and of a copper(II) dioxo intermediate complex formed in the absence of substrate are determined, providing a new structural description of oxygen activation by substrate free lytic polysaccharide monooxygenases.
Abstract: Lytic polysaccharide monooxygenases have attracted vast attention owing to their abilities to disrupt glycosidic bonds via oxidation instead of hydrolysis and to enhance enzymatic digestion of recalcitrant substrates including chitin and cellulose. We have determined high-resolution X-ray crystal structures of an enzyme from Neurospora crassa in the resting state and of a copper(II) dioxo intermediate complex formed in the absence of substrate. X-ray crystal structures also revealed “pre-bound” molecular oxygen adjacent to the active site. An examination of protonation states enabled by neutron crystallography and density functional theory calculations identified a role for a conserved histidine in promoting oxygen activation. These results provide a new structural description of oxygen activation by substrate free lytic polysaccharide monooxygenases and provide insights that can be extended to reactivity in the enzyme–substrate complex.
66 citations
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TL;DR: The Bio-SANS instrument of the High Flux Isotope Reactor of Oak Ridge National Laboratory (ORNL) is a high-flux low-background SANS instrument that is, uniquely among SANS instruments, dedicated to serving the needs of the structural biology and biomaterials communities as an open-access user facility as discussed by the authors.
Abstract: Small-angle neutron scattering (SANS) is a powerful tool for characterizing complex disordered materials, including biological materials. The Bio-SANS instrument of the High Flux Isotope Reactor of Oak Ridge National Laboratory (ORNL) is a high-flux low-background SANS instrument that is, uniquely among SANS instruments, dedicated to serving the needs of the structural biology and biomaterials communities as an open-access user facility. Here, the technical specifications and performance of the Bio-SANS are presented. Sample environments developed to address the needs of the user program of the instrument are also presented. Further, the isotopic labeling and sample preparation capabilities available in the Bio-Deuteration Laboratory for users of the Bio-SANS and other neutron scattering instruments at ORNL are described. Finally, a brief survey of research performed using the Bio-SANS is presented, which demonstrates the breadth of the research that the instrument's user community engages in.
62 citations
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TL;DR: The Macromolecular Neutron Diffractometer (MaNDi) is located on beamline 11B at Oak Ridge National Laboratory as discussed by the authors, which is optimized to collect diffraction data from single crystals.
Abstract: The Macromolecular Neutron Diffractometer (MaNDi) is located on beamline 11B of the Spallation Neutron Source at Oak Ridge National Laboratory. The instrument is a neutron time-of-flight wavelength-resolved Laue diffractometer optimized to collect diffraction data from single crystals. The instrument has been designed to provide flexibility in several instrumental parameters, such as beam divergence and wavelength bandwidth, to allow data collection from a range of macromolecular systems.
55 citations
References
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TL;DR: The PHENIX software for macromolecular structure determination is described and its uses and benefits are described.
Abstract: Macromolecular X-ray crystallography is routinely applied to understand biological processes at a molecular level. However, significant time and effort are still required to solve and complete many of these structures because of the need for manual interpretation of complex numerical data using many software packages and the repeated use of interactive three-dimensional graphics. PHENIX has been developed to provide a comprehensive system for macromolecular crystallographic structure solution with an emphasis on the automation of all procedures. This has relied on the development of algorithms that minimize or eliminate subjective input, the development of algorithms that automate procedures that are traditionally performed by hand and, finally, the development of a framework that allows a tight integration between the algorithms.
15,827 citations
"The IMAGINE instrument: first neutr..." refers methods in this paper
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TL;DR: An overview of the CCP4 software suite for macromolecular crystallography is given.
Abstract: The CCP4 (Collaborative Computational Project, Number 4) software suite is a collection of programs and associated data and software libraries which can be used for macromolecular structure determination by X-ray crystallography. The suite is designed to be flexible, allowing users a number of methods of achieving their aims. The programs are from a wide variety of sources but are connected by a common infrastructure provided by standard file formats, data objects and graphical interfaces. Structure solution by macromolecular crystallography is becoming increasingly automated and the CCP4 suite includes several automation pipelines. After giving a brief description of the evolution of CCP4 over the last 30 years, an overview of the current suite is given. While detailed descriptions are given in the accompanying articles, here it is shown how the individual programs contribute to a complete software package.
9,516 citations
"The IMAGINE instrument: first neutr..." refers methods in this paper
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TL;DR: A small nonheme iron protein which can substitute for ferredoxin as an electron carrier in several reactions mediated by extracts of Clostridium pasteurianum is isolated and tentatively named rubredoxin.
Abstract: The nature and role of nonheme iron proteins which participate in a variety of biological oxidations has recently come under intensive scrutiny. Such study received considerable impetus from the discovery and characterization of ferredoxins. 1-3 During the isolation of ferredoxin a red protein has been observed in extracts of Clostridium pasteurianum.4' 5 We have now isolated this protein in crystalline form and tentatively named it rubredoxin.6 It is a small nonheme iron protein which can substitute for ferredoxin as an electron carrier in several reactions mediated by extracts of C. pasteurianum. Rubredoxin differs from the ferredoxins spectrally and chemically. The properties of purified rubredoxin are the subject of this communication.
183 citations
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TL;DR: In this article, the authors used the SRS wiggler from a protein, pea lectin, and small-molecule crystals to record transmission Laue diffraction images.
Abstract: Transmission Laue diffraction photographs can be recorded with short exposure times from stationary macromolecular and small-molecule crystals. With the use of a broad wavelength band a very large number of reflections is stimulated in a single `snapshot' of large regions of reciprocal space. Processing software has been developed which allows quantitation of the Laue data without resort to monochromatic data. The procedures have been developed and the software strategies optimized by using test data recorded on the SRS wiggler from a protein, pea lectin, and small-molecule crystals. These latter include an organic molecule, trimethyl-1H-2,1,3-benzophosphadiazine-4(3H)-thione 2,2-disulfide, referred to as BPD, and a rhodium complex, [Rh6(CO)14(dppm)], where dppm is Ph2PCH2PPh2, referred to as RHCOP. Monochromatic data were available for comparison.
177 citations
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01 Oct 1994-Nuclear Instruments & Methods in Physics Research Section A-accelerators Spectrometers Detectors and Associated Equipment
TL;DR: In this paper, two kinds of imaging plate neutron detectors (IP-ND) were developed, where the neutron converters, 6 Li and Gd, were mixed with photostimulated luminescence (PSL) materials on a flexible plastic support, and the dynamic range and spatial resolution of their IP-ND were successfully obtained as 1:10 5 and less than 0.2 mm, respectively.
Abstract: We have developed two kinds of imaging plate neutron detectors (IP-ND), where the neutron converters, 6 Li (tentatively, Nat Li was used) and Gd were mixed with photostimulated luminescence (PSL) materials on a flexible plastic support, and the dynamic range and spatial resolution of our IP-ND were successfully obtained as 1:10 5 and less than 0.2 mm, respectively, which are comparable to the ones of X-ray. The distinctive features of the IP-ND, where the IP-NDs of different neutron detection efficiency and converters were positively used, were found and discussed. The application of the IP-ND for neutron radiography was demonstrated.
166 citations
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