I03-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
I04-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
|
Patrick
Rabe
,
Jos J. A. G.
Kamps
,
Kyle D.
Sutherlin
,
James D. S.
Linyard
,
Pierre
Aller
,
Cindy C.
Pham
,
Mikako
Makita
,
Ian
Clifton
,
Michael A.
Mcdonough
,
Thomas M.
Leissing
,
Denis
Shutin
,
Pauline A.
Lang
,
Agata
Butryn
,
Jurgen
Brem
,
Sheraz
Gul
,
Franklin D.
Fuller
,
In-Sik
Kim
,
Mun Hon
Cheah
,
Thomas
Fransson
,
Asmit
Bhowmick
,
Iris D.
Young
,
Lee
O'Riordan
,
Aaron S.
Brewster
,
Ilaria
Pettinati
,
Margaret
Doyle
,
Yasumasa
Joti
,
Shigeki
Owada
,
Kensuke
Tono
,
Alexander
Batyuk
,
Mark S.
Hunter
,
Roberto
Alonso-Mori
,
Uwe
Bergmann
,
Robin L.
Owen
,
Nicholas K.
Sauter
,
Timothy D. W.
Claridge
,
Carol V.
Robinson
,
Vittal K.
Yachandra
,
Junko
Yano
,
Jan F.
Kern
,
Allen M.
Orville
,
Christopher J.
Schofield
Diamond Proposal Number(s):
[23459, 19458]
Open Access
Abstract: Isopenicillin N synthase (IPNS) catalyzes the unique reaction of L-δ-(α-aminoadipoyl)-L-cysteinyl-D-valine (ACV) with dioxygen giving isopenicillin N (IPN), the precursor of all natural penicillins and cephalosporins. X-ray free-electron laser studies including time-resolved crystallography and emission spectroscopy reveal how reaction of IPNS:Fe(II):ACV with dioxygen to yield an Fe(III) superoxide causes differences in active site volume and unexpected conformational changes that propagate to structurally remote regions. Combined with solution studies, the results reveal the importance of protein dynamics in regulating intermediate conformations during conversion of ACV to IPN. The results have implications for catalysis by multiple IPNS-related oxygenases, including those involved in the human hypoxic response, and highlight the power of serial femtosecond crystallography to provide insight into long-range enzyme dynamics during reactions presently impossible for nonprotein catalysts.
|
Aug 2021
|
|
I24-Microfocus Macromolecular Crystallography
|
Agata
Butryn
,
Philipp S.
Simon
,
Pierre
Aller
,
Philip
Hinchliffe
,
Ramzi N.
Massad
,
Gabriel
Leen
,
Catherine L.
Tooke
,
Isabel
Bogacz
,
In-Sik
Kim
,
Asmit
Bhowmick
,
Aaron S.
Brewster
,
Nicholas E.
Devenish
,
Jurgen
Brem
,
Jos J. A. G.
Kamps
,
Pauline A.
Lang
,
Patrick
Rabe
,
Danny
Axford
,
John H.
Beale
,
Bradley
Davy
,
Ali
Ebrahim
,
Julien
Orlans
,
Selina L. S.
Storm
,
Tiankun
Zhou
,
Shigeki
Owada
,
Rie
Tanaka
,
Kensuke
Tono
,
Gwyndaf
Evans
,
Robin L.
Owen
,
Frances A.
Houle
,
Nicholas K.
Sauter
,
Christopher J.
Schofield
,
James
Spencer
,
Vittal K.
Yachandra
,
Junko
Yano
,
Jan F.
Kern
,
Allen M.
Orville
Diamond Proposal Number(s):
[19458, 25260]
Open Access
Abstract: Serial femtosecond crystallography has opened up many new opportunities in structural biology. In recent years, several approaches employing light-inducible systems have emerged to enable time-resolved experiments that reveal protein dynamics at high atomic and temporal resolutions. However, very few enzymes are light-dependent, whereas macromolecules requiring ligand diffusion into an active site are ubiquitous. In this work we present a drop-on-drop sample delivery system that enables the study of enzyme-catalyzed reactions in microcrystal slurries. The system delivers ligand solutions in bursts of multiple picoliter-sized drops on top of a larger crystal-containing drop inducing turbulent mixing and transports the mixture to the X-ray interaction region with temporal resolution. We demonstrate mixing using fluorescent dyes, numerical simulations and time-resolved serial femtosecond crystallography, which show rapid ligand diffusion through microdroplets. The drop-on-drop method has the potential to be widely applicable to serial crystallography studies, particularly of enzyme reactions with small molecule substrates.
|
Jul 2021
|
|
|
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.
|
Jul 2020
|
|
|
Mohamed
Ibrahim
,
Thomas
Fransson
,
Ruchira
Chatterjee
,
Mun Hon
Cheah
,
Rana
Hussein
,
Louise
Lassalle
,
Kyle D.
Sutherlin
,
Iris D.
Young
,
Franklin D.
Fuller
,
Sheraz
Gul
,
In-Sik
Kim
,
Philipp S.
Simon
,
Casper
De Lichtenberg
,
Petko
Chernev
,
Isabel
Bogacz
,
Cindy C.
Pham
,
Allen M.
Orville
,
Nicholas
Saichek
,
Trent
Northen
,
Alexander
Batyuk
,
Sergio
Carbajo
,
Roberto
Alonso-Mori
,
Kensuke
Tono
,
Shigeki
Owada
,
Asmit
Bhowmick
,
Robert
Bolotovsky
,
Derek
Mendez
,
Nigel W.
Moriarty
,
James M.
Holton
,
Holger
Dobbek
,
Aaron S.
Brewster
,
Paul D.
Adams
,
Nicholas K.
Sauter
,
Uwe
Bergmann
,
Athina
Zouni
,
Johannes
Messinger
,
Jan
Kern
,
Vittal K.
Yachandra
,
Junko
Yano
Open Access
Abstract: 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, S1, S2, S3, and S0, showing that a water molecule is inserted during the S2 → S3 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 O2 formation, we recorded diffraction and Mn X-ray emission spectroscopy (XES) data at several time points during the S2 → S3 transition. At the electron acceptor site, changes due to the two-electron redox chemistry at the quinones, QA and QB, are observed. At the donor site, tyrosine YZ 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 OX(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 S2 → S3 transition mirrors the appearance of OX 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.
|
May 2020
|
|
I24-Microfocus Macromolecular Crystallography
|
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.
|
Dec 2019
|
|
|
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.
|
Nov 2018
|
|
|
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.
|
Feb 2017
|
|
|
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.
|
Nov 2016
|
|