I24-Microfocus Macromolecular Crystallography
|
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.
|
Sep 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
|
|
I03-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
I04-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
|
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.
|
Aug 2017
|
|
|
|
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
|
|
I04-Macromolecular Crystallography
|
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.
|
Nov 2016
|
|
|
|
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
|
|
|
|
Abstract: X-ray diffraction from crystals of membrane proteins very often yields incomplete datasets due to, among other things, severe radiation damage. Multiple crystals are thus required to form complete datasets, provided the crystals themselves are isomorphous. Selection and combination of data from multiple crystals is a difficult and tedious task that can be facilitated by purpose-built software. BLEND, in the CCP4 suite of programs for macromolecular crystallography (MX), has been created exactly for this reason. In this chapter the program is described and its workings illustrated by means of data from two membrane proteins.
|
Aug 2016
|
|
I24-Microfocus Macromolecular Crystallography
VMXi-Versatile Macromolecular Crystallography in situ
|
Open Access
Abstract: Examples are shown of protein crystallization in, and data collection from, solutions sandwiched between thin polymer films using vapour-diffusion and batch methods. The crystallization platform is optimal for both visualization and in situ data collection, with the need for traditional harvesting being eliminated. In wells constructed from the thinnest plastic and with a minimum of aqueous liquid, flash-cooling to 100 K is possible without significant ice formation and without any degradation in crystal quality. The approach is simple; it utilizes low-cost consumables but yields high-quality data with minimal sample intervention and, with the very low levels of background X-ray scatter that are observed, is optimal for microcrystals.
|
Apr 2016
|
|
I02-Macromolecular Crystallography
I03-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
I04-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
|
Raymond J.
Owens
,
Pierre
Aller
,
Juan
Sanchez-weatherby
,
James
Foadi
,
Graeme
Winter
,
Carina
Lobley
,
Danny
Axford
,
Alun
Ashton
,
Domenico
Bellini
,
Jose
Brandao-neto
,
Simone
Culurgioni
,
Alice
Douangamath
,
Ramona
Duman
,
Gwyndaf
Evans
,
Stuart
Fisher
,
Ralf
Flaig
,
Dave
Hall
,
Petra
Lukacik
,
Marco
Mazzorana
,
Katherine
Mcauley
Abstract: Macromolecular crystallography (MX) is the most powerful technique available to structural biologists to visualize in atomic detail the macromolecular machinery of the cell. Since the emergence of structural genomics initiatives, significant advances have been made in all key steps of the structure determination process. In particular, third-generation synchrotron sources and the application of highly automated approaches to data acquisition and analysis at these facilities have been the major factors in the rate of increase of macromolecular structures determined annually. A plethora of tools are now available to users of synchrotron beamlines to enable rapid and efficient evaluation of samples, collection of the best data, and in favorable cases structure solution in near real time. Here, we provide a short overview of the emerging use of collecting X-ray diffraction data directly from the crystallization experiment. These in situ experiments are now routinely available to users at a number of synchrotron MX beamlines. A practical guide to the use of the method on the MX suite of beamlines at Diamond Light Source is given.
|
Nov 2015
|
|
I04-Macromolecular Crystallography
Optics
|
Open Access
Abstract: The measurement of diffraction data from macromolecular crystal samples held in vacuo holds the promise of a very low X-ray background and zero absorption of incident and scattered beams, leading to better data and the potential for accessing very long X-ray wavelengths (>3 Å) for native sulfur phasing. Maintaining the hydration of protein crystals under vacuum is achieved by the use of liquid jets, as with serial data collection at free-electron lasers, or is side-stepped by cryocooling the samples, as implemented at new synchrotron beamlines. Graphene has been shown to protect crystals from dehydration by creating an extremely thin layer that is impermeable to any exchanges with the environment. Furthermore, owing to its hydrophobicity, most of the aqueous solution surrounding the crystal is excluded during sample preparation, thus eliminating most of the background caused by liquid. Here, it is shown that high-quality data can be recorded at room temperature from graphene-wrapped protein crystals in a rough vacuum. Furthermore, it was observed that graphene protects crystals exposed to different relative humidities and a chemically harsh environment
|
Oct 2015
|
|
