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Instruments / Technical Area	Publication Details	Published
Krios I-Titan Krios I at Diamond Krios III-Titan Krios III at Diamond Krios V-Titan Krios V at Diamond	Structural basis for tuning activity and membrane specificity of bacterial cytolysins Nita R. Shah , Tomas B. Voisin , Edward S. Parsons , Courtney M. Boyd , Bart W. Hoogenboom , Doryen Bubeck Nature Communications 11 DOI: 10.1038/s41467-020-19482-6 Diamond Proposal Number(s): [18659] Open Access Show Abstract ▼ Abstract: Cholesterol-dependent cytolysins (CDCs) are pore-forming proteins that serve as major virulence factors for pathogenic bacteria. They target eukaryotic cells using different mechanisms, but all require the presence of cholesterol to pierce lipid bilayers. How CDCs use cholesterol to selectively lyse cells is essential for understanding virulence strategies of several pathogenic bacteria, and for repurposing CDCs to kill new cellular targets. Here we address that question by trapping an early state of pore formation for the CDC intermedilysin, bound to the human immune receptor CD59 in a nanodisc model membrane. Our cryo electron microscopy map reveals structural transitions required for oligomerization, which include the lateral movement of a key amphipathic helix. We demonstrate that the charge of this helix is crucial for tuning lytic activity of CDCs. Furthermore, we discover modifications that overcome the requirement of cholesterol for membrane rupture, which may facilitate engineering the target-cell specificity of pore-forming proteins.	Nov 2020
I22-Small angle scattering & Diffraction	Pressure-temperature phase behavior of mixtures of natural sphingomyelin and ceramide extracts Hanna Barriga , Edward Parsons , Nicola Mccarthy , Oscar Ces , John Seddon , Robert Law , Nick Brooks Langmuir 31 (12) DOI: 10.1021/la504935c Diamond Proposal Number(s): [8366, 9150, 9756] Show Abstract ▼ Abstract: Ceramides are a group of sphingolipids that act as highly important signaling molecules in a variety of cellular processes including differentiation and apoptosis. The predominant in vivo synthetic pathway for ceramide formation is via sphingomyelinase catalyzed hydrolysis of sphingomyelin. The biochemistry of this essential pathway has been studied in detail; however, there is currently a lack of information on the structural behavior of sphingomyelin- and ceramide-rich model membrane systems, which is essential for developing a bottom-up understanding of ceramide signaling and platform formation. We have studied the lyotropic phase behavior of sphingomyelin−ceramide mixtures in excess water as a function of temperature (30−70 °C) and pressure (1−200 MPa) by small- and wide-angle X-ray scattering. At low ceramide concentrations the mixtures form the ripple gel phase (Pβ′) below the gel transition temperature for sphingomyelin, and this observation has been confirmed by atomic force microscopy. Formation of the ripple gel phase can also be induced at higher temperatures via the application of hydrostatic pressure. At high ceramide concentration an inverse hexagonal phase (HII) is formed coexisting with a cubic phase.	Mar 2015
I22-Small angle scattering & Diffraction	Electrostatic swelling of bicontinuous cubic lipid phases Arwen Tyler , Hanna Barriga , Edward Parsons , Nicola Mccarthy , Oscar Ces , Robert Law , John Seddon , Nick Brooks Soft Matter DOI: 10.1039/C5SM00311C Diamond Proposal Number(s): [9150, 9756] Show Abstract ▼ Abstract: Lipid bicontinuous cubic phases have attracted enormous interest as bio-compatible scaffolds for use in a wide range of applications including membrane protein crystallisation, drug delivery and biosensing. One of the major bottlenecks that has hindered exploitation of these structures is an inability to create targeted highly swollen bicontinuous cubic structures with large and tunable pore sizes. In contrast, cubic structures found in-vivo have periodicities approaching the micron scale. We have been able to engineer and control highly swollen bicontinuous cubic phases of spacegroup Im3m containing only lipids by a) increasing the bilayer stiffness by adding cholesterol and b) inducing electrostatic repulsion across the water channels by addition of anionic lipids to monoolein. By controlling the composition of the ternary mixtures we have been able to achieve lattice parameters up to 470 Å, which is 5 times that observed in pure monoolein and nearly twice the size of any lipidic cubic phase reported previously. These lattice parameters significantly exceed the predicted maximum swelling for bicontinuous cubic lipid structures, which suggest that thermal fluctuations should destroy such phases for lattice parameters larger than 300 Å.	Mar 2015

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