I24-Microfocus Macromolecular Crystallography
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James
Baxter
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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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I24-Microfocus Macromolecular Crystallography
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Richard J.
Gildea
,
James
Beilsten-Edmands
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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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Abstract: The SARS-CoV-2’s endoribonuclease (NendoU) nsp15, is an Mn2+ dependent endoribonuclease specific to uridylate that SARS-CoV-2 uses to avoid the innate immune response by managing the stray RNA generated during replication. As of the writing of this review 20 structures of SARS-CoV-2 nsp15 have been deposited into the PDB, largely solved using X-ray crystallography and some through Cryo-EM. These structures show that an nsp15 monomer consist of three conserved domains, the N-terminal oligomerization domain, the middle domain, and the catalytic NendoU domain. Enzymatically active nsp15 forms a hexamer through a dimer of trimers (point group 32), whose assembly is facilitated by the oligomerization domain. This review summarises the structural and functional information gained from SARs-CoV-2, SARs-CoV and MERS-CoV nsp15 structures, compiles the current structure-based drug design efforts, and complementary knowledge with a view to provide a clear starting point for downstream structure users interested in studying nsp15 as a novel drug target to treat COVID-19.
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Jun 2022
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Tristan I.
Croll
,
Kay
Diederichs
,
Florens
Fischer
,
Cameron D.
Fyfe
,
Yunyun
Gao
,
Sam
Horrell
,
Agnel Praveen
Joseph
,
Luise
Kandler
,
Oliver
Kippes
,
Ferdinand
Kirsten
,
Konstantin
Müller
,
Kristopher
Nolte
,
Alexander M.
Payne
,
Matthew
Reeves
,
Jane S.
Richardson
,
Gianluca
Santoni
,
Sabrina
Stäb
,
Dale E.
Tronrud
,
Lea C.
Von Soosten
,
Christopher J.
Williams
,
Andrea
Thorn
Abstract: Structural biology plays a crucial role in the fight against COVID-19, permitting us to ‘see’ and understand SARS-CoV-2. However, the macromolecular structures of SARS-CoV-2 proteins that were solved with great speed and urgency can contain errors that may hinder drug design. The Coronavirus Structural Task Force has been working behind the scenes to evaluate and improve these structures, making the results freely available at https://insidecorona.net/.
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May 2021
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Jose Ramon
Macias
,
Ruben
Sanchez-Garcia
,
Pablo
Conesa
,
Erney
Ramirez-Aportela
,
Marta
Martinez Gonzalez
,
Carlos
Wert-Carvajal
,
Alberto M.
Parra-Perez
,
Joan
Segura Mora
,
Sam
Horrell
,
Andrea
Thorn
,
Carlos O. S.
Sorzano
,
Jose Maria
Carazo
Open Access
Abstract: The web platform 3DBionotes-WS integrates multiple Web Services and an interactive Web Viewer to provide a unified environment in which biological annotations can be analyzed in their structural context. Since the COVID-19 outbreak, new structural data from many viral proteins have been provided at a very fast pace. This effort includes many cryogenic Electron Microscopy (cryo-EM) studies, together with more traditional ones (X-rays, NMR), using several modeling approaches and complemented with structural predictions. At the same time, a plethora of new genomics and interactomics information (including fragment screening and structure-based virtual screening efforts) have been made available from different servers. In this context we have developed 3DBionotes-COVID-19 as an answer to: (1) The need to explore multi-omics data in a unified context with a special focus on structural information and (2) the drive to incorporate quality measurements, especially in the form of advanced validation metrics for cryogenic Electron Microscopy.
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May 2021
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I24-Microfocus Macromolecular Crystallography
Data acquisition
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Open Access
Abstract: Serial data collection is a relatively new technique for synchrotron users. A user manual for fixed target data collection at I24, Diamond Light Source is presented with detailed step-by-step instructions, figures, and videos for smooth data collection.
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Feb 2021
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I24-Microfocus Macromolecular Crystallography
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Sam
Horrell
,
Demet
Kekilli
,
Kakali
Sen
,
Robin L.
Owen
,
Florian S. N.
Dworkowski
,
Svetlana V.
Antonyuk
,
Thomas W.
Keal
,
Chin W.
Yong
,
Robert R.
Eady
,
S. Samar
Hasnain
,
Richard W.
Strange
,
Mike
Hough
Diamond Proposal Number(s):
[11175]
Open Access
Abstract: High-resolution crystal structures of enzymes in relevant redox states have transformed our understanding of enzyme catalysis. Recent developments have demonstrated that X-rays can be used, via the generation of solvated electrons, to drive reactions in crystals at cryogenic temperatures (100 K) to generate `structural movies' of enzyme reactions. However, a serious limitation at these temperatures is that protein conformational motion can be significantly supressed. Here, the recently developed MSOX (multiple serial structures from one crystal) approach has been applied to nitrite-bound copper nitrite reductase at room temperature and at 190 K, close to the glass transition. During both series of multiple structures, nitrite was initially observed in a `top-hat' geometry, which was rapidly transformed to a `side-on' configuration before conversion to side-on NO, followed by dissociation of NO and substitution by water to reform the resting state. Density functional theory calculations indicate that the top-hat orientation corresponds to the oxidized type 2 copper site, while the side-on orientation is consistent with the reduced state. It is demonstrated that substrate-to-product conversion within the crystal occurs at a lower radiation dose at 190 K, allowing more of the enzyme catalytic cycle to be captured at high resolution than in the previous 100 K experiment. At room temperature the reaction was very rapid, but it remained possible to generate and characterize several structural states. These experiments open up the possibility of obtaining MSOX structural movies at multiple temperatures (MSOX-VT), providing an unparallelled level of structural information during catalysis for redox enzymes.
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May 2018
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I02-Macromolecular Crystallography
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Diamond Proposal Number(s):
[13467]
Open Access
Abstract: Microbial nitrite reductases are denitrifying enzymes that are a major component of the global nitrogen cycle. Multiple structures measured from one crystal (MSOX data) of copper nitrite reductase at 240 K, together with molecular-dynamics simulations, have revealed protein dynamics at the type 2 copper site that are significant for its catalytic properties and for the entry and exit of solvent or ligands to and from the active site. Molecular-dynamics simulations were performed using different protonation states of the key catalytic residues (AspCAT and HisCAT) involved in the nitrite-reduction mechanism of this enzyme. Taken together, the crystal structures and simulations show that the AspCAT protonation state strongly influences the active-site solvent accessibility, while the dynamics of the active-site `capping residue' (IleCAT), a determinant of ligand binding, are influenced both by temperature and by the protonation state of AspCAT. A previously unobserved conformation of IleCAT is seen in the elevated temperature series compared with 100 K structures. DFT calculations also show that the loss of a bound water ligand at the active site during the MSOX series is consistent with reduction of the type 2 Cu atom.
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Jul 2017
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I02-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
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Diamond Proposal Number(s):
[11175, 8663, 8889]
Open Access
Abstract: The bacterial second messenger cyclic di-3′,5′-guanosine monophosphate (c-di-GMP) is a key regulator of bacterial motility and virulence. As high levels of c-di-GMP are associated with the biofilm lifestyle, c-di-GMP hydrolysing phosphodiesterases (PDEs) have been identified as key targets to aid development of novel strategies to treat chronic infection by exploiting biofilm dispersal. We have studied the EAL signature motif-containing phosphodiesterase domains from the Pseudomonas aeruginosa proteins PA3825 (PA3825EAL) and PA1727 (MucREAL). Different dimerisation interfaces allow us to identify interface independent principles of enzyme regulation. Unlike previously characterised two-metal binding EAL-phosphodiesterases, PA3825EAL in complex with pGpG provides a model for a third metal site. The third metal is positioned to stabilise the negative charge of the 5′-phosphate, and thus three metals could be required for catalysis in analogy to other nucleases. This newly uncovered variation in metal coordination may provide a further level of bacterial PDE regulation.
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Feb 2017
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I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[11740]
Open Access
Abstract: Relating individual protein crystal structures to an enzyme mechanism remains a major and challenging goal for structural biology. Serial crystallography using multiple crystals has recently been reported in both synchrotron-radiation and X-ray free-electron laser experiments. In this work, serial crystallography was used to obtain multiple structures serially from one crystal (MSOX) to study in crystallo enzyme catalysis. Rapid, shutterless X-ray detector technology on a synchrotron MX beamline was exploited to perform low-dose serial crystallography on a single copper nitrite reductase crystal, which survived long enough for 45 consecutive 100 K X-ray structures to be collected at 1.07–1.62 Å resolution, all sampled from the same crystal volume. This serial crystallography approach revealed the gradual conversion of the substrate bound at the catalytic type 2 Cu centre from nitrite to nitric oxide, following reduction of the type 1 Cu electron-transfer centre by X-ray-generated solvated electrons. Significant, well defined structural rearrangements in the active site are evident in the series as the enzyme moves through its catalytic cycle, namely nitrite reduction, which is a vital step in the global denitrification process. It is proposed that such a serial crystallography approach is widely applicable for studying any redox or electron-driven enzyme reactions from a single protein crystal. It can provide a `catalytic reaction movie' highlighting the structural changes that occur during enzyme catalysis. The anticipated developments in the automation of data analysis and modelling are likely to allow seamless and near-real-time analysis of such data on-site at some of the powerful synchrotron crystallographic beamlines.
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Jul 2016
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