I24-Microfocus Macromolecular Crystallography
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Diamond Proposal Number(s):
[31420]
Open Access
Abstract: Hemoglycin, a 1494 Da polymer composed of iron and glycine has been detected in several carbonaceous meteorites. Iron atoms close out the ends of a 5nm anti-parallel glycine beta sheet and contribute visible and near infrared absorptions that are not present with glycine alone. The 483nm absorption of hemoglycin was discovered in theory and then observed on beamline I24 at Diamond Light Source. Light absorption in a molecule involves a coupled lower set of states receiving light energy that causes a transition into an upper set of states. In the reverse process some energy source, such as an X-ray beam, populates the upper set of molecular states, which then radiates light as it returns to the lower "ground" set of states. We report on visible light re-emission during X-ray irradiation of a hemoglycin crystal. The emission is dominated by bands centered at 489nm and 551nm.
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Feb 2023
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I24-Microfocus Macromolecular Crystallography
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James
Baxter
,
Christopher D. M.
Hutchison
,
Karim
Maghlaoui
,
Violeta
Cordon-Preciado
,
R. Marc L.
Morgan
,
Pierre
Aller
,
Agata
Butryn
,
Danny
Axford
,
Sam
Horrell
,
Robin L.
Owen
,
Selina L. S.
Storm
,
Nicholas E.
Devenish
,
Jasper J.
Van Thor
Diamond Proposal Number(s):
[17221]
Open Access
Abstract: The chromophores of reversibly switchable fluorescent proteins (rsFPs) undergo photoisomerization of both the trans and cis forms. Concurrent with cis/trans photoisomerisation, rsFPs typically become protonated on the phenolic oxygen resulting in a blue shift of the absorption. A synthetic rsFP referred to as rsEospa, derived from EosFP family, displays the same spectroscopic behavior as the GFP-like rsFP Dronpa at pH 8.4 and involves the photoconversion between nonfluorescent neutral and fluorescent anionic chromophore states. Millisecond time-resolved synchrotron serial crystallography of rsEospa at pH 8.4 shows that photoisomerization is accompanied by rearrangements of the same three residues as seen in Dronpa. However, at pH 5.5 we observe that the OFF state is identified as the cationic chromophore with additional protonation of the imidazolinone nitrogen which is concurrent with a newly formed hydrogen bond with the Glu212 carboxylate side chain. FTIR spectroscopy resolves the characteristic up-shifted carbonyl stretching frequency at 1713 cm–1 for the cationic species. Electronic spectroscopy furthermore distinguishes the cationic absorption band at 397 nm from the neutral species at pH 8.4 seen at 387 nm. The observation of photoisomerization of the cationic chromophore state demonstrates the conical intersection for the electronic configuration, where previously fluorescence was proposed to be the main decay route for states containing imidazolinone nitrogen protonation. We present the full time-resolved room-temperature X-ray crystallographic, FTIR, and UV/vis assignment and photoconversion modeling of rsEospa.
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Nov 2022
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I11-High Resolution Powder Diffraction
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Diamond Proposal Number(s):
[23209]
Open Access
Abstract: X-ray characterisation methods have undoubtedly enabled cutting-edge advances in all aspects of materials research. Despite the enormous breadth of information that can be extracted from these techniques, the challenge of radiation-induced sample change and damage remains prevalent. This is largely due to the emergence of modern, high-intensity X-ray source technologies and the growing potential to carry out more complex, longer duration in situ or in operando studies. The tunability of synchrotron beamlines enables the routine application of photon energy-dependent experiments. This work explores the structural stability of [Rh(COD)Cl]2, a widely used catalyst and precursor in the chemical industry, across a range of beamline parameters that target X-ray energies of 8 keV, 15 keV, 18 keV and 25 keV, on a powder X-ray diffraction synchrotron beamline at room temperature. Structural changes are discussed with respect to absorbed X-ray dose at each experimental setting associated with the respective photon energy. In addition, the X-ray radiation hardness of the catalyst is discussed, by utilising the diffraction data collected at the different energies to determine a dose limit, which is often considered in protein crystallography and typically overlooked in small molecule crystallography. This work not only gives fundamental insight into how damage manifests in this organometallic catalyst, but will encourage careful consideration of experimental X-ray parameters before conducting diffraction on similar radiation-sensitive organometallic materials.
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Nov 2022
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I24-Microfocus Macromolecular Crystallography
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Diamond Proposal Number(s):
[25108, 28583]
Open Access
Abstract: Controlling the reactivity of high-valent Fe(IV)–O catalytic intermediates, Compounds I and II, generated in heme enzymes upon reaction with dioxygen or hydrogen peroxide, is important for function. It has been hypothesized that the presence (wet) or absence (dry) of distal heme pocket water molecules can influence whether Compound I undergoes sequential one-electron additions or a concerted two-electron reduction. To test this hypothesis, we investigate the role of water in the heme distal pocket of a dye-decolorizing peroxidase utilizing a combination of serial femtosecond crystallography and rapid kinetic studies. In a dry distal heme site, Compound I reduction proceeds through a mechanism in which Compound II concentration is low. This reaction shows a strong deuterium isotope effect, indicating that reduction is coupled to proton uptake. The resulting protonated Compound II (Fe(IV)–OH) rapidly reduces to the ferric state, giving the appearance of a two-electron transfer process. In a wet site, reduction of Compound I is faster, has no deuterium effect, and yields highly populated Compound II, which is subsequently reduced to the ferric form. This work provides a definitive experimental test of the hypothesis advanced in the literature that relates sequential or concerted electron transfer to Compound I in wet or dry distal heme sites.
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Oct 2022
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I24-Microfocus Macromolecular Crystallography
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Tadeo
Moreno-Chicano
,
Leiah M.
Carey
,
Danny
Axford
,
John H.
Beale
,
R. Bruce
Doak
,
Helen M. E.
Duyvesteyn
,
Ali
Ebrahim
,
Robert W.
Henning
,
Diana C. F.
Monteiro
,
Dean A.
Myles
,
Shigeki
Owada
,
Darren A.
Sherrell
,
Megan L.
Straw
,
Vukica
Šrajer
,
Hiroshi
Sugimoto
,
Kensuke
Tono
,
Takehiko
Tosha
,
Ivo
Tews
,
Martin
Trebbin
,
Richard W.
Strange
,
Kevin L.
Weiss
,
Jonathan A. R.
Worrall
,
Flora
Meilleur
,
Robin L.
Owen
,
Reza A.
Ghiladi
,
Michael A.
Hough
Diamond Proposal Number(s):
[14493]
Open Access
Abstract: Room-temperature macromolecular crystallography allows protein structures to be determined under close-to-physiological conditions, permits dynamic freedom in protein motions and enables time-resolved studies. In the case of metalloenzymes that are highly sensitive to radiation damage, such room-temperature experiments can present challenges, including increased rates of X-ray reduction of metal centres and site-specific radiation-damage artefacts, as well as in devising appropriate sample-delivery and data-collection methods. It can also be problematic to compare structures measured using different crystal sizes and light sources. In this study, structures of a multifunctional globin, dehaloperoxidase B (DHP-B), obtained using several methods of room-temperature crystallographic structure determination are described and compared. Here, data were measured from large single crystals and multiple microcrystals using neutrons, X-ray free-electron laser pulses, monochromatic synchrotron radiation and polychromatic (Laue) radiation light sources. These approaches span a range of 18 orders of magnitude in measurement time per diffraction pattern and four orders of magnitude in crystal volume. The first room-temperature neutron structures of DHP-B are also presented, allowing the explicit identification of the hydrogen positions. The neutron data proved to be complementary to the serial femtosecond crystallography data, with both methods providing structures free of the effects of X-ray radiation damage when compared with standard cryo-crystallography. Comparison of these room-temperature methods demonstrated the large differences in sample requirements, data-collection time and the potential for radiation damage between them. With regard to the structure and function of DHP-B, despite the results being partly limited by differences in the underlying structures, new information was gained on the protonation states of active-site residues which may guide future studies of DHP-B.
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Sep 2022
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I24-Microfocus Macromolecular Crystallography
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Gyorgy
Babnigg
,
Darren A.
Sherrell
,
Youngchang
Kim
,
Jessica L.
Johnson
,
Boguslaw
Nocek
,
Kemin
Tan
,
Danny
Axford
,
Hui
Li
,
Lance
Bigelow
,
Lukas
Welk
,
Michael
Endres
,
Robin L.
Owen
,
Andrzej
Joachimiak
Abstract: Protein crystals grown in microfluidic droplets have been shown to be an effective and robust platform for storage, transport and serial crystallography data collection with a minimal impact on diffraction quality. Single macromolecular microcrystals grown in nanolitre-sized droplets allow the very efficient use of protein samples and can produce large quantities of high-quality samples for data collection. However, there are challenges not only in growing crystals in microfluidic droplets, but also in delivering the droplets into X-ray beams, including the physical arrangement, beamline and timing constraints and ease of use. Here, the crystallization of two human gut microbial hydrolases in microfluidic droplets is described: a sample-transport and data-collection approach that is inexpensive, is convenient, requires small amounts of protein and is forgiving. It is shown that crystals can be grown in 50–500 pl droplets when the crystallization conditions are compatible with the droplet environment. Local and remote data-collection methods are described and it is shown that crystals grown in microfluidics droplets and housed as an emulsion in an Eppendorf tube can be shipped from the US to the UK using a FedEx envelope, and data can be collected successfully. Details of how crystals were delivered to the X-ray beam by depositing an emulsion of droplets onto a silicon fixed-target serial device are provided. After three months of storage at 4°C, the crystals endured and diffracted well, showing only a slight decrease in diffracting power, demonstrating a suitable way to grow crystals, and to store and collect the droplets with crystals for data collection. This sample-delivery and data-collection strategy allows crystal droplets to be shipped and set aside until beamtime is available.
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Aug 2022
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I24-Microfocus Macromolecular Crystallography
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Richard J.
Gildea
,
James
Beilsten-Edmands
,
Danny
Axford
,
Sam
Horrell
,
Pierre
Aller
,
James
Sandy
,
Juan
Sanchez-Weatherby
,
C. David
Owen
,
Petra
Lukacik
,
Claire
Strain-Damerell
,
Robin L.
Owen
,
Martin A.
Walsh
,
Graeme
Winter
Diamond Proposal Number(s):
[26986, 27088]
Open Access
Abstract: In macromolecular crystallography, radiation damage limits the amount of data that can be collected from a single crystal. It is often necessary to merge data sets from multiple crystals; for example, small-wedge data collections from micro-crystals, in situ room-temperature data collections and data collection from membrane proteins in lipidic mesophases. Whilst the indexing and integration of individual data sets may be relatively straightforward with existing software, merging multiple data sets from small wedges presents new challenges. The identification of a consensus symmetry can be problematic, particularly in the presence of a potential indexing ambiguity. Furthermore, the presence of non-isomorphous or poor-quality data sets may reduce the overall quality of the final merged data set. To facilitate and help to optimize the scaling and merging of multiple data sets, a new program, xia2.multiplex, has been developed which takes data sets individually integrated with DIALS and performs symmetry analysis, scaling and merging of multi-crystal data sets. xia2.multiplex also performs analysis of various pathologies that typically affect multi-crystal data sets, including non-isomorphism, radiation damage and preferential orientation. After the description of a number of use cases, the benefit of xia2.multiplex is demonstrated within a wider autoprocessing framework in facilitating a multi-crystal experiment collected as part of in situ room-temperature fragment-screening experiments on the SARS-CoV-2 main protease.
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Jun 2022
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Open Access
Abstract: An estimated half of all proteins contain a metal, with these being essential for a tremendous variety of biological functions. X-ray crystallography is the major method for obtaining structures at high resolution of these metalloproteins, but there are considerable challenges to obtain intact structures due to the effects of radiation damage. Serial crystallography offers the prospect of determining low-dose synchrotron or effectively damage free XFEL structures at room temperature and enables time-resolved or dose-resolved approaches. Complementary spectroscopic data can validate redox and or ligand states within metalloprotein crystals. In this opinion, we discuss developments in the application of serial crystallographic approaches to metalloproteins and comment on future directions.
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Dec 2021
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I24-Microfocus Macromolecular Crystallography
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Open Access
Abstract: X-ray-induced radiation damage is a limiting factor for the macromolecular crystallographer and data must often be merged from many crystals to yield complete data sets for the structure solution of challenging samples. Increasing the X-ray energy beyond the typical 10–15 keV range promises to provide an extension of crystal lifetime via an increase in diffraction efficiency. To date, however, hardware limitations have negated any possible gains. Through the first use of a cadmium telluride EIGER2 detector and a beamline optimized for high-energy data collection, it is shown that at higher energies fewer crystals will be required to obtain complete data, as the diffracted intensity per unit dose increases by a factor of more than two between 12.4 and 25 keV. Additionally, these higher energy data can provide more information, as shown by a systematic increase in the high-resolution cutoff of the data collected. Taken together, these gains point to a high-energy future for synchrotron-based macromolecular crystallography.
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Nov 2021
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I24-Microfocus Macromolecular Crystallography
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Diamond Proposal Number(s):
[18565]
Open Access
Abstract: Structure determination of proteins and enzymes by X-ray crystallography remains the most widely used approach to complement functional and mechanistic studies. Capturing the structures of intact redox states in metalloenzymes is critical for assigning the chemistry carried out by the metal in the catalytic cycle. Unfortunately, X-rays interact with protein crystals to generate solvated photoelectrons that can reduce redox active metals and hence change the coordination geometry and the coupled protein structure. Approaches to mitigate such site-specific radiation damage continue to be developed, but nevertheless application of such approaches to metalloenzymes in combination with mechanistic studies are often overlooked. In this review, we summarize our recent structural and kinetic studies on a set of three heme peroxidases found in the bacterium Streptomyces lividans that each belong to the dye decolourizing peroxidase (DyP) superfamily. Kinetically, each of these DyPs has a distinct reactivity with hydrogen peroxide. Through a combination of low dose synchrotron X-ray crystallography and zero dose serial femtosecond X-ray crystallography using an X-ray free electron laser (XFEL), high-resolution structures with unambiguous redox state assignment of the ferric and ferryl (FeIV = O) heme species have been obtained. Experiments using stopped-flow kinetics, solvent-isotope exchange and site-directed mutagenesis with this set of redox state validated DyP structures have provided the first comprehensive kinetic and structural framework for how DyPs can modulate their distal heme pocket Asp/Arg dyad to use either the Asp or the Arg to facilitate proton transfer and rate enhancement of peroxide heterolysis.
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Sep 2021
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