I24-Microfocus Macromolecular Crystallography
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Maia
Kinnebrew
,
Rachel E.
Woolley
,
T. Bertie
Ansell
,
Eamon F. X.
Byrne
,
Sara
Frigui
,
Giovanni
Luchetti
,
Ria
Sircar
,
Sigrid
Nachtergaele
,
Laurel
Mydock-Mcgrane
,
Kathiresan
Krishnan
,
Simon
Newstead
,
Mark S. P.
Sansom
,
Douglas F.
Covey
,
Christian
Siebold
,
Rajat
Rohatgi
Diamond Proposal Number(s):
[14744, 19946]
Open Access
Abstract: Smoothened (SMO) transduces the Hedgehog (Hh) signal across the plasma membrane in response to accessible cholesterol. Cholesterol binds SMO at two sites: one in the extracellular cysteine-rich domain (CRD) and a second in the transmembrane domain (TMD). How these two sterol-binding sites mediate SMO activation in response to the ligand Sonic Hedgehog (SHH) remains unknown. We find that mutations in the CRD (but not the TMD) reduce the fold increase in SMO activity triggered by SHH. SHH also promotes the photocrosslinking of a sterol analog to the CRD in intact cells. In contrast, sterol binding to the TMD site boosts SMO activity regardless of SHH exposure. Mutational and computational analyses show that these sites are in allosteric communication despite being 45 angstroms apart. Hence, sterols function as both SHH-regulated orthosteric ligands at the CRD and allosteric ligands at the TMD to regulate SMO activity and Hh signaling.
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Jun 2022
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NONE-No attached Diamond beamline
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Charles J.
Buchanan
,
Ben
Gaunt
,
Peter J.
Harrison
,
Yun
Yang
,
Jiwei
Liu
,
Aziz
Khan
,
Andrew M.
Giltrap
,
Audrey
Le Bas
,
Philip N.
Ward
,
Kapil
Gupta
,
Maud
Dumoux
,
Tiong Kit
Tan
,
Lisa
Schimaski
,
Sergio
Daga
,
Nicola
Picchiotti
,
Margherita
Baldassarri
,
Elisa
Benetti
,
Chiara
Fallerini
,
Francesca
Fava
,
Annarita
Giliberti
,
Panagiotis I.
Koukos
,
Matthew J.
Davy
,
Abirami
Lakshminarayanan
,
Xiaochao
Xue
,
Georgios
Papadakis
,
Lachlan P.
Deimel
,
Virgínia
Casablancas-Antràs
,
Timothy D. W.
Claridge
,
Alexandre M. J. J.
Bonvin
,
Quentin J.
Sattentau
,
Simone
Furini
,
Marco
Gori
,
Jiandong
Huo
,
Raymond J.
Owens
,
Christiane
Schaffitzel
,
Imre
Berger
,
Alessandra
Renieri
,
James H.
Naismith
,
Andrew J.
Baldwin
,
Benjamin G.
Davis
Open Access
Abstract: Many pathogens exploit host cell-surface glycans. However, precise analyses of glycan ligands binding with heavily-modified pathogen proteins can be confounded by overlapping sugar signals and/or compound with known experimental constraints. ‘Universal saturation transfer analysis’ (uSTA) builds on existing nuclear magnetic resonance spectroscopy to provide an automated workflow for quantitating protein-ligand interactions. uSTA reveals that early-pandemic, B-origin lineage SARS-CoV-2 spike trimer binds sialoside sugars in an ‘end-on’ manner. uSTA-guided modelling and a high-resolution cryo-electron microscopy structure implicate the spike N-terminal domain (NTD) and confirm end-on binding. This finding rationalizes the effect of NTD mutations that abolish sugar-binding in SARS CoV 2 variants of concern. Together with genetic variance analyses in early pandemic patient cohorts, this binding implicates a sialylated polylactosamine motif found on tetraantennary N-linked glycoproteins in deeper human lung as potentially relevant to virulence and/or zoonosis.
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Jun 2022
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I04-1-Macromolecular Crystallography (fixed wavelength)
I04-Macromolecular Crystallography
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Maurice
Michel
,
Carlos
Benítez-Buelga
,
Patricia A.
Calvo
,
Bishoy M. F.
Hanna
,
Oliver
Mortusewicz
,
Geoffrey
Masuyer
,
Jonathan
Davies
,
Olov
Wallner
,
Sanjiv
Kumar
,
Julian J.
Albers
,
Sergio
Castañeda-Zegarra
,
Ann-Sofie
Jemth
,
Torkild
Visnes
,
Ana
Sastre-Perona
,
Akhilesh N.
Danda
,
Evert J.
Homan
,
Karthick
Marimuthu
,
Zhao
Zhenjun
,
Celestine N.
Chi
,
Antonio
Sarno
,
Elisée
Wiita
,
Catharina
Von Nicolai
,
Anna J.
Komor
,
Varshni
Rajagopal
,
Sarah
Müller
,
Emily C.
Hank
,
Marek
Varga
,
Emma R.
Scaletti
,
Monica
Pandey
,
Stella
Karsten
,
Hanne
Haslene-Hox
,
Simon
Loevenich
,
Petra
Marttila
,
Azita
Rasti
,
Kirill
Mamonov
,
Florian
Ortis
,
Fritz
Schömberg
,
Olga
Loseva
,
Josephine
Stewart
,
Nicholas
D’arcy-Evans
,
Tobias
Koolmeister
,
Martin
Henriksson
,
Dana
Michel
,
Ana
De Ory
,
Lucia
Acero
,
Oriol
Calvete
,
Martin
Scobie
,
Christian
Hertweck
,
Ivan
Vilotijevic
,
Christina
Kalderén
,
Ana
Osorio
,
Rosario
Perona
,
Alexandra
Stolz
,
Pal
Stenmark
,
Ulrika
Warpman Berglund
,
Miguel
De Vega
,
Thomas
Helleday
Diamond Proposal Number(s):
[15806, 21625]
Abstract: Oxidative DNA damage is recognized by 8-oxoguanine (8-oxoG) DNA glycosylase 1 (OGG1), which excises 8-oxoG, leaving a substrate for apurinic endonuclease 1 (APE1) and initiating repair. Here, we describe a small molecule (TH10785) that interacts with the phenylalanine-319 and glycine-42 amino acids of OGG1, increases the enzyme activity 10-fold, and generates a previously undescribed β,δ-lyase enzymatic function. TH10785 controls the catalytic activity mediated by a nitrogen base within its molecular structure. In cells, TH10785 increases OGG1 recruitment to and repair of oxidative DNA damage. This alters the repair process, which no longer requires APE1 but instead is dependent on polynucleotide kinase phosphatase (PNKP1) activity. The increased repair of oxidative DNA lesions with a small molecule may have therapeutic applications in various diseases and aging.
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Jun 2022
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I07-Surface & interface diffraction
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Diamond Proposal Number(s):
[8225, 8733]
Open Access
Abstract: Exposure to the secondary pollutant ozone in ambient air is associated with adverse health effects when inhaled. In this work we use surface pressure measurements, combined with X-ray and neutron reflection, to observe changes in a layer of lung surfactant at the air water interface when exposed to gas phase ozone. The results demonstrate that the layer reacts with ozone changing its physical characteristics. A slight loss of material, a significant thinning of the layer and increased hydration of the surfactant material is observed. The results support the hypothesis that unsaturated lipids present in lung surfactant are still susceptible to rapid reaction with ozone and the reaction changes the properties of the interfacial layer.
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Jun 2022
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I13-2-Diamond Manchester Imaging
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Diamond Proposal Number(s):
[16205]
Open Access
Abstract: Gas bubble in aquatic sediments has a significant effect on its geophysical and geomechanical properties. Recent studies have shown that methane gas and hydrate can coexist in gas hydrate–bearing sediments. Accurate calibration and understanding of the fundamental processes regarding such coexisting gas bubble dynamics is essential for geophysical characterization and hazard mitigation. We conducted high-resolution synchrotron imaging of methane hydrate formation from methane gas in water-saturated sand. While previous hydrate synchrotron imaging has focused on hydrate evolution, here we focus on the gas bubble dynamics. We used a novel semantic segmentation technique based on convolutional neural networks to observe bubble dynamics before and during hydrate formation. Our results show that bubbles change shape and size even before hydrate formation. Hydrate forms on the outer surface of the bubbles, leading to reduction in bubble size, connectivity of bubbles, and the development of nano-to micro-sized bubbles. Interestingly, methane gas bubble size does not monotonously decrease with hydrate formation; rather, we observe some bubbles being completely used up during hydrate formation, while bubbles originate from hydrates in other parts. This indicates the dynamic nature of gas and hydrate formation. We also used an effective medium model including gas bubble resonance effects to study how these bubble sizes affect the geophysical properties. Gas bubble resonance modeling for field or experimental data generally considers an average or equivalent bubble size. We use synchrotron imaging data to extract individual gas bubble volumes and equivalent spherical radii from the segmented images and implement this into the rock physics model. Our modeling results show that using actual bubble size distribution has a different effect on the geophysical properties compared to the using mean and median bubble size distributions. Our imaging and modeling studies show that the existence of these small gas bubbles of a specific size range, compared to a bigger bubble of equivalent volume, may give rise to significant uncertainties in the geophysical inversion of gas quantification.
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Jun 2022
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B21-High Throughput SAXS
I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[14739, 29790, 20229]
Open Access
Abstract: IMP dehydrogenase(IMPDH) is an essential enzyme that catalyzes the rate-limiting step in the guanine nucleotide pathway. In eukaryotic cells, GTP binding to the regulatory domain allosterically controls the activity of IMPDH by a mechanism that is fine-tuned by post-translational modifications and enzyme polymerization. Nonetheless, the mechanisms of regulation of IMPDH in bacterial cells remain unclear. Using biochemical, structural, and evolutionary analyses, we demonstrate that, in most bacterial phyla, (p)ppGpp compete with ATP to allosterically modulate IMPDH activity by binding to a, previously unrecognized, conserved high affinity pocket within the regulatory domain. This pocket was lost during the evolution of Proteobacteria, making their IMPDHs insensitive to these alarmones. Instead, most proteobacterial IMPDHs evolved to be directly modulated by the balance between ATP and GTP that compete for the same allosteric binding site. Altogether, we demonstrate that the activity of bacterial IMPDHs is allosterically modulated by a universally conserved nucleotide-controlled conformational switch that has divergently evolved to adapt to the specific particularities of each organism. These results reconcile the reported data on the crosstalk between (p)ppGpp signaling and the guanine nucleotide biosynthetic pathway and reinforce the essential role of IMPDH allosteric regulation on bacterial GTP homeostasis.
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May 2022
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I24-Microfocus Macromolecular Crystallography
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Chih-Jen
Wei
,
Wei
Bu
,
Laura A.
Nguyen
,
Joseph D.
Batchelor
,
Junghyun
Kim
,
Stefania
Pittaluga
,
James R.
Fuller
,
Hanh
Nguyen
,
Te-Hui
Chou
,
Jeffrey I.
Cohen
,
Gary J.
Nabel
Diamond Proposal Number(s):
[22894]
Abstract: Epstein-Barr virus (EBV) is the major cause of infectious mononucleosis and is associated with several human cancers and, more recently, multiple sclerosis. Despite its prevalence and health impact, there are currently no vaccines or treatments. Four viral glycoproteins (gp), gp350 and gH/gL/gp42, mediate entry into the major sites of viral replication, B cells, and epithelial cells. Here, we designed a nanoparticle vaccine displaying these proteins and showed that it elicits potent neutralizing antibodies that protect against infection in vivo. We designed single-chain gH/gL and gH/gL/gp42 proteins that were each fused to bacterial ferritin to form a self-assembling nanoparticle. Structural analysis revealed that single-chain gH/gL and gH/gL/gp42 adopted a similar conformation to the wild-type proteins, and the protein spikes were observed by electron microscopy. Single-chain gH/gL or gH/gL/gp42 nanoparticle vaccines were constructed to ensure product homogeneity needed for clinical development. These vaccines elicited neutralizing antibodies in mice, ferrets, and nonhuman primates that inhibited EBV entry into both B cells and epithelial cells. When mixed with a previously reported gp350 nanoparticle vaccine, gp350D123, no immune competition was observed. To confirm its efficacy in vivo, humanized mice were challenged with EBV after passive transfer of IgG from mice vaccinated with control, gH/gL/gp42+gp350D123, or gH/gL+gp350D123 nanoparticles. Although all control animals were infected, only one mouse in each vaccine group that received immune IgG had detectable transient viremia. Furthermore, no EBV lymphomas were detected in immune animals. This bivalent EBV nanoparticle vaccine represents a promising candidate to prevent EBV infection and EBV-related malignancies in humans.
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May 2022
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I03-Macromolecular Crystallography
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Moshe
Giladi
,
Michal
Lisnyansky Bar-El
,
Pavla
Vaňková
,
Alisa
Ferofontov
,
Emelia
Melvin
,
Suha
Alkaderi
,
Daniel
Kavan
,
Boris
Redko
,
Elvira
Haimov
,
Reuven
Wiener
,
Petr
Man
,
Yoni
Haitin
Diamond Proposal Number(s):
[19884]
Open Access
Abstract: Isoprenoids are synthesized by the prenyltransferase superfamily, which is subdivided according to the product stereoisomerism and length. In short- and medium-chain isoprenoids, product length correlates with active site volume. However, enzymes synthesizing long-chain products and rubber synthases fail to conform to this paradigm, because of an unexpectedly small active site. Here, we focused on the human cis-prenyltransferase complex (hcis-PT), residing at the endoplasmic reticulum membrane and playing a crucial role in protein glycosylation. Crystallographic investigation of hcis-PT along the reaction cycle revealed an outlet for the elongating product. Hydrogen-deuterium exchange mass spectrometry analysis showed that the hydrophobic active site core is flanked by dynamic regions consistent with separate inlet and outlet orifices. Last, using a fluorescence substrate analog, we show that product elongation and membrane association are closely correlated. Together, our results support direct membrane insertion of the elongating isoprenoid during catalysis, uncoupling active site volume from product length.
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May 2022
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I22-Small angle scattering & Diffraction
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Diamond Proposal Number(s):
[23096]
Open Access
Abstract: Atmospheric aerosol particles can be coated with organic material, impacting on aerosol atmospheric lifetime and urban air quality. Coatings of organic material are also found on indoor surfaces such as window glass. Oleic acid is a fatty acid surfactant which is abundant in cooking and marine aerosol emissions. Under ambient conditions it can self-assemble into lamellar bilayers (stacks) with its sodium salt. We found that nano-scale oleic acid-sodium oleate films spin-coated onto solid silicon substrates form a mixed-phase area of lamellar stacks and amorphous film. The coatings were subjected to simulated atmospheric ageing (ozonolysis and humidity changes) while the surface structure was followed by neutron reflectometry. We found that the orientation of lamellar stacks, which is known to affect the diffusivity of small molecules through them, was sensitive to humidity both in oxidised and pristine films. Lamellar bilayer stacks in oxidised films acquired ~11-fold more water in humid conditions (> 80 % relative humidity) compared to the unoxidised film, demonstrating a significant increase in film hygroscopicity after oxidation. Lamellar stacks, consisting only of starting materials, persisted at the end of simulated atmospheric ageing. These findings for atmospherically relevant nano-scale films corroborate previous work on micrometre-scale layers, thus demonstrating that fatty acid self-assembly could significantly increase the atmospheric lifetime of these molecules. The persistence of such semi-solid surfactant arrangements in the atmosphere has implications for the climate as well as urban and indoor air pollution.
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May 2022
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Open Access
Abstract: Phosphides are interesting candidates for hole transport materials and p-type transparent conducting applications, capable of achieving greater valence band dispersion than their oxide counterparts due to the higher lying energy and increased size of the P 3p orbital. After computational identification of the indirect-gap semiconductor CaCuP as a promising candidate, we now report reactive sputter deposition of phase-pure p-type CaCuP thin films. Their intrinsic hole concentration and hole mobility exceed 1 × 1020 cm−3 and 35 cm2 V−1 s−1 at room temperature, respectively. Transport calculations indicate potential for even higher mobilities. Copper vacancies are identified as the main source of conductivity, displaying markedly different behaviour compared to typical p-type transparent conductors, leading to improved electronic properties. The optical transparency of CaCuP films is lower than expected from first principles calculations of phonon-mediated indirect transitions. This discrepancy could be partly attributed to crystalline imperfections within the films, increasing the strength of indirect transitions. We determine the transparent conductor figure of merit of CaCuP films as a function of composition, revealing links between stoichiometry, crystalline quality, and opto-electronic properties. These findings provide a promising initial assessment of the viability of CaCuP as a p-type transparent contact.
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May 2022
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