I12-JEEP: Joint Engineering, Environmental and Processing
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Diamond Proposal Number(s):
[24685]
Open Access
Abstract: This paper presents a diamond gammavoltaic cell – a solid state device which converts gamma radiation into electricity - with a novel design and promising capabilities. Gammavoltaics pose a unique challenge among radiovoltaics due to the highly penetrating nature of gamma rays. Adapting existing radiovoltaic and dosimeter designs by increasing their thickness risks throttling the flowing current, due to an attendant increase in series resistance. The presented design partially decouples this relationship, by creating a low-coverage hydrogen-terminated collection volume around the device, exploiting the transfer doping effect. This paper proves that hydrogen termination is necessary for the gammavoltaism exhibited. Data are then presented from current-voltage curves taken using synchrotron radiation, over the range 50 – 150 keV. A drop in series resistance over the range is discovered, and linked to the transition from the photoelectric effect to Compton scattering. The cell produces an open-circuit voltage VOC = 0.8 V. Its short-circuit current ISC and maximum power Pmax are found to also depend on photon energy, reaching maxima at ∼ 150 keV, where ISC > 10 μA and Pmax > 3 μW, normalised in flux to 2 × 1011 γ.s-1. Groundwork is hence laid for developing this type of cell for micropower applications.
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Feb 2021
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I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[17251]
Open Access
Abstract: Acquisition of foreign DNA by Staphylococcus aureus, including vancomycin resistance genes, is thwarted by the ATP-dependent endonuclease SauUSI. Deciphering the mechanism of action of SauUSI could unravel the reason how it singularly plays a major role in preventing horizontal gene transfer (HGT) in S. aureus. Here, we report a detailed biochemical and structural characterization of SauUSI, which reveals that in the presence of ATP, the enzyme can cleave DNA having a single or multiple target site/s. Remarkably, in the case of multiple target sites, the entire region of DNA flanked by two target sites is shred into smaller fragments by SauUSI. Crystal structure of SauUSI reveals a stable dimer held together by the nuclease domains, which are spatially arranged to hydrolyze the phosphodiester bonds of both strands of the duplex. Thus, the architecture of the dimeric SauUSI facilitates cleavage of either single-site or multi-site DNA. The structure also provides insights into the molecular basis of target recognition by SauUSI. We show that target recognition activates ATP hydrolysis by the helicase-like ATPase domain, which powers active directional movement (translocation) of SauUSI along the DNA. We propose that a pile-up of multiple translocating SauUSI molecules against a stationary SauUSI bound to a target site catalyzes random double-stranded breaks causing shredding of the DNA between two target sites. The extensive and irreparable damage of the foreign DNA by shredding makes SauUSI a potent barrier against HGT.
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Feb 2021
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I02-Macromolecular Crystallography
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Abstract: Anisotropic environments can drastically alter the spectroscopy and photochemistry of molecules, leading to complex structure‐function relationships. We examined this using fluorescent proteins as easy‐to‐modify model systems. Starting from a single scaffold, we have developed a range of 27 photochromic fluorescent proteins that cover a broad range of spectroscopic properties, including the determination of 43 crystal structures. Correlation and principal component analysis confirmed the complex relationship between structure and spectroscopy, but also allowed us to identify consistent trends and to relate these to the spatial organization. We find that changes in spectroscopic properties can come about through multiple underlying mechanisms, of which polarity, hydrogen bonding and presence of water molecules are key modulators. We anticipate that our findings and rich structure/spectroscopy dataset can open opportunities for the development and evaluation of new and existing protein engineering methods.
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Feb 2021
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B21-High Throughput SAXS
I04-1-Macromolecular Crystallography (fixed wavelength)
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Diamond Proposal Number(s):
[20229]
Open Access
Abstract: Thioredoxin reductases control the redox state of thioredoxins (Trxs)—ubiquitous proteins that regulate a spectrum of enzymes by dithiol-disulfide exchange reactions. In most organisms, Trx is reduced by NADPH via a thioredoxin reductase flavoenzyme (NTR), but in oxygenic photosynthetic organisms, this function can also be performed by an iron-sulfur ferredoxin (Fdx)-dependent thioredoxin reductase (FTR) that links light to metabolic regulation. We have recently found that some cyanobacteria, such as the thylakoid-less Gloeobacter and the ocean-dwelling green oxyphotobacterium Prochlorococcus, lack NTR and FTR but contain a thioredoxin reductase flavoenzyme (formerly tentatively called deeply-rooted thioredoxin reductase or DTR), whose electron donor remained undefined. Here we demonstrate that Fdx functions in this capacity and report the crystallographic structure of the transient complex between the plant-type Fdx1 and the thioredoxin reductase flavoenzyme from Gloeobacter violaceus. Thereby, our data demonstrate that this cyanobacterial enzyme belongs to the Fdx flavin-thioredoxin reductase (FFTR) family, originally described in the anaerobic bacterium Clostridium pasteurianum. Accordingly, the enzyme hitherto termed DTR is renamed FFTR. Our experiments further show that the redox sensitive peptide CP12 is modulated in vitro by the FFTR/Trx system, demonstrating that FFTR functionally substitutes for FTR in light-linked enzyme regulation in Gloeobacter. Altogether, we demonstrate the FFTR is spread within the cyanobacteria phylum and propose that, by substituting for FTR, it connects the reduction of target proteins to photosynthesis. Besides, the results indicate that FFTR acquisition constitutes a mechanism of evolutionary adaptation in marine phytoplankton such as Prochlorococcus that live in low-iron environments.
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Feb 2021
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I14-Hard X-ray Nanoprobe
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Kazuya
Morooka
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Eitaro
Kurihara
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Masato
Takehara
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Ryu
Takami
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Kazuki
Fueda
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Kenji
Horie
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Mami
Takehara
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Shinya
Yamasaki
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Toshihiko
Ohnuki
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Bernd
Grambow
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Gareth T. W.
Law
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Joyce W. I.
Ang
,
William R.
Bower
,
Julia
Parker
,
Rodney
Ewings
,
Satoshi
Utsunomiya
Diamond Proposal Number(s):
[21246]
Abstract: A contaminated zone elongated toward Futaba Town, north-northwest of the Fukushima Daiichi Nuclear Power Plant (FDNPP), contains highly radioactive particles released from reactor Unit 1. There are uncertainties associated with the physio-chemical properties and environmental impacts of these particles. In this study, 31 radioactive particles were isolated from surface soils collected 3.9 km north-northwest of the FDNPP. Two of these particles have the highest particle-associated 134+137Cs activity ever reported for Fukushima (6.1 × 105 and 2.5 × 106 Bq per particle after decay-correction to March, 2011). The new, highly-radioactive particle labeled FTB1 is an aggregate of flaky silicate nanoparticles with an amorphous structure containing ~0.8 wt% Cs, occasionally associated with SiO2 and TiO2 inclusions. FTB1 likely originates from the reactor building, which was damaged by a H2 explosion, after adsorbing volatilized Cs. The 134+137Cs activity in the other highly radioactive particle labeled FTB26 exceeded 106 Bq. FTB26 has a glassy carbon core and a surface that is embedded with numerous micro-particles: Pb–Sn alloy, fibrous Al-silicate, Ca-carbonate or hydroxide, and quartz. The isotopic signatures of the micro-particles indicate neutron capture by B, Cs volatilization, and adsorption of natural Ba. The composition of the micro-particles on FTB26 reflects the composition of airborne particles at the moment of the H2 explosion. Owing to their large size, the health effects of the highly radioactive particles are likely limited to external radiation during static contact with skin; the highly radioactive particles are thus expected to have negligible health impacts for humans. By investigating the mobility of the highly radioactive particles, we can better understand how the radiation dose transfers through environments impacted by Unit 1. The highly radioactive particles also provide insights into the atmospheric conditions at the time of the Unit 1 explosion and the physio-chemical phenomena that occurred during reactor meltdown.
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Feb 2021
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I03-Macromolecular Crystallography
I04-Macromolecular Crystallography
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Emerald S.
Ellis
,
Daniel J.
Hinchen
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Alissa
Bleem
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Lintao
Bu
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Sam J. B.
Mallinson
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Mark D.
Allen
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Bennett R.
Streit
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Melodie M.
Machovina
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Quinlan V.
Doolin
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William E.
Michener
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Christopher W.
Johnson
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Brandon C.
Knott
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Gregg T.
Beckham
,
John E.
Mcgeehan
,
Jennifer L.
Dubois
Diamond Proposal Number(s):
[17212, 23269]
Open Access
Abstract: Biological funneling of lignin-derived aromatic compounds is a promising approach for valorizing its catalytic depolymerization products. Industrial processes for aromatic bioconversion will require efficient enzymes for key reactions, including demethylation of O-methoxy-aryl groups, an essential and often rate-limiting step. The recently characterized GcoAB cytochrome P450 system comprises a coupled monoxygenase (GcoA) and reductase (GcoB) that catalyzes oxidative demethylation of the O-methoxy-aryl group in guaiacol. Here, we evaluate a series of engineered GcoA variants for their ability to demethylate o-and p-vanillin, which are abundant lignin depolymerization products. Two rationally designed, single amino acid substitutions, F169S and T296S, are required to convert GcoA into an efficient catalyst toward the o- and p-isomers of vanillin, respectively. Gain-of-function in each case is explained in light of an extensive series of enzyme-ligand structures, kinetic data, and molecular dynamics simulations. Using strains of Pseudomonas putida KT2440 already optimized for p-vanillin production from ferulate, we demonstrate demethylation by the T296S variant in vivo. This work expands the known aromatic O-demethylation capacity of cytochrome P450 enzymes toward important lignin-derived aromatic monomers.
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Feb 2021
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I03-Macromolecular Crystallography
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Piyada
Supasa
,
Daming
Zhou
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Wanwisa
Dejnirattisai
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Chang
Liu
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Alexander J.
Mentzer
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Helen M.
Ginn
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Yuguang
Zhao
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Helen M. E.
Duyvesteyn
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Rungtiwa
Nutalai
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Aekkachai
Tuekprakhon
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Beibei
Wang
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Guido
Paesen
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Jose
Slon-campos
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César
López-camacho
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Bassam
Hallis
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Naomi
Coombes
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Kevin
Bewley
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Sue
Charlton
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Thomas S.
Walter
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Eleanor
Barnes
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Susanna J.
Dunachie
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Donal
Skelly
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Sheila F.
Lumley
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Natalie
Baker
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Imam
Shaik
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Holly
Humphries
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Kerry
Godwin
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Nick
Gent
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Alex
Sienkiewicz
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Christina
Dold
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Robert
Levin
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Tao
Dong
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Andrew J.
Pollard
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Julian C.
Knight
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Paul
Klenerman
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Derrick
Crook
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Teresa
Lambe
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Elizabeth
Clutterbuck
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Sagida
Bibi
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Amy
Flaxman
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Mustapha
Bittaye
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Sandra
Belij-rammerstorfer
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Sarah
Gilbert
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Dave R.
Hall
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Mark
Williams
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Neil G.
Paterson
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William
James
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Miles W.
Carroll
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Elizabeth E.
Fry
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Juthathip
Mongkolsapaya
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Jingshan
Ren
,
David I.
Stuart
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Gavin R.
Screaton
Diamond Proposal Number(s):
[27009]
Open Access
Abstract: SARS-CoV-2 has caused over 2M deaths in little over a year. Vaccines are being deployed at scale, aiming to generate responses against the virus spike. The scale of the pandemic and error-prone virus replication is leading to the appearance of mutant viruses and potentially escape from antibody responses. Variant B.1.1.7, now dominant in the UK, with increased transmission, harbours 9 amino-acid changes in the spike, including N501Y in the ACE2 interacting-surface. We examine the ability of B.1.1.7 to evade antibody responses elicited by natural SARS-CoV-2 infection or vaccination. We map the impact of N501Y by structure/function analysis of a large panel of well-characterised monoclonal antibodies. B.1.1.7 is harder to neutralize than parental virus, compromising neutralization by some members of a major class of public antibodies through light chain contacts with residue 501. However, widespread escape from monoclonal antibodies or antibody responses generated by natural infection or vaccination was not observed.
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Feb 2021
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I03-Macromolecular Crystallography
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Diamond Proposal Number(s):
[23620]
Open Access
Abstract: UTP-glucose-1-phosphate uridylyltransferases (UGPases) are enzymes that produce UDP-glucose from UTP and glucose-1-phosphate. In Bacillus subtilis 168, UDP-glucose is required for the decoration of wall teichoic acid (WTA) with glucose residues and the formation of glucolipids. The B. subtilis UGPase GtaB is essential for UDP-glucose production under standard aerobic growth conditions, and gtaB mutants display severe growth and morphological defects. However, bioinformatics predictions indicate that two other UGPases, are present in B. subtilis. Here, we investigated the function of one of them named YngB. The crystal structure of YngB revealed that the protein has the typical fold and all necessary active site features of a functional UGPase. Furthermore, UGPase activity could be demonstrated in vitro using UTP and glucose-1-phosphate as substrates. Expression of YngB from a synthetic promoter in a B. subtilis gtaB mutant resulted in the reintroduction of glucose residues on WTA and production of glycolipids, demonstrating that the enzyme can function as UGPase in vivo. When wild-type and mutant B. subtilis strains were grown under anaerobic conditions, YngB-dependent glycolipid production and glucose decorations on WTA could be detected, revealing that YngB is expressed from its native promoter under anaerobic condition. Based on these findings, along with the structure of the operon containing yngB and the transcription factor thought to be required for its expression, we propose that besides WTA, potentially other cell wall components might be decorated with glucose residues during oxygen limited growth condition.
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Feb 2021
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B22-Multimode InfraRed imaging And Microspectroscopy
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Diamond Proposal Number(s):
[17044]
Abstract: Bis(2-ethylhexyl) phosphate (BEHP) was exposed to carbon steel surfaces from dry and water-saturated dodecane. The resulting changes to the surfaces were characterised using spectroscopic techniques (energy dispersive X-ray (EDX), X-ray photoelectron (XPS), and far-infrared reflection absorption (RAIRS) spectroscopies) and polarised neutron reflectometry (PNR). Although there was no observable affinity of BEHP to the steel surface in dry solvent, a layer of rough iron (III) phosphate formed in water-saturated dodecane. The phosphate-reacted steel surface showed some resistance to corrosion by seawater, suggesting the formation of a cohesive barrier against corrosive species. The results support the use of BEHP as an anti-corrosion additive and a viable phosphating agent for steel surfaces.
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Feb 2021
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I03-Macromolecular Crystallography
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Diamond Proposal Number(s):
[13467]
Open Access
Abstract: Microbial plant pathogens secrete effector proteins which manipulate the host to promote infection. Effectors can be recognised by plant intracellular nucleotide-binding leucine-rich repeat (NLR) receptors, initiating an immune response. The AVR-Pik effector from the rice blast fungus Magnaporthe oryzae is recognised by a pair of rice NLR receptors, Pik-1 and Pik-2. Pik-1 contains a non-canonical integrated heavy metal-associated (HMA) domain, which directly binds AVR-Pik to activate plant defences. The host targets of AVR-Pik are also HMA domain-containing proteins, namely heavy metal-associated isoprenylated plant proteins (HIPPs) and heavy metal-associated plant proteins (HPPs). Here, we demonstrate that one of these targets interacts with a wider set of AVR-Pik variants compared to the Pik-1 HMA domains. We define the biochemical and structural basis of the interaction between AVR-Pik and OsHIPP19, and compare the interaction to that formed with the HMA domain of Pik-1. Using analytical gel filtration and surface plasmon resonance, we show that multiple AVR-Pik variants, including the stealthy variants AVR-PikC and AVR-PikF which do not interact with any characterised Pik-1 alleles, bind to OsHIPP19 with nanomolar affinity. The crystal structure of OsHIPP19 in complex with AVR-PikF reveals differences at the interface that underpin high-affinity binding of OsHIPP19-HMA to a wider set of AVR-Pik variants than achieved by the integrated HMA domain of Pik-1. Our results provide a foundation for engineering the HMA domain of Pik-1 to extend binding to currently unrecognised AVR-Pik variants and expand disease resistance in rice to divergent pathogen strains.
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Feb 2021
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