I24-Microfocus Macromolecular Crystallography
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Patrick
Rabe
,
John
Beale
,
Agata
Butryn
,
Pierre
Aller
,
Anna
Dirr
,
Pauline A.
Lang
,
Danny N.
Axford
,
Stephen
Carr
,
Thomas M.
Leissing
,
Michael A.
Mcdonough
,
Bradley
Davy
,
Ali
Ebrahim
,
Julien
Orlans
,
Selina L. S.
Storm
,
Allen M.
Orville
,
Christopher J.
Schofield
,
Robin L.
Owen
Diamond Proposal Number(s):
[19458]
Open Access
Abstract: 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 nitrogen cryo-stream at 100 K) enable, is data collection of dioxygen-sensitive samples. Although not strictly anaerobic, at low temperatures the vitreous ice conditions severely restrict O2 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 dioxygen-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 hydroxylase 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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Sep 2020
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I04-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
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Diamond Proposal Number(s):
[14449, 16818]
Open Access
Abstract: 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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Aug 2020
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Vivek
Srinivas
,
Rahul
Banerjee
,
Hugo
Lebrette
,
Jason C.
Jones
,
Oskar
Aurelius
,
In-sik
Kim
,
Cindy C.
Pham
,
Sheraz
Gul
,
Kyle
Sutherlin
,
Asmit
Bhowmick
,
Juliane
John
,
Esra
Bozkurt
,
Thomas
Fransson
,
Pierre
Aller
,
Agata
Butryn
,
Isabel
Bogacz
,
Philipp Stefan
Simon
,
Stephen
Keable
,
Alexander
Britz
,
Kensuke
Tono
,
Kyung-sook
Kim
,
Sang-youn
Park
,
Sang-jae
Lee
,
Jaehyun
Park
,
Roberto
Alonso-mori
,
Franklin
Fuller
,
Alexander
Batyuk
,
Aaron S.
Brewster
,
Uwe
Bergmann
,
Nicholas
Sauter
,
Allen M.
Orville
,
Vittal K.
Yachandra
,
Junko
Yano
,
John D.
Lipscomb
,
Jan F.
Kern
,
Martin
Högbom
Abstract: 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 O2 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 pure oxidation states. Here microcrystals of the sMMOH:MMOB complex from Methylosinus trichosporium 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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Jul 2020
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I24-Microfocus Macromolecular Crystallography
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E. Sethe
Burgie
,
Jonathan A.
Clinger
,
Mitchell D.
Miller
,
Aaron S.
Brewster
,
Pierre
Aller
,
Agata
Butryn
,
Franklin D.
Fuller
,
Sheraz
Gul
,
Iris D.
Young
,
Cindy C.
Pham
,
In-sik
Kim
,
Asmit
Bhowmick
,
Lee J.
O’riordan
,
Kyle D.
Sutherlin
,
Joshua V.
Heinemann
,
Alexander
Batyuk
,
Roberto
Alonso-mori
,
Mark S.
Hunter
,
Jason E.
Koglin
,
Junko
Yano
,
Vittal K.
Yachandra
,
Nicholas K.
Sauter
,
Aina E.
Cohen
,
Jan
Kern
,
Allen M.
Orville
,
George N.
Phillips
,
Richard D.
Vierstra
Diamond Proposal Number(s):
[19458]
Open Access
Abstract: 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 Thermosynechococcus elongatus 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 Fobs − Fobs 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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Dec 2019
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I24-Microfocus Macromolecular Crystallography
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Diamond Proposal Number(s):
[14493, 19458]
Open Access
Abstract: 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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Sep 2019
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Jan
Kern
,
Ruchira
Chatterjee
,
Iris D.
Young
,
Franklin D.
Fuller
,
Louise
Lassalle
,
Mohamed
Ibrahim
,
Sheraz
Gul
,
Thomas
Fransson
,
Aaron S.
Brewster
,
Roberto
Alonso-mori
,
Rana
Hussein
,
Miao
Zhang
,
Lacey
Douthit
,
Casper
De Lichtenberg
,
Mun Hon
Cheah
,
Dmitry
Shevela
,
Julia
Wersig
,
Ina
Seuffert
,
Dimosthenis
Sokaras
,
Ernest
Pastor
,
Clemens
Weninger
,
Thomas
Kroll
,
Raymond G.
Sierra
,
Pierre
Aller
,
Agata
Butryn
,
Allen M.
Orville
,
Mengning
Liang
,
Alexander
Batyuk
,
Jason E.
Koglin
,
Sergio
Carbajo
,
Sébastien
Boutet
,
Nigel W.
Moriarty
,
James M.
Holton
,
Holger
Dobbek
,
Paul D.
Adams
,
Uwe
Bergmann
,
Nicholas K.
Sauter
,
Athina
Zouni
,
Johannes
Messinger
,
Junko
Yano
,
Vittal K.
Yachandra
Abstract: 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 cycle1,2. The model comprises four (meta)stable intermediates (S0, S1, S2 and S3) and one transient S4 state, which precedes dioxygen formation occurring in a concerted reaction from two water-derived oxygens bound at an oxo-bridged tetra manganese calcium (Mn4CaO5) cluster in the oxygen-evolving complex3,4,5,6,7. This reaction is coupled to the two-step reduction and protonation of the mobile plastoquinone QB 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 S2→S3 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 S3 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 O2 release. Thus, our results exclude peroxo-bond formation in the S3 state, and the nucleophilic attack of W3 onto W2 is unlikely.
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Nov 2018
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I03-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
I04-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
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Diamond Proposal Number(s):
[8031, 8681, 5005, 4982]
Open Access
Abstract: The present article describes how to use the computer program BLEND to help assemble complete datasets for the solution of macromolecular structures, starting from partial or complete datasets, derived from data collection from multiple crystals. The program is demonstrated on more than two hundred X-ray diffraction datasets obtained from 50 crystals of a complex formed between the SRF transcription factor, its cognate DNA, and a peptide from the SRF cofactor MRTF-A. This structure is currently in the process of being fully solved. While full details of the structure are not yet available, the repeated application of BLEND on data from this structure, as they have become available, has made it possible to produce electron density maps clear enough to visualise the potential location of MRTF sequences.
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Aug 2017
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Franklin D
Fuller
,
Sheraz
Gul
,
Ruchira
Chatterjee
,
E. Sethe
Burgie
,
Iris D.
Young
,
Hugo
Lebrette
,
Vivek
Srinivas
,
Aaron
Brewster
,
Tara
Michels-clark
,
Jonathan A
Clinger
,
Babak
Andi
,
Mohamed
Ibrahim
,
Ernest
Pastor
,
Casper
De Lichtenberg
,
Rana
Hussein
,
Christopher J
Pollock
,
Miao
Zhang
,
Claudiu A
Stan
,
Thomas
Kroll
,
Thomas
Fransson
,
Clemens
Weninger
,
Markus
Kubin
,
Pierre
Aller
,
Louise
Lassalle
,
Philipp
Braeuer
,
Mitchell D.
Miller
,
Muhamed
Amin
,
Sergey
Koroidov
,
Christian G.
Roessler
,
Marc
Allaire
,
Raymond G
Sierra
,
Peter T.
Docker
,
James M.
Glownia
,
Silke
Nelson
,
Jason E
Koglin
,
Diling
Zhu
,
Matthieu
Chollet
,
Sanghoon
Song
,
Henrik
Lemke
,
Mengning
Liang
,
Dimosthenis
Sokaras
,
Roberto
Alonso-mori
,
Athina
Zouni
,
Johannes
Messinger
,
Uwe
Bergmann
,
Amie K.
Boal
,
J. Martin
Bollinger
,
Carsten
Krebs
,
Martin
Högbom
,
George N.
Phillips
,
Richard D.
Vierstra
,
Nicholas K
Sauter
,
Allen M.
Orville
,
Jan
Kern
,
Vittal K
Yachandra
,
Junko
Yano
Abstract: 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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Feb 2017
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I04-Macromolecular Crystallography
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Hanna
Kwon
,
Jaswir
Basran
,
Cecilia M.
Casadei
,
Alistair J.
Fielding
,
Tobias E.
Schrader
,
Andreas
Ostermann
,
Juliette M.
Devos
,
Pierre
Aller
,
Matthew P.
Blakeley
,
P. C. E.
Moody
,
Emma L.
Raven
Diamond Proposal Number(s):
[10369]
Open Access
Abstract: Catalytic heme enzymes carry out a wide range of oxidations in biology. They have in common a mechanism that requires formation of highly oxidized ferryl intermediates. It is these ferryl intermediates that provide the catalytic engine to drive the biological activity. Unravelling the nature of the ferryl species is of fundamental and widespread importance. The essential question is whether the ferryl is best described as a Fe(IV)=O or a Fe(IV)–OH species, but previous spectroscopic and X-ray crystallographic studies have not been able to unambiguously differentiate between the two species. Here we use a different approach. We report a neutron crystal structure of the ferryl intermediate in Compound II of a heme peroxidase; the structure allows the protonation states of the ferryl heme to be directly observed. This, together with pre-steady state kinetic analyses, electron paramagnetic resonance spectroscopy and single crystal X-ray fluorescence, identifies a Fe(IV)–OH species as the reactive intermediate. The structure establishes a precedent for the formation of Fe(IV)–OH in a peroxidase.
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Nov 2016
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Iris D.
Young
,
Mohamed
Ibrahim
,
Ruchira
Chatterjee
,
Sheraz
Gul
,
Franklin D.
Fuller
,
Sergey
Koroidov
,
Aaron S.
Brewster
,
Rosalie
Tran
,
Roberto
Alonso-mori
,
Thomas
Kroll
,
Tara
Michels-clark
,
Hartawan
Laksmono
,
Raymond G.
Sierra
,
Claudiu A.
Stan
,
Rana
Hussein
,
Miao
Zhang
,
Lacey
Douthit
,
Markus
Kubin
,
Casper
De Lichtenberg
,
Long
Vo Pham
,
Håkan
Nilsson
,
Mun Hon
Cheah
,
Dmitriy
Shevela
,
Claudio
Saracini
,
Mackenzie A.
Bean
,
Ina
Seuffert
,
Dimosthenis
Sokaras
,
Tsu-chien
Weng
,
Ernest
Pastor
,
Clemens
Weninger
,
Thomas
Fransson
,
Louise
Lassalle
,
Philipp
Bräuer
,
Pierre
Aller
,
Peter T.
Docker
,
Babak
Andi
,
Allen M.
Orville
,
James M.
Glownia
,
Silke
Nelson
,
Marcin
Sikorski
,
Diling
Zhu
,
Mark S.
Hunter
,
Thomas J.
Lane
,
Andy
Aquila
,
Jason E.
Koglin
,
Joseph
Robinson
,
Mengning
Liang
,
Sébastien
Boutet
,
Artem Y.
Lyubimov
,
Monarin
Uervirojnangkoorn
,
Nigel W.
Moriarty
,
Dorothee
Liebschner
,
Pavel V.
Afonine
,
David G.
Waterman
,
Gwyndaf
Evans
,
Philippe
Wernet
,
Holger
Dobbek
,
William I.
Weis
,
Axel T.
Brunger
,
Petrus H.
Zwart
,
Paul D.
Adams
,
Athina
Zouni
,
Johannes
Messinger
,
Uwe
Bergmann
,
Nicholas K.
Sauter
,
Jan
Kern
,
Vittal K.
Yachandra
,
Junko
Yano
Abstract: 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 Mn4CaO5 cluster in the oxygen-evolving complex (OEC). Under illumination, the OEC cycles through five intermediate S-states (S0 to S4)1, in which S1 is the dark-stable state and S3 is the last semi-stable state before O–O bond formation and O2 evolution2, 3. 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 site4, 5, 6. 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 (S1), two-flash illuminated (2F; S3-enriched), and ammonia-bound two-flash illuminated (2F-NH3; S3-enriched) PS II. Although the recent 1.95 Å resolution structure of PS II at cryogenic temperature using an XFEL7 provided a damage-free view of the S1 state, measurements at room temperature are required to study the structural landscape of proteins under functional conditions8, 9, 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 Mn4CaO5 cluster in the S2 and S3 states10. Since the ammonia-bound OEC is active, the ammonia-binding Mn site is not a substrate water site10, 11, 12, 13. 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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Nov 2016
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