Publications by authors named "Allen M Orville"

51 Publications

PGRP-LB: An Inside View into the Mechanism of the Amidase Reaction.

Int J Mol Sci 2021 May 7;22(9). Epub 2021 May 7.

Univ Lyon, INSA Lyon, INRAE, BF2I, UMR 203, 69621 Villeurbanne, France.

Peptidoglycan recognition proteins (PGRPs) are ubiquitous among animals and play pivotal functions in insect immunity. Non-catalytic PGRPs are involved in the activation of immune pathways by binding to the peptidoglycan (PGN), whereas amidase PGRPs are capable of cleaving the PGN into non-immunogenic compounds. PGRP-LB belongs to the amidase PGRPs and downregulates the immune deficiency (IMD) pathway by cleaving -2,6-diaminopimelic (-DAP or DAP)-type PGN. While the recognition process is well analyzed for the non-catalytic PGRPs, little is known about the enzymatic mechanism for the amidase PGRPs, despite their essential function in immune homeostasis. Here, we analyzed the specific activity of different isoforms of PGRP-LB towards various PGN substrates to understand their specificity and role in immunity. We show that these isoforms have similar activity towards the different compounds. To analyze the mechanism of the amidase activity, we performed site directed mutagenesis and solved the X-ray structures of wild-type PGRP-LB and its mutants, with one of these structures presenting a protein complexed with the tracheal cytotoxin (TCT), a muropeptide derived from the PGN. Only the Y78F mutation abolished the PGN cleavage while other mutations reduced the activity solely. Together, our findings suggest the dynamic role of the residue Y78 in the amidase mechanism by nucleophilic attack through a water molecule to the carbonyl group of the amide function destabilized by Zn.
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http://dx.doi.org/10.3390/ijms22094957DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8124813PMC
May 2021

Dynamic Structural Biology Experiments at XFEL or Synchrotron Sources.

Methods Mol Biol 2021 ;2305:203-228

Diamond Light Source Ltd, Didcot, UK.

Macromolecular crystallography (MX) leverages the methods of physics and the language of chemistry to reveal fundamental insights into biology. Often beautifully artistic images present MX results to support profound functional hypotheses that are vital to entire life science research community. Over the past several decades, synchrotrons around the world have been the workhorses for X-ray diffraction data collection at many highly automated beamlines. The newest tools include X-ray-free electron lasers (XFELs) located at facilities in the USA, Japan, Korea, Switzerland, and Germany that deliver about nine orders of magnitude higher brightness in discrete femtosecond long pulses. At each of these facilities, new serial femtosecond crystallography (SFX) strategies exploit slurries of micron-size crystals by rapidly delivering individual crystals into the XFEL X-ray interaction region, from which one diffraction pattern is collected per crystal before it is destroyed by the intense X-ray pulse. Relatively simple adaptions to SFX methods produce time-resolved data collection strategies wherein reactions are triggered by visible light illumination or by chemical diffusion/mixing. Thus, XFELs provide new opportunities for high temporal and spatial resolution studies of systems engaged in function at physiological temperature. In this chapter, we summarize various issues related to microcrystal slurry preparation, sample delivery into the X-ray interaction region, and some emerging strategies for time-resolved SFX data collection.
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http://dx.doi.org/10.1007/978-1-0716-1406-8_11DOI Listing
January 2021

Faropenem reacts with serine and metallo-β-lactamases to give multiple products.

Eur J Med Chem 2021 Apr 9;215:113257. Epub 2021 Feb 9.

Chemistry Research Laboratory, The Department of Chemistry and the Ineos Oxford Institute for Antimicrobial Research, 12 Mansfield Road, Oxford, OX1 3TA, United Kingdom. Electronic address:

Penems have demonstrated potential as antibacterials and β-lactamase inhibitors; however, their clinical use has been limited, especially in comparison with the structurally related carbapenems. Faropenem is an orally active antibiotic with a C-2 tetrahydrofuran (THF) ring, which is resistant to hydrolysis by some β-lactamases. We report studies on the reactions of faropenem with carbapenem-hydrolysing β-lactamases, focusing on the class A serine β-lactamase KPC-2 and the metallo β-lactamases (MBLs) VIM-2 (a subclass B1 MBL) and L1 (a B3 MBL). Kinetic studies show that faropenem is a substrate for all three β-lactamases, though it is less efficiently hydrolysed by KPC-2. Crystallographic analyses on faropenem-derived complexes reveal opening of the β-lactam ring with formation of an imine with KPC-2, VIM-2, and L1. In the cases of the KPC-2 and VIM-2 structures, the THF ring is opened to give an alkene, but with L1 the THF ring remains intact. Solution state studies, employing NMR, were performed on L1, KPC-2, VIM-2, VIM-1, NDM-1, OXA-23, OXA-10, and OXA-48. The solution results reveal, in all cases, formation of imine products in which the THF ring is opened; formation of a THF ring-closed imine product was only observed with VIM-1 and VIM-2. An enamine product with a closed THF ring was also observed in all cases, at varying levels. Combined with previous reports, the results exemplify the potential for different outcomes in the reactions of penems with MBLs and SBLs and imply further structure-activity relationship studies are worthwhile to optimise the interactions of penems with β-lactamases. They also exemplify how crystal structures of β-lactamase substrate/inhibitor complexes do not always reflect reaction outcomes in solution.
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http://dx.doi.org/10.1016/j.ejmech.2021.113257DOI Listing
April 2021

Recent results in time resolved serial femtosecond crystallography at XFELs.

Authors:
Allen M Orville

Curr Opin Struct Biol 2020 12 10;65:193-208. Epub 2020 Oct 10.

Diamond Light Source Ltd, Harwell Science and Innovation Campus, Didcot, OX11 0DE, United Kingdom; Research Complex at Harwell, Rutherford Appleton Laboratory, Didcot, OX11 0FA, United Kingdom. Electronic address:

Time-resolved serial femtosecond crystallography (tr-SFX) methods exploit slurries of crystalline samples that range in size from hundreds of nanometers to a few tens of micrometers, at near-physiological temperature and pressure, to generate atomic resolution models and probe authentic function with the same experiment. 'Dynamic structural biology' is often used to encompass the research philosophy and techniques. Reaction cycles for tr-SFX studies are initiated by photons or ligand addition/mixing strategies, wherein the latter are potentially generalizable across enzymology. Thus, dynamic structural biology often creates stop-motion molecular movies of macromolecular function. In metal-dependent systems, complementary spectroscopic information can also be collected from the same samples and X-ray pulses, which provides even more detailed mechanistic insights. These types of experimental data also complement quantum mechanical and classical dynamics numerical calculations. Correlated structural-functional results will yield more detailed mechanistic insights and will likely translate into better drugs and treatments impacting human health, and better catalysis for clean energy and agriculture.
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http://dx.doi.org/10.1016/j.sbi.2020.08.011DOI Listing
December 2020

Anaerobic fixed-target serial crystallography.

IUCrJ 2020 Sep 21;7(Pt 5):901-912. Epub 2020 Aug 21.

Diamond Light Source, Harwell Science and Innovation Campus, Didcot, Oxfordshire OX11 0DE, United Kingdom.

Cryogenic X-ray diffraction is a powerful tool for crystallographic studies on enzymes including oxygenases and oxidases. Amongst the benefits that cryo-conditions (usually employing a nitro-gen cryo-stream at 100 K) enable, is data collection of di-oxy-gen-sensitive samples. Although not strictly anaerobic, at low temperatures the vitreous ice conditions severely restrict O diffusion into and/or through the protein crystal. Cryo-conditions limit chemical reactivity, including reactions that require significant conformational changes. By contrast, data collection at room temperature imposes fewer restrictions on diffusion and reactivity; room-temperature serial methods are thus becoming common at synchrotrons and XFELs. However, maintaining an anaerobic environment for di-oxy-gen-dependent enzymes has not been explored for serial room-temperature data collection at synchrotron light sources. This work describes a methodology that employs an adaptation of the 'sheet-on-sheet' sample mount, which is suitable for the low-dose room-temperature data collection of anaerobic samples at synchrotron light sources. The method is characterized by easy sample preparation in an anaerobic glovebox, gentle handling of crystals, low sample consumption and preservation of a localized anaerobic environment over the timescale of the experiment (<5 min). The utility of the method is highlighted by studies with three X-ray-radiation-sensitive Fe(II)-containing model enzymes: the 2-oxoglutarate-dependent l-arginine hy-droxy-lase VioC and the DNA repair enzyme AlkB, as well as the oxidase isopenicillin N synthase (IPNS), which is involved in the biosynthesis of all penicillin and cephalosporin antibiotics.
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http://dx.doi.org/10.1107/S2052252520010374DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7467159PMC
September 2020

High-throughput in situ experimental phasing.

Acta Crystallogr D Struct Biol 2020 Aug 28;76(Pt 8):790-801. Epub 2020 Jul 28.

Diamond Light Source, Harwell Science and Innovation Campus, Didcot OX11 0DE, United Kingdom.

In this article, a new approach to experimental phasing for macromolecular crystallography (MX) at synchrotrons is introduced and described for the first time. It makes use of automated robotics applied to a multi-crystal framework in which human intervention is reduced to a minimum. Hundreds of samples are automatically soaked in heavy-atom solutions, using a Labcyte Inc. Echo 550 Liquid Handler, in a highly controlled and optimized fashion in order to generate derivatized and isomorphous crystals. Partial data sets obtained on MX beamlines using an in situ setup for data collection are processed with the aim of producing good-quality anomalous signal leading to successful experimental phasing.
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http://dx.doi.org/10.1107/S2059798320009109DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7397491PMC
August 2020

High-Resolution XFEL Structure of the Soluble Methane Monooxygenase Hydroxylase Complex with its Regulatory Component at Ambient Temperature in Two Oxidation States.

J Am Chem Soc 2020 08 5;142(33):14249-14266. Epub 2020 Aug 5.

Molecular Biophysics and Integrated Bioimaging Division, Lawrence Berkeley National Laboratory, Berkeley, California 94720, United States.

Soluble methane monooxygenase (sMMO) is a multicomponent metalloenzyme that catalyzes the conversion of methane to methanol at ambient temperature using a nonheme, oxygen-bridged dinuclear iron cluster in the active site. Structural changes in the hydroxylase component (sMMOH) containing the diiron cluster caused by complex formation with a regulatory component (MMOB) and by iron reduction are important for the regulation of O activation and substrate hydroxylation. Structural studies of metalloenzymes using traditional synchrotron-based X-ray crystallography are often complicated by partial X-ray-induced photoreduction of the metal center, thereby obviating determination of the structure of the enzyme in pure oxidation states. Here, microcrystals of the sMMOH:MMOB complex from OB3b were serially exposed to X-ray free electron laser (XFEL) pulses, where the ≤35 fs duration of exposure of an individual crystal yields diffraction data before photoreduction-induced structural changes can manifest. Merging diffraction patterns obtained from thousands of crystals generates radiation damage-free, 1.95 Å resolution crystal structures for the fully oxidized and fully reduced states of the sMMOH:MMOB complex for the first time. The results provide new insight into the manner by which the diiron cluster and the active site environment are reorganized by the regulatory protein component in order to enhance the steps of oxygen activation and methane oxidation. This study also emphasizes the value of XFEL and serial femtosecond crystallography (SFX) methods for investigating the structures of metalloenzymes with radiation sensitive metal active sites.
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http://dx.doi.org/10.1021/jacs.0c05613DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7457426PMC
August 2020

Untangling the sequence of events during the S → S transition in photosystem II and implications for the water oxidation mechanism.

Proc Natl Acad Sci U S A 2020 06 20;117(23):12624-12635. Epub 2020 May 20.

Institut für Biologie, Humboldt-Universität zu Berlin, D-10115 Berlin, Germany.

In oxygenic photosynthesis, light-driven oxidation of water to molecular oxygen is carried out by the oxygen-evolving complex (OEC) in photosystem II (PS II). Recently, we reported the room-temperature structures of PS II in the four (semi)stable S-states, S, S, S, and S, showing that a water molecule is inserted during the S → S transition, as a new bridging O(H)-ligand between Mn1 and Ca. To understand the sequence of events leading to the formation of this last stable intermediate state before O formation, we recorded diffraction and Mn X-ray emission spectroscopy (XES) data at several time points during the S → S transition. At the electron acceptor site, changes due to the two-electron redox chemistry at the quinones, Q and Q, are observed. At the donor site, tyrosine Y and His190 H-bonded to it move by 50 µs after the second flash, and Glu189 moves away from Ca. This is followed by Mn1 and Mn4 moving apart, and the insertion of O(H) at the open coordination site of Mn1. This water, possibly a ligand of Ca, could be supplied via a "water wheel"-like arrangement of five waters next to the OEC that is connected by a large channel to the bulk solvent. XES spectra show that Mn oxidation (τ of ∼350 µs) during the S → S transition mirrors the appearance of O electron density. This indicates that the oxidation state change and the insertion of water as a bridging atom between Mn1 and Ca are highly correlated.
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http://dx.doi.org/10.1073/pnas.2000529117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7293653PMC
June 2020

Photoreversible interconversion of a phytochrome photosensory module in the crystalline state.

Proc Natl Acad Sci U S A 2020 01 18;117(1):300-307. Epub 2019 Dec 18.

Department of Biology, Washington University in St. Louis, St. Louis, MO 63130;

A major barrier to defining the structural intermediates that arise during the reversible photointerconversion of phytochromes between their biologically inactive and active states has been the lack of crystals that faithfully undergo this transition within the crystal lattice. Here, we describe a crystalline form of the cyclic GMP phosphodiesterases/adenylyl cyclase/FhlA (GAF) domain from the cyanobacteriochrome PixJ in assembled with phycocyanobilin that permits reversible photoconversion between the blue light-absorbing Pb and green light-absorbing Pg states, as well as thermal reversion of Pg back to Pb. The X-ray crystallographic structure of Pb matches previous models, including autocatalytic conversion of phycocyanobilin to phycoviolobilin upon binding and its tandem thioether linkage to the GAF domain. Cryocrystallography at 150 K, which compared diffraction data from a single crystal as Pb or after irradiation with blue light, detected photoconversion product(s) based on F - F difference maps that were consistent with rotation of the bonds connecting pyrrole rings C and D. Further spectroscopic analyses showed that phycoviolobilin is susceptible to X-ray radiation damage, especially as Pg, during single-crystal X-ray diffraction analyses, which could complicate fine mapping of the various intermediate states. Fortunately, we found that PixJ crystals are amenable to serial femtosecond crystallography (SFX) analyses using X-ray free-electron lasers (XFELs). As proof of principle, we solved by room temperature SFX the GAF domain structure of Pb to 1.55-Å resolution, which was strongly congruent with synchrotron-based models. Analysis of these crystals by SFX should now enable structural characterization of the early events that drive phytochrome photoconversion.
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http://dx.doi.org/10.1073/pnas.1912041116DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6955287PMC
January 2020

Molecular basis for GTP recognition by light-activated guanylate cyclase RhGC.

FEBS J 2020 07 20;287(13):2797-2807. Epub 2019 Dec 20.

Diamond Light Source Limited, Didcot, UK.

Cyclic guanosine 3',5'-monophosphate (cGMP) is an intracellular signalling molecule involved in many sensory and developmental processes. Synthesis of cGMP from GTP is catalysed by guanylate cyclase (GC) in a reaction analogous to cAMP formation by adenylate cyclase (AC). Although detailed structural information is available on the catalytic region of nucleotidyl cyclases (NCs) in various states, these atomic models do not provide a sufficient explanation for the substrate selectivity between GC and AC family members. Detailed structural information on the GC domain in its active conformation is largely missing, and no crystal structure of a GTP-bound wild-type GC domain has been published to date. Here, we describe the crystal structure of the catalytic domain of rhodopsin-GC (RhGC) from Catenaria anguillulae in complex with GTP at 1.7 Å resolution. Our study reveals the organization of a eukaryotic GC domain in its active conformation. We observe that the binding mode of the substrate GTP is similar to that of AC-ATP interaction, although surprisingly not all of the interactions predicted to be responsible for base recognition are present. The structure provides insights into potential mechanisms of substrate discrimination and activity regulation that may be common to all class III purine NCs. DATABASE: Structural data are available in Protein Data Bank database under the accession number 6SIR. ENZYMES: EC4.6.1.2.
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http://dx.doi.org/10.1111/febs.15167DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7384201PMC
July 2020

Reducing sample consumption for serial crystallography using acoustic drop ejection.

J Synchrotron Radiat 2019 Sep 16;26(Pt 5):1820-1825. Epub 2019 Aug 16.

Diamond Light Source, Harwell Science and Innovation Campus, Didcot, Oxfordshire OX11 0DE, UK.

Efficient sample delivery is an essential aspect of serial crystallography at both synchrotrons and X-ray free-electron lasers. Rastering fixed target chips through the X-ray beam is an efficient method for serial delivery from the perspectives of both sample consumption and beam time usage. Here, an approach for loading fixed targets using acoustic drop ejection is presented that does not compromise crystal quality, can reduce sample consumption by more than an order of magnitude and allows serial diffraction to be collected from a larger proportion of the crystals in the slurry.
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http://dx.doi.org/10.1107/S1600577519009329DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6730619PMC
September 2019

Structures of the intermediates of Kok's photosynthetic water oxidation clock.

Nature 2018 11 7;563(7731):421-425. Epub 2018 Nov 7.

LCLS, SLAC National Accelerator Laboratory, Menlo Park, CA, USA.

Inspired by the period-four oscillation in flash-induced oxygen evolution of photosystem II discovered by Joliot in 1969, Kok performed additional experiments and proposed a five-state kinetic model for photosynthetic oxygen evolution, known as Kok's S-state clock or cycle. The model comprises four (meta)stable intermediates (S, S, S and S) and one transient S state, which precedes dioxygen formation occurring in a concerted reaction from two water-derived oxygens bound at an oxo-bridged tetra manganese calcium (MnCaO) cluster in the oxygen-evolving complex. This reaction is coupled to the two-step reduction and protonation of the mobile plastoquinone Q at the acceptor side of PSII. Here, using serial femtosecond X-ray crystallography and simultaneous X-ray emission spectroscopy with multi-flash visible laser excitation at room temperature, we visualize all (meta)stable states of Kok's cycle as high-resolution structures (2.04-2.08 Å). In addition, we report structures of two transient states at 150 and 400 µs, revealing notable structural changes including the binding of one additional 'water', Ox, during the S→S state transition. Our results suggest that one water ligand to calcium (W3) is directly involved in substrate delivery. The binding of the additional oxygen Ox in the S state between Ca and Mn1 supports O-O bond formation mechanisms involving O5 as one substrate, where Ox is either the other substrate oxygen or is perfectly positioned to refill the O5 position during O release. Thus, our results exclude peroxo-bond formation in the S state, and the nucleophilic attack of W3 onto W2 is unlikely.
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http://dx.doi.org/10.1038/s41586-018-0681-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6485242PMC
November 2018

Megahertz serial crystallography.

Nat Commun 2018 10 2;9(1):4025. Epub 2018 Oct 2.

European XFEL GmbH, Holzkoppel 4, 22869, Schenefeld, Germany.

The new European X-ray Free-Electron Laser is the first X-ray free-electron laser capable of delivering X-ray pulses with a megahertz inter-pulse spacing, more than four orders of magnitude higher than previously possible. However, to date, it has been unclear whether it would indeed be possible to measure high-quality diffraction data at megahertz pulse repetition rates. Here, we show that high-quality structures can indeed be obtained using currently available operating conditions at the European XFEL. We present two complete data sets, one from the well-known model system lysozyme and the other from a so far unknown complex of a β-lactamase from K. pneumoniae involved in antibiotic resistance. This result opens up megahertz serial femtosecond crystallography (SFX) as a tool for reliable structure determination, substrate screening and the efficient measurement of the evolution and dynamics of molecular structures using megahertz repetition rate pulses available at this new class of X-ray laser source.
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http://dx.doi.org/10.1038/s41467-018-06156-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6168542PMC
October 2018

Entering an era of dynamic structural biology….

Authors:
Allen M Orville

BMC Biol 2018 05 31;16(1):55. Epub 2018 May 31.

Diamond Light Source, Research Complex at Harwell, and University of Oxford, Oxfordshire, OX11 0DE, UK.

A recent paper in BMC Biology presents a general method for mix-and-inject serial crystallography, to facilitate the visualization of enzyme intermediates via time-resolved serial femtosecond crystallography (tr-SFX). They apply their method to resolve in near atomic detail the cleavage and inactivation of the antibiotic ceftriaxone by a β-lactamase enzyme from Mycobacterium tuberculosis. Their work demonstrates the general applicability of time-resolved crystallography, from which dynamic structures, at atomic resolution, can be obtained.See research article: https://bmcbiol.biomedcentral.com/articles/10.1186/s12915-018-0524-5 .
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http://dx.doi.org/10.1186/s12915-018-0533-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5977480PMC
May 2018

Where is crystallography going?

Acta Crystallogr D Struct Biol 2018 Feb 1;74(Pt 2):152-166. Epub 2018 Feb 1.

Science Division, Diamond Light Source, Harwell Science and Innovation Campus, Didcot OX11 0DE, England.

Macromolecular crystallography (MX) has been a motor for biology for over half a century and this continues apace. A series of revolutions, including the production of recombinant proteins and cryo-crystallography, have meant that MX has repeatedly reinvented itself to dramatically increase its reach. Over the last 30 years synchrotron radiation has nucleated a succession of advances, ranging from detectors to optics and automation. These advances, in turn, open up opportunities. For instance, a further order of magnitude could perhaps be gained in signal to noise for general synchrotron experiments. In addition, X-ray free-electron lasers offer to capture fragments of reciprocal space without radiation damage, and open up the subpicosecond regime of protein dynamics and activity. But electrons have recently stolen the limelight: so is X-ray crystallography in rude health, or will imaging methods, especially single-particle electron microscopy, render it obsolete for the most interesting biology, whilst electron diffraction enables structure determination from even the smallest crystals? We will lay out some information to help you decide.
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http://dx.doi.org/10.1107/S2059798317016709DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5947779PMC
February 2018

Drop-on-demand sample delivery for studying biocatalysts in action at X-ray free-electron lasers.

Nat Methods 2017 Apr 27;14(4):443-449. Epub 2017 Feb 27.

Molecular Biophysics and Integrated Bioimaging Division, Lawrence Berkeley National Laboratory, Berkeley, California, USA.

X-ray crystallography at X-ray free-electron laser sources is a powerful method for studying macromolecules at biologically relevant temperatures. Moreover, when combined with complementary techniques like X-ray emission spectroscopy, both global structures and chemical properties of metalloenzymes can be obtained concurrently, providing insights into the interplay between the protein structure and dynamics and the chemistry at an active site. The implementation of such a multimodal approach can be compromised by conflicting requirements to optimize each individual method. In particular, the method used for sample delivery greatly affects the data quality. We present here a robust way of delivering controlled sample amounts on demand using acoustic droplet ejection coupled with a conveyor belt drive that is optimized for crystallography and spectroscopy measurements of photochemical and chemical reactions over a wide range of time scales. Studies with photosystem II, the phytochrome photoreceptor, and ribonucleotide reductase R2 illustrate the power and versatility of this method.
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http://dx.doi.org/10.1038/nmeth.4195DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5376230PMC
April 2017

Structure of photosystem II and substrate binding at room temperature.

Nature 2016 12 21;540(7633):453-457. Epub 2016 Nov 21.

Stanford PULSE Institute, SLAC National Accelerator Laboratory, Menlo Park, CA 94025, USA.

Light-induced oxidation of water by photosystem II (PS II) in plants, algae and cyanobacteria has generated most of the dioxygen in the atmosphere. PS II, a membrane-bound multi-subunit pigment protein complex, couples the one-electron photochemistry at the reaction centre with the four-electron redox chemistry of water oxidation at the MnCaO cluster in the oxygen-evolving complex (OEC). Under illumination, the OEC cycles through five intermediate S-states (S to S), in which S is the dark-stable state and S is the last semi-stable state before O-O bond formation and O evolution. A detailed understanding of the O-O bond formation mechanism remains a challenge, and will require elucidation of both the structures of the OEC in the different S-states and the binding of the two substrate waters to the catalytic site. Here we report the use of femtosecond pulses from an X-ray free electron laser (XFEL) to obtain damage-free, room temperature structures of dark-adapted (S), two-flash illuminated (2F; S-enriched), and ammonia-bound two-flash illuminated (2F-NH; S-enriched) PS II. Although the recent 1.95 Å resolution structure of PS II at cryogenic temperature using an XFEL provided a damage-free view of the S state, measurements at room temperature are required to study the structural landscape of proteins under functional conditions, and also for in situ advancement of the S-states. To investigate the water-binding site(s), ammonia, a water analogue, has been used as a marker, as it binds to the MnCaO cluster in the S and S states. Since the ammonia-bound OEC is active, the ammonia-binding Mn site is not a substrate water site. This approach, together with a comparison of the native dark and 2F states, is used to discriminate between proposed O-O bond formation mechanisms.
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http://dx.doi.org/10.1038/nature20161DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5201176PMC
December 2016

Acoustic Injectors for Drop-On-Demand Serial Femtosecond Crystallography.

Structure 2016 Apr 17;24(4):631-640. Epub 2016 Mar 17.

Department of Chemistry, Boston University, Boston, MA 02215-2521, USA.

X-ray free-electron lasers (XFELs) provide very intense X-ray pulses suitable for macromolecular crystallography. Each X-ray pulse typically lasts for tens of femtoseconds and the interval between pulses is many orders of magnitude longer. Here we describe two novel acoustic injection systems that use focused sound waves to eject picoliter to nanoliter crystal-containing droplets out of microplates and into the X-ray pulse from which diffraction data are collected. The on-demand droplet delivery is synchronized to the XFEL pulse scheme, resulting in X-ray pulses intersecting up to 88% of the droplets. We tested several types of samples in a range of crystallization conditions, wherein the overall crystal hit ratio (e.g., fraction of images with observable diffraction patterns) is a function of the microcrystal slurry concentration. We report crystal structures from lysozyme, thermolysin, and stachydrine demethylase (Stc2). Additional samples were screened to demonstrate that these methods can be applied to rare samples.
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http://dx.doi.org/10.1016/j.str.2016.02.007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4920001PMC
April 2016

The structure of the giant haemoglobin from Glossoscolex paulistus.

Acta Crystallogr D Biol Crystallogr 2015 Jun 14;71(Pt 6):1257-71. Epub 2015 May 14.

Instituto de Física de São Carlos, Universidade de São Paulo, São Carlos, Brazil.

The sequences of all seven polypeptide chains from the giant haemoglobin of the free-living earthworm Glossoscolex paulistus (HbGp) are reported together with the three-dimensional structure of the 3.6 MDa complex which they form. The refinement of the full particle, which has been solved at 3.2 Å resolution, the highest resolution reported to date for a hexagonal bilayer haemoglobin composed of 12 protomers, is reported. This has allowed a more detailed description of the contacts between subunits which are essential for particle stability. Interpretation of features in the electron-density maps suggests the presence of metal-binding sites (probably Zn(2+) and Ca(2+)) and glycosylation sites, some of which have not been reported previously. The former appear to be important for the integrity of the particle. The crystal structure of the isolated d chain (d-HbGp) at 2.1 Å resolution shows different interchain contacts between d monomers compared with those observed in the full particle. Instead of forming trimers, as seen in the complex, the isolated d chains associate to form dimers across a crystallographic twofold axis. These observations eliminate the possibility that trimers form spontaneously in solution as intermediates during the formation of the dodecameric globin cap and contribute to understanding of the possible ways in which the particle self-assembles.
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http://dx.doi.org/10.1107/S1399004715005453DOI Listing
June 2015

X-ray radiation induces deprotonation of the bilin chromophore in crystalline D. radiodurans phytochrome.

J Am Chem Soc 2015 Mar 18;137(8):2792-5. Epub 2015 Feb 18.

Photon Sciences Directorate and ∥Biosciences Department, Brookhaven National Laboratory , Upton, New York 11973, United States.

We report that in the red light-absorbing (Pr) state, the bilin chromophore of the Deinococcus radiodurans proteobacterial phytochrome (DrBphP) is hypersensitive to X-ray photons used in typical synchrotron X-ray protein crystallography experiments. This causes the otherwise fully protonated chromophore to deprotonate without additional major structural changes. These results have major implications for our understanding of the structural and chemical characteristics of the resting and intermediate states of phytochromes and other photoreceptor proteins.
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http://dx.doi.org/10.1021/ja510923mDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5660918PMC
March 2015

Acoustic transfer of protein crystals from agarose pedestals to micromeshes for high-throughput screening.

Acta Crystallogr D Biol Crystallogr 2015 Jan 1;71(Pt 1):94-103. Epub 2015 Jan 1.

Photon Sciences Directorate, Brookhaven National Laboratory, Upton, NY 11973-5000, USA.

Acoustic droplet ejection (ADE) is an emerging technology with broad applications in serial crystallography such as growing, improving and manipulating protein crystals. One application of this technology is to gently transfer crystals onto MiTeGen micromeshes with minimal solvent. Once mounted on a micromesh, each crystal can be combined with different chemicals such as crystal-improving additives or a fragment library. Acoustic crystal mounting is fast (2.33 transfers s(-1)) and all transfers occur in a sealed environment that is in vapor equilibrium with the mother liquor. Here, a system is presented to retain crystals near the ejection point and away from the inaccessible dead volume at the bottom of the well by placing the crystals on a concave agarose pedestal (CAP) with the same chemical composition as the crystal mother liquor. The bowl-shaped CAP is impenetrable to crystals. Consequently, gravity will gently move the crystals into the optimal location for acoustic ejection. It is demonstrated that an agarose pedestal of this type is compatible with most commercially available crystallization conditions and that protein crystals are readily transferred from the agarose pedestal onto micromeshes with no loss in diffraction quality. It is also shown that crystals can be grown directly on CAPs, which avoids the need to transfer the crystals from the hanging drop to a CAP. This technology has been used to combine thermolysin and lysozyme crystals with an assortment of anomalously scattering heavy atoms. The results point towards a fast nanolitre method for crystal mounting and high-throughput screening.
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http://dx.doi.org/10.1107/S1399004714013728DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4304690PMC
January 2015

Crystallographic and spectroscopic snapshots reveal a dehydrogenase in action.

Nat Commun 2015 Jan 7;6:5935. Epub 2015 Jan 7.

1] Department of Chemistry, Georgia State University, Atlanta, Georgia 30303, USA [2] Molecular Basis of Disease Area of Focus Program, Georgia State University, Atlanta, Georgia 30303, USA.

Aldehydes are ubiquitous intermediates in metabolic pathways and their innate reactivity can often make them quite unstable. There are several aldehydic intermediates in the metabolic pathway for tryptophan degradation that can decay into neuroactive compounds that have been associated with numerous neurological diseases. An enzyme of this pathway, 2-aminomuconate-6-semialdehyde dehydrogenase, is responsible for 'disarming' the final aldehydic intermediate. Here we show the crystal structures of a bacterial analogue enzyme in five catalytically relevant forms: resting state, one binary and two ternary complexes, and a covalent, thioacyl intermediate. We also report the crystal structures of a tetrahedral, thiohemiacetal intermediate, a thioacyl intermediate and an NAD(+)-bound complex from an active site mutant. These covalent intermediates are characterized by single-crystal and solution-state electronic absorption spectroscopy. The crystal structures reveal that the substrate undergoes an E/Z isomerization at the enzyme active site before an sp(3)-to-sp(2) transition during enzyme-mediated oxidation.
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http://dx.doi.org/10.1038/ncomms6935DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4286809PMC
January 2015

Solvent minimization induces preferential orientation and crystal clustering in serial micro-crystallography on micro-meshes, in situ plates and on a movable crystal conveyor belt.

J Synchrotron Radiat 2014 Nov 9;21(Pt 6):1231-9. Epub 2014 Oct 9.

Photon Sciences Directorate, Brookhaven National Laboratory, Upton, NY 11973, USA.

X-ray diffraction data were obtained at the National Synchrotron Light Source from insulin and lysozyme crystals that were densely deposited on three types of surfaces suitable for serial micro-crystallography: MiTeGen MicroMeshes™, Greiner Bio-One Ltd in situ micro-plates, and a moving kapton crystal conveyor belt that is used to deliver crystals directly into the X-ray beam. 6° wedges of data were taken from ∼100 crystals mounted on each material, and these individual data sets were merged to form nine complete data sets (six from insulin crystals and three from lysozyme crystals). Insulin crystals have a parallelepiped habit with an extended flat face that preferentially aligned with the mounting surfaces, impacting the data collection strategy and the design of the serial crystallography apparatus. Lysozyme crystals had a cuboidal habit and showed no preferential orientation. Preferential orientation occluded regions of reciprocal space when the X-ray beam was incident normal to the data-collection medium surface, requiring a second pass of data collection with the apparatus inclined away from the orthogonal. In addition, crystals measuring less than 20 µm were observed to clump together into clusters of crystals. Clustering required that the X-ray beam be adjusted to match the crystal size to prevent overlapping diffraction patterns. No additional problems were encountered with the serial crystallography strategy of combining small randomly oriented wedges of data from a large number of specimens. High-quality data able to support a realistic molecular replacement solution were readily obtained from both crystal types using all three serial crystallography strategies.
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http://dx.doi.org/10.1107/S1600577514017731DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4211130PMC
November 2014

A linear relationship between crystal size and fragment binding time observed crystallographically: implications for fragment library screening using acoustic droplet ejection.

PLoS One 2014 2;9(7):e101036. Epub 2014 Jul 2.

Photon Sciences Directorate, Brookhaven National Laboratory, Upton, New York, United States of America.

High throughput screening technologies such as acoustic droplet ejection (ADE) greatly increase the rate at which X-ray diffraction data can be acquired from crystals. One promising high throughput screening application of ADE is to rapidly combine protein crystals with fragment libraries. In this approach, each fragment soaks into a protein crystal either directly on data collection media or on a moving conveyor belt which then delivers the crystals to the X-ray beam. By simultaneously handling multiple crystals combined with fragment specimens, these techniques relax the automounter duty-cycle bottleneck that currently prevents optimal exploitation of third generation synchrotrons. Two factors limit the speed and scope of projects that are suitable for fragment screening using techniques such as ADE. Firstly, in applications where the high throughput screening apparatus is located inside the X-ray station (such as the conveyor belt system described above), the speed of data acquisition is limited by the time required for each fragment to soak into its protein crystal. Secondly, in applications where crystals are combined with fragments directly on data acquisition media (including both of the ADE methods described above), the maximum time that fragments have to soak into crystals is limited by evaporative dehydration of the protein crystals during the fragment soak. Here we demonstrate that both of these problems can be minimized by using small crystals, because the soak time required for a fragment hit to attain high occupancy depends approximately linearly on crystal size.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0101036PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4079544PMC
February 2015

Hitting the target: fragment screening with acoustic in situ co-crystallization of proteins plus fragment libraries on pin-mounted data-collection micromeshes.

Acta Crystallogr D Biol Crystallogr 2014 May 30;70(Pt 5):1177-89. Epub 2014 Apr 30.

Photon Sciences Directorate, Brookhaven National Laboratory, Upton, NY 11973-5000, USA.

Acoustic droplet ejection (ADE) is a powerful technology that supports crystallographic applications such as growing, improving and manipulating protein crystals. A fragment-screening strategy is described that uses ADE to co-crystallize proteins with fragment libraries directly on MiTeGen MicroMeshes. Co-crystallization trials can be prepared rapidly and economically. The high speed of specimen preparation and the low consumption of fragment and protein allow the use of individual rather than pooled fragments. The Echo 550 liquid-handling instrument (Labcyte Inc., Sunnyvale, California, USA) generates droplets with accurate trajectories, which allows multiple co-crystallization experiments to be discretely positioned on a single data-collection micromesh. This accuracy also allows all components to be transferred through small apertures. Consequently, the crystallization tray is in equilibrium with the reservoir before, during and after the transfer of protein, precipitant and fragment to the micromesh on which crystallization will occur. This strict control of the specimen environment means that the crystallography experiments remain identical as the working volumes are decreased from the few microlitres level to the few nanolitres level. Using this system, lysozyme, thermolysin, trypsin and stachydrine demethylase crystals were co-crystallized with a small 33-compound mini-library to search for fragment hits. This technology pushes towards a much faster, more automated and more flexible strategy for structure-based drug discovery using as little as 2.5 nl of each major component.
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http://dx.doi.org/10.1107/S1399004713034603DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4014116PMC
May 2014

Macromolecular crystallography beamline X25 at the NSLS.

J Synchrotron Radiat 2014 May 8;21(Pt 3):627-32. Epub 2014 Apr 8.

Photon Sciences Directorate, Brookhaven National Laboratory, PO Box 5000, Upton, NY 11973-5000, USA.

Beamline X25 at the NSLS is one of the five beamlines dedicated to macromolecular crystallography operated by the Brookhaven National Laboratory Macromolecular Crystallography Research Resource group. This mini-gap insertion-device beamline has seen constant upgrades for the last seven years in order to achieve mini-beam capability down to 20 µm × 20 µm. All major components beginning with the radiation source, and continuing along the beamline and its experimental hutch, have changed to produce a state-of-the-art facility for the scientific community.
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http://dx.doi.org/10.1107/S1600577514003415DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3998817PMC
May 2014

Acoustic methods for high-throughput protein crystal mounting at next-generation macromolecular crystallographic beamlines.

J Synchrotron Radiat 2013 Sep 8;20(Pt 5):805-8. Epub 2013 Aug 8.

Photon Sciences Directorate, Brookhaven National Laboratory, Upton, NY 11973, USA.

To take full advantage of advanced data collection techniques and high beam flux at next-generation macromolecular crystallography beamlines, rapid and reliable methods will be needed to mount and align many samples per second. One approach is to use an acoustic ejector to eject crystal-containing droplets onto a solid X-ray transparent surface, which can then be positioned and rotated for data collection. Proof-of-concept experiments were conducted at the National Synchrotron Light Source on thermolysin crystals acoustically ejected onto a polyimide `conveyor belt'. Small wedges of data were collected on each crystal, and a complete dataset was assembled from a well diffracting subset of these crystals. Future developments and implementation will focus on achieving ejection and translation of single droplets at a rate of over one hundred per second.
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http://dx.doi.org/10.1107/S0909049513020372DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3747951PMC
September 2013

Quaternary ammonium oxidative demethylation: X-ray crystallographic, resonance Raman, and UV-visible spectroscopic analysis of a Rieske-type demethylase.

J Am Chem Soc 2012 Feb 26;134(5):2823-34. Epub 2012 Jan 26.

Department of Physiology and Biophysics, Boston University School of Medicine, Boston, Massachusetts 02218, USA.

Herein, the structure resulting from in situ turnover in a chemically challenging quaternary ammonium oxidative demethylation reaction was captured via crystallographic analysis and analyzed via single-crystal spectroscopy. Crystal structures were determined for the Rieske-type monooxygenase, stachydrine demethylase, in the unliganded state (at 1.6 Å resolution) and in the product complex (at 2.2 Å resolution). The ligand complex was obtained from enzyme aerobically cocrystallized with the substrate stachydrine (N,N-dimethylproline). The ligand electron density in the complex was interpreted as proline, generated within the active site at 100 K by the absorption of X-ray photon energy and two consecutive demethylation cycles. The oxidation state of the Rieske iron-sulfur cluster was characterized by UV-visible spectroscopy throughout X-ray data collection in conjunction with resonance Raman spectra collected before and after diffraction data. Shifts in the absorption band wavelength and intensity as a function of absorbed X-ray dose demonstrated that the Rieske center was reduced by solvated electrons generated by X-ray photons; the kinetics of the reduction process differed dramatically for the liganded complex compared to unliganded demethylase, which may correspond to the observed turnover in the crystal.
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http://dx.doi.org/10.1021/ja2111898DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5718839PMC
February 2012

Acoustically mounted microcrystals yield high-resolution X-ray structures.

Biochemistry 2011 May 9;50(21):4399-401. Epub 2011 May 9.

Biology Department, Brookhaven National Laboratory, Upton, NY 11973-5000, USA.

We demonstrate a general strategy for determining structures from showers of microcrystals. It uses acoustic droplet ejection to transfer 2.5 nL droplets from the surface of microcrystal slurries, through the air, onto mounting micromesh pins. Individual microcrystals are located by raster-scanning a several-micrometer X-ray beam across the cryocooled micromeshes. X-ray diffraction data sets merged from several micrometer-sized crystals are used to determine 1.8 Ǻ resolution crystal structures.
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http://dx.doi.org/10.1021/bi200549xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3144476PMC
May 2011

Correlated single-crystal electronic absorption spectroscopy and X-ray crystallography at NSLS beamline X26-C.

J Synchrotron Radiat 2011 May 19;18(Pt 3):358-66. Epub 2011 Mar 19.

Biology Department, Brookhaven National Laboratory, Upton, NY 11973-5000, USA.

The research philosophy and new capabilities installed at NSLS beamline X26-C to support electronic absorption and Raman spectroscopies coupled with X-ray diffraction are reviewed. This beamline is dedicated full time to multidisciplinary studies with goals that include revealing the relationship between the electronic and atomic structures in macromolecules. The beamline instrumentation has been fully integrated such that optical absorption spectra and X-ray diffraction images are interlaced. Therefore, optical changes induced by X-ray exposure can be correlated with X-ray diffraction data collection. The installation of Raman spectroscopy into the beamline is also briefly reviewed. Data are now routinely generated almost simultaneously from three complementary types of experiments from the same sample. The beamline is available now to the NSLS general user population.
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http://dx.doi.org/10.1107/S0909049511006315DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3083912PMC
May 2011