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Instruments / Technical Area	Publication Details	Published
	Accessibility to peptidoglycan is important for the recognition of gram-positive bacteria in Drosophila Filipa Vaz , Ilias Kounatidis , Gonçalo Covas , Richard M. Parton , Maria Harkiolaki , Ilan Davis , Sergio Raposo Filipe , Petros Ligoxygakis Cell Reports 27, 2480 - 2492.e6 DOI: 10.1016/j.celrep.2019.04.103 Open Access Show Abstract ▼ Abstract: In Drosophila, it is thought that peptidoglycan recognition proteins (PGRPs) SA and LC structurally discriminate between bacterial peptidoglycans with lysine (Lys) or diaminopimelic (DAP) acid, respectively, thus inducing differential antimicrobial transcription response. Here, we find that accessibility to PG at the cell wall plays a central role in immunity to infection. When wall teichoic acids (WTAs) are genetically removed from S. aureus (Lys type) and Bacillus subtilis (DAP type), thus increasing accessibility, the binding of both PGRPs to either bacterium is increased. PGRP-SA and -LC double mutant flies are more susceptible to infection with both WTA-less bacteria. In addition, WTA-less bacteria grow better in PGRP-SA/-LC double mutant flies. Finally, infection with WTA-less bacteria abolishes any differential activation of downstream antimicrobial transcription. Our results indicate that accessibility to cell wall PG is a major factor in PGRP-mediated immunity and may be the cause for discrimination between classes of pathogens.	May 2019
Optics	Compensation of x-ray mirror distortion by cooling temperature control Matthew Hand , Hongchang Wang , Maria Harkiolaki , Federica Venturini , Rosa Arrigo , Pilar Ferrer Escorihuela , Simon Alcock , Ioana Nistea , Andy Marshall , Stewart Scott , Liz Duke , Georg Held , Kawal Sawhney Proceedings of the 13th International Conference on Synchrotron Radiation Instrumentation – SRI2018 DOI: 10.1063/1.5084675 Open Access Show Abstract ▼ Abstract: Synchrotron radiation is emitted from a bending magnet source in a wide ray fan which is collected by the first optical element in a beamline. In order to maximize angular acceptance, and hence flux, it is beneficial to increase the length of this mirror and optical design requirements may necessitate that the optical surface be over 1 m in length. Such mirrors also require cooling as they may be subject to high heat loads from the incident radiation. Two beamlines, B07 and B24, at Diamond Light Source, UK, use 1.4 m long toroidal mirrors which utilize a similar side-clamped cooling manifold design. While this scheme has been successful in providing effective cooling of the mirror, it has also been discovered that it introduces deformation of the radius of curvature which is sufficient to alter the focusing characteristics of the mirror. At both beamlines, the horizontal focus of the beam was found to differ by up to several meter from the design position at the exit slit which resulted in poor flux throughput, reduced energy resolution and other side effects. A pencil beam scan method has been used to diagnose this issue and infer the position of the focus and mirror shape. Through the use of a standalone chiller to alter the temperature of the water within the cooling loop, it has been possible to correct the distortion of the radius and restore the focus to its nominal position.	Jan 2019
B24-Cryo Soft X-ray Tomography	Correlation of cryo soft x-ray tomography with cryo fluorescence microscopy to characterise cellular organelles at beamline B24, Diamond Light Source Matthew C. Spink , Michele C. Darrow , Hannah Fisher , Alister Burt , Karen Marshall , Imanol Luengo , Maria Harkiolaki , Liz Duke Microscopy And Microanalysis 24, 376 - 377 Microscopy & Microanalysis 2018 DOI: 10.1017/S1431927618014162 Show Abstract ▼ Abstract: Cryo soft-X-ray tomography (Cryo–SXT) is a 3D imaging technique that allows us to image whole vitrified biological cells and their contents in a near native state. Cryo-SXT utilises the natural contrast of cellular structures by imaging in an energy range termed the ‘water window’ The water window is between the K absorption edges of Carbon 284 eV and Oxygen 543 eV. Imaging between these energies results in higher X-ray absorption by carbon rich cellular structures than oxygen containing molecules such as vitrified water. The resulting contrast allows us to see cellular structures (Figure 1A) at a resolution of ~40nm, depending on the sample and microscope components.	Aug 2018
	Cryo-soft X-ray tomography: using soft X-rays to explore the ultrastructure of whole cells Maria Harkiolaki , Michele C. Darrow , Matthew C. Spink , Ewelina Kosior , Kyle Dent , Elisabeth Duke Emerging Topics In Life Sciences DOI: 10.1042/ETLS20170086 Open Access Show Abstract ▼ Abstract: Cryo-soft X-ray tomography is an imaging technique that addresses the need for mesoscale imaging of cellular ultrastructure of relatively thick samples without the need for staining or chemical modification. It allows the imaging of cellular ultrastructure to a resolution of 25–40 nm and can be used in correlation with other imaging modalities, such as electron tomography and fluorescence microscopy, to further enhance the information content derived from biological samples. An overview of the technique, discussion of sample suitability and information about sample preparation, data collection and data analysis is presented here. Recent developments and future outlook are also discussed.	Mar 2018
B24-Cryo Soft X-ray Tomography	Parasitophorous vacuole poration precedes its rupture and rapid host erythrocyte cytoskeleton collapse in Plasmodium falciparum egress Victoria Hale , Jean M. Watermeyer , Fiona Hackett , Gema Vizcay-barrena , Christiaan Van Ooij , James A. Thomas , Matthew C. Spink , Maria Harkiolaki , Elizabeth Duke , Roland A. Fleck , Michael J. Blackman , Helen Saibil Proceedings Of The National Academy Of Sciences DOI: 10.1073/pnas.1619441114 Diamond Proposal Number(s): [14410] Show Abstract ▼ Abstract: In the asexual blood stages of malarial infection, merozoites invade erythrocytes and replicate within a parasitophorous vacuole to form daughter cells that eventually exit (egress) by sequential rupture of the vacuole and erythrocyte membranes. The current model is that PKG, a malarial cGMP-dependent protein kinase, triggers egress, activating malarial proteases and other effectors. Using selective inhibitors of either PKG or cysteine proteases to separately inhibit the sequential steps in membrane perforation, combined with video microscopy, electron tomography, electron energy loss spectroscopy, and soft X-ray tomography of mature intracellular Plasmodium falciparum parasites, we resolve intermediate steps in egress. We show that the parasitophorous vacuole membrane (PVM) is permeabilized 10–30 min before its PKG-triggered breakdown into multilayered vesicles. Just before PVM breakdown, the host red cell undergoes an abrupt, dramatic shape change due to the sudden breakdown of the erythrocyte cytoskeleton, before permeabilization and eventual rupture of the erythrocyte membrane to release the parasites. In contrast to the previous view of PKG-triggered initiation of egress and a gradual dismantling of the host erythrocyte cytoskeleton over the course of schizont development, our findings identify an initial step in egress and show that host cell cytoskeleton breakdown is restricted to a narrow time window within the final stages of egress.	Mar 2017
I02-Macromolecular Crystallography	The structure of Bacillus subtilis SP[beta] prophage dUTPase and its complexes with two nucleotides Javier García-nafría , Maria Harkiolaki , Rebecca Persson , Mark Fogg , Keith Wilson Biological Crystallography 67 (3), 167-175 DOI: 10.1107/S0907444911003234 PMID: 21358047 Show Abstract ▼ Abstract: dUTPases are housekeeping enzymes which catalyse the hydrolysis of dUTP to dUMP in an ion-dependent manner. Bacillus subtilis has both a genomic and an SP[beta] prophage homotrimeric dUTPase. Here, structure determination of the prophage apoenzyme and of its complexes with dUDP and dUpNHpp-Mg2+ is described at 1.75, 1.9 and 2.55 Å resolution, respectively. The C-terminal extension, which carries the conserved motif V, is disordered in all three structures. Unlike all other trimeric dUTPases for which structures are available, with the exception of the Bacillus genomic enzyme, the aromatic residue covering the uridine and acting as the Phe-lid is close to motif III in the sequence rather than in motif V. This is in spite of the presence of an aromatic amino acid at the usual Phe-lid position in motif V. The alternative position of the Phe-lid requires a reconsideration of its role in the catalytic cycle of the enzyme. In the dUpNHpp-Mg2+ complex a water can be seen at the position expected for nucleophilic attack on the [alpha]-phosphate, in spite of motif V being disordered. Differences in the active site between the free enzyme and the dUDP and dUpNHpp-Mg2+ complexes shows that the triphosphate moiety needs to be in the gauche conformation to trigger the conformational changes that can be seen in both B. subtilis dUTPases.	Mar 2011
I03-Macromolecular Crystallography	Distinct Binding Modes of Two Epitopes in Gab2 that Interact with the SH3C Domain of Grb2 Maria Harkiolaki , Theodora Tsirka , Marc Lewitzky , Philip Simister , Dhira Joshi , Louise Bird , E. Yvonne Jones , Nicola O'reilly , Stephen Feller Structure 17 (6), 809-822 DOI: 10.1016/j.str.2009.03.017 PMID: 19523899 Show Abstract ▼ Abstract: Grb2 and Gab2 form a complex implicated in normal cell signaling and cancer development. Binding of the Grb2SH3C domain to Gab2 is essential for the interaction, but molecular details remained undefined. Using peptide arrays and isothermal titration calorimetry, two Grb2SH3C binding sites in Gab2 (Gab2a and Gab2b) were confirmed and characterized. Gab2a bears similarity to a p27Kip1 epitope that also binds Grb2SH3C. Crystal structures of both Gab2 epitopes complexed with Grb2SH3C reveal that Gab2b contains a 310 helix that positions the arginine and lysine of the core-binding motif RxxK in parallel orientation. In contrast, the Gab2a RxxK motif is embedded in a PPII helix with Arg and Lys in staggered orientation. A similar interaction mode is also present in a new complex of Mona/GadsSH3C with an RxxxxK epitope from the putative phosphatase HD-PTP. In summary, our study reveals interaction types of SH3 domains, highlighting their great versatility.	Jun 2009

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