I23-Long wavelength MX
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Open Access
Abstract: AlphaFold2 has revolutionized structural biology by offering unparalleled accuracy in predicting protein structures. Traditional methods for determining protein structures, such as X-ray crystallography and cryo-electron microscopy, are often time-consuming and resource-intensive. AlphaFold2 provides models that are valuable for molecular replacement, aiding in model building and docking into electron density or potential maps. However, despite its capabilities, models from AlphaFold2 do not consistently match the accuracy of experimentally determined structures, need to be validated experimentally and currently miss some crucial information, such as post-translational modifications, ligands and bound ions. In this paper, the advantages are explored of collecting X-ray anomalous data to identify chemical elements, such as metal ions, which are key to understanding certain structures and functions of proteins. This is achieved through methods such as calculating anomalous difference Fourier maps or refining the imaginary component of the anomalous scattering factor f′′. Anomalous data can serve as a valuable complement to the information provided by AlphaFold2 models and this is particularly significant in elucidating the roles of metal ions.
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Oct 2024
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I23-Long wavelength MX
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Diamond Proposal Number(s):
[31800]
Open Access
Abstract: Metal ions have important roles in supporting the catalytic activity of DNA-regulating enzymes such as topoisomerases (topos). Bacterial type II topos, gyrases and topo IV, are primary drug targets for fluoroquinolones, a class of clinically relevant antibacterials requiring metal ions for efficient drug binding. While the presence of metal ions in topos has been elucidated in biochemical studies, accurate location and assignment of metal ions in structural studies have historically posed significant challenges. Recent advances in X-ray crystallography address these limitations by extending the experimental capabilities into the long-wavelength range, exploiting the anomalous contrast from light elements of biological relevance. This breakthrough enables us to confirm experimentally the locations of Mg2+ in the fluoroquinolone-stabilized Streptococcus pneumoniae topo IV complex. Moreover, we can unambiguously identify the presence of K+ and Cl- ions in the complex with one pair of K+ ions functioning as an additional intersubunit bridge. Overall, our data extend current knowledge on the functional and structural roles of metal ions in type II topos.
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Oct 2024
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I03-Macromolecular Crystallography
I23-Long wavelength MX
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Kamel
El Omari
,
Ramona
Duman
,
Vitaliy
Mykhaylyk
,
Christian M.
Orr
,
Merlyn
Latimer-Smith
,
Graeme
Winter
,
Vinay
Grama
,
Feng
Qu
,
Kiran
Bountra
,
Hok Sau
Kwong
,
Maria
Romano
,
Rosana
Reis
,
Lutz
Vogeley
,
Luca
Vecchia
,
C. David
Owen
,
Sina
Wittmann
,
Max
Renner
,
Miki
Senda
,
Naohiro
Matsugaki
,
Yoshiaki
Kawano
,
Thomas A.
Bowden
,
Isabel
Moraes
,
Jonathan M.
Grimes
,
Erika J.
Mancini
,
Martin A.
Walsh
,
Cristiane R.
Guzzo
,
Raymond J.
Owens
,
E. Yvonne
Jones
,
David G.
Brown
,
Dave I.
Stuart
,
Konstantinos
Beis
,
Armin
Wagner
Open Access
Abstract: Despite recent advances in cryo-electron microscopy and artificial intelligence-based model predictions, a significant fraction of structure determinations by macromolecular crystallography still requires experimental phasing, usually by means of single-wavelength anomalous diffraction (SAD) techniques. Most synchrotron beamlines provide highly brilliant beams of X-rays of between 0.7 and 2 Å wavelength. Use of longer wavelengths to access the absorption edges of biologically important lighter atoms such as calcium, potassium, chlorine, sulfur and phosphorus for native-SAD phasing is attractive but technically highly challenging. The long-wavelength beamline I23 at Diamond Light Source overcomes these limitations and extends the accessible wavelength range to λ = 5.9 Å. Here we report 22 macromolecular structures solved in this extended wavelength range, using anomalous scattering from a range of elements which demonstrate the routine feasibility of lighter atom phasing. We suggest that, in light of its advantages, long-wavelength crystallography is a compelling option for experimental phasing.
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Oct 2023
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I23-Long wavelength MX
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Alisia
Fadini
,
Christopher D. M.
Hutchison
,
Dmitry
Morozov
,
Jeffrey
Chang
,
Karim
Maghlaoui
,
Samuel
Perrett
,
Fangjia
Luo
,
Jeslyn C. X.
Kho
,
Matthew G.
Romei
,
R. Marc L.
Morgan
,
Christian
Orr
,
Violeta
Cordon-Preciado
,
Takaaki
Fujiwara
,
Nipawan
Nuemket
,
Takehiko
Tosha
,
Rie
Tanaka
,
Shigeki
Owada
,
Kensuke
Tono
,
So
Iwata
,
Steven G.
Boxer
,
Gerrit
Groenhof
,
Eriko
Nango
,
Jasper J.
Van Thor
Diamond Proposal Number(s):
[23620]
Open Access
Abstract: Chromophore cis/trans photoisomerization is a fundamental process in chemistry and in the activation of many photosensitive proteins. A major task is understanding the effect of the protein environment on the efficiency and direction of this reaction compared to what is observed in the gas and solution phases. In this study, we set out to visualize the hula twist (HT) mechanism in a fluorescent protein, which is hypothesized to be the preferred mechanism in a spatially constrained binding pocket. We use a chlorine substituent to break the twofold symmetry of the embedded phenolic group of the chromophore and unambiguously identify the HT primary photoproduct. Through serial femtosecond crystallography, we then track the photoreaction from femtoseconds to the microsecond regime. We observe signals for the photoisomerization of the chromophore as early as 300 fs, obtaining the first experimental structural evidence of the HT mechanism in a protein on its femtosecond-to-picosecond timescale. We are then able to follow how chromophore isomerization and twisting lead to secondary structure rearrangements of the protein β-barrel across the time window of our measurements.
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Jul 2023
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I23-Long wavelength MX
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Open Access
Abstract: Despite being fundamental to multiple biological processes, phosphorus (P) availability in marine environments is often growth-limiting, with generally low surface concentrations. Picocyanobacteria strains encode a putative ABC-type phosphite/phosphate/phosphonate transporter, phnDCE, thought to provide access to an alternative phosphorus pool. This, however, is paradoxical given most picocyanobacterial strains lack known phosphite degradation or carbon-phosphate lyase pathway to utilise alternate phosphorus pools. To understand the function of the PhnDCE transport system and its ecological consequences, we characterised the PhnD1 binding proteins from four distinct marine Synechococcus isolates (CC9311, CC9605, MITS9220, and WH8102). We show the Synechococcus PhnD1 proteins selectively bind phosphorus compounds with a stronger affinity for phosphite than for phosphate or methyl phosphonate. However, based on our comprehensive ligand screening and growth experiments showing Synechococcus strains WH8102 and MITS9220 cannot utilise phosphite or methylphosphonate as a sole phosphorus source, we hypothesise that the picocyanobacterial PhnDCE transporter is a constitutively expressed, medium-affinity phosphate transporter, and the measured affinity of PhnD1 to phosphite or methyl phosphonate is fortuitous. Our MITS9220_PhnD1 structure explains the comparatively lower affinity of picocyanobacterial PhnD1 for phosphate, resulting from a more limited H-bond network. We propose two possible physiological roles for PhnD1. First, it could function in phospholipid recycling, working together with the predicted phospholipase, TesA, and alkaline phosphatase. Second, by having multiple transporters for P (PhnDCE and Pst), picocyanobacteria could balance the need for rapid transport during transient episodes of higher P availability in the environment, with the need for efficient P utilisation in typical phosphate-deplete conditions.
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Apr 2023
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Xiaojie
Yu
,
Christian M.
Orr
,
H. T. Claude
Chan
,
Sonya
James
,
Christine A.
Penfold
,
Jinny
Kim
,
Tatyana
Inzhelevskaya
,
C. Ian
Mockridge
,
Kerry L.
Cox
,
Jonathan W.
Essex
,
Ivo
Tews
,
Martin J.
Glennie
,
Mark S.
Cragg
Abstract: Antibody responses during infection and vaccination typically undergo affinity maturation to achieve high-affinity binding for efficient neutralization of pathogens1,2. Similarly, high affinity is routinely the goal for therapeutic antibody generation. However, in contrast to naturally occurring or direct-targeting therapeutic antibodies, immunomodulatory antibodies, which are designed to modulate receptor signalling, have not been widely examined for their affinity–function relationship. Here we examine three separate immunologically important receptors spanning two receptor superfamilies: CD40, 4-1BB and PD-1. We show that low rather than high affinity delivers greater activity through increased clustering. This approach delivered higher immune cell activation, in vivo T cell expansion and antitumour activity in the case of CD40. Moreover, an inert anti-4-1BB monoclonal antibody was transformed into an agonist. Low-affinity variants of the clinically important antagonistic anti-PD-1 monoclonal antibody nivolumab also mediated more potent signalling and affected T cell activation. These findings reveal a new paradigm for augmenting agonism across diverse receptor families and shed light on the mechanism of antibody-mediated receptor signalling. Such affinity engineering offers a rational, efficient and highly tuneable solution to deliver antibody-mediated receptor activity across a range of potencies suitable for translation to the treatment of human disease.
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Feb 2023
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B21-High Throughput SAXS
I03-Macromolecular Crystallography
I23-Long wavelength MX
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Eugene
Kuatsjah
,
Michael
Zahn
,
Xiangyang
Chen
,
Ryo
Kato
,
Daniel J.
Hinchen
,
Mikhail O.
Konev
,
Rui
Katahira
,
Christian
Orr
,
Armin
Wagner
,
Yike
Zou
,
Stefan J.
Haugen
,
Kelsey J.
Ramirez
,
Joshua K.
Michener
,
Andrew R.
Pickford
,
Naofumi
Kamimura
,
Eiji
Masai
,
Kendall N.
Houk
,
John
Mcgeehan
,
Gregg T.
Beckham
Diamond Proposal Number(s):
[23269]
Open Access
Abstract: Lignin valorization is being intensely pursued via tandem catalytic depolymerization and biological funneling to produce single products. In many lignin depolymerization processes, aromatic dimers and oligomers linked by carbon–carbon bonds remain intact, necessitating the development of enzymes capable of cleaving these compounds to monomers. Recently, the catabolism of erythro-1,2-diguaiacylpropane-1,3-diol (erythro-DGPD), a ring-opened lignin-derived β-1 dimer, was reported in Novosphingobium aromaticivorans. The first enzyme in this pathway, LdpA (formerly LsdE), is a member of the nuclear transport factor 2 (NTF-2)-like structural superfamily that converts erythro-DGPD to lignostilbene through a heretofore unknown mechanism. In this study, we performed biochemical, structural, and mechanistic characterization of the N. aromaticivorans LdpA and another homolog identified in Sphingobium sp. SYK-6, for which activity was confirmed in vivo. For both enzymes, we first demonstrated that formaldehyde is the C1 reaction product, and we further demonstrated that both enantiomers of erythro-DGPD were transformed simultaneously, suggesting that LdpA, while diastereomerically specific, lacks enantioselectivity. We also show that LdpA is subject to a severe competitive product inhibition by lignostilbene. Three-dimensional structures of LdpA were determined using X-ray crystallography, including substrate-bound complexes, revealing several residues that were shown to be catalytically essential. We used density functional theory to validate a proposed mechanism that proceeds via dehydroxylation and formation of a quinone methide intermediate that serves as an electron sink for the ensuing deformylation. Overall, this study expands the range of chemistry catalyzed by the NTF-2-like protein family to a prevalent lignin dimer through a cofactorless deformylation reaction.
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Jan 2023
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Open Access
Abstract: Historically, solving the structure of a protein required deep knowledge of crystallography and the ability to produce protein crystals of suitable quality to generate high-quality diffraction data. Over the years, as beamline optics, end-stations, detectors, and data collection strategies have improved, it has become more feasible to extract high-quality diffraction data from ever smaller or less perfect protein crystals and from very large arrays of crystals for techniques such as serial synchrotron crystallography and fragment-based drug discovery. At Diamond, these improvements have been coupled with highly integrated automated pipelines for data reduction and structure solution using techniques such as molecular replacement and experimental phasing. This has led to the dichotomy, and benefits, of being able to do increasingly challenging experiments requiring deep crystallographic knowledge with facility staff support at the same time as lowering the barrier to entry where automated structure solution tools of the facility perform this task for those scientists with less experience. This enables users to focus on the science rather than the process.
Diamond Light Source, the UK’s national synchrotron, has a suite of instruments dedicated to solving the 3D structure of large biological molecules, including seven macromolecular crystallography (MX) beamlines. Solved 3D structures are deposited into the publicly available Protein Data Bank (PDB) and the depositions are released on a weekly basis. In 2020, following 13 years of operation, Diamond hit the milestone of 10,000 structures deposited in the PDB. Two years on, this number is now more than 12,000. Thanks to decades of work across the world, there is an ocean of information in the PDB that serves as an invaluable reference when solving the structures of new proteins.
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Oct 2022
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I03-Macromolecular Crystallography
I04-Macromolecular Crystallography
I23-Long wavelength MX
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Diamond Proposal Number(s):
[17221]
Open Access
Abstract: Orange Carotenoid protein (OCP) is the only known photoreceptor which uses carotenoid for its activation. It is found exclusively in cyanobacteria, where it functions to control light-harvesting of the photosynthetic machinery. However, the photochemical reactions and structural dynamics of this unique photosensing process are not yet resolved. We present time-resolved crystal structures at second-to-minute delays under bright illumination, capturing the early photoproduct and structures of the subsequent reaction intermediates. The first stable photoproduct shows concerted isomerization of C9’-C8’ and C7’-C6’ single bonds in the bicycle-pedal (s-BP) manner and structural changes in the N-terminal domain with minute timescale kinetics. These are followed by a thermally-driven recovery of the s-BP isomer to the dark state carotenoid configuration. Structural changes propagate to the C-terminal domain, resulting, at later time, in the H-bond rupture of the carotenoid keto group with protein residues. Solution FTIR and UV/Vis spectroscopy support the single bond isomerization of the carotenoid in the s-BP manner and subsequent thermal structural reactions as the basis of OCP photoreception.
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Oct 2022
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I23-Long wavelength MX
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Diamond Proposal Number(s):
[20281]
Open Access
Abstract: The introduction of phosphorothioate (PS) linkages to the backbone of therapeutic nucleic acids substantially increases their stability and potency. It also affects their interactions with cellular proteins, but the molecular mechanisms that underlie this effect are poorly understood. Here, we report structural and biochemical studies of interactions between annexin A2, a protein that does not possess any known canonical DNA binding domains, and phosphorothioate-modified antisense oligonucleotides. We show that a unique mode of hydrophobic interactions between a sulfur atom of the phosphorothioate group and lysine and arginine residues account for the enhanced affinity of modified nucleic acid for the protein. Our results demonstrate that this mechanism of interaction is observed not only for nucleic acid-binding proteins but can also account for the association of PS oligonucleotides with other proteins. Using the anomalous diffraction of sulfur, we showed that preference for phosphorothioate stereoisomers is determined by the hydrophobic environment around the PS linkage that comes not only from protein but also from additional structural features within the ASO such as 5-Me groups on cytosine nucleobases.
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Sep 2022
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