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Instruments / Technical Area	Publication Details	Published
I03-Macromolecular Crystallography I04-1-Macromolecular Crystallography (fixed wavelength) I04-Macromolecular Crystallography	Directed evolution of an efficient and thermostable PET depolymerase Elizabeth L. Bell , Ross Smithson , Siobhan Kilbride , Jake Foster , Florence J. Hardy , Saranarayanan Ramachandran , Aleksander A. Tedstone , Sarah J. Haigh , Arthur A. Garforth , Philip J. R. Day , Colin Levy , Michael P. Shaver , Anthony P. Green Nature Catalysis 3 DOI: 10.1038/s41929-022-00821-3 Diamond Proposal Number(s): [12788, 17773] Show Abstract ▼ Abstract: The recent discovery of IsPETase, a hydrolytic enzyme that can deconstruct poly(ethylene terephthalate) (PET), has sparked great interest in biocatalytic approaches to recycle plastics. Realization of commercial use will require the development of robust engineered enzymes that meet the demands of industrial processes. Although rationally engineered PETases have been described, enzymes that have been experimentally optimized via directed evolution have not previously been reported. Here, we describe an automated, high-throughput directed evolution platform for engineering polymer degrading enzymes. Applying catalytic activity at elevated temperatures as a primary selection pressure, a thermostable IsPETase variant (HotPETase, Tm = 82.5 °C) was engineered that can operate at the glass transition temperature of PET. HotPETase can depolymerize semicrystalline PET more rapidly than previously reported PETases and can selectively deconstruct the PET component of a laminated multimaterial. Structural analysis of HotPETase reveals interesting features that have emerged to improve thermotolerance and catalytic performance. Our study establishes laboratory evolution as a platform for engineering useful plastic degrading enzymes.	Aug 2022
	Occlusion of micron-sized algae cells and synthetic hollow mineral spheres within single crystals Bartosz Marzec , Jessica Walker , Yasmeen Jhons , Fiona C. Meldrum , Michael P. Shaver , Fabio Nudelman Faraday Discussions DOI: 10.1039/D1FD00095K Open Access Show Abstract ▼ Abstract: Incorporating additives within host single crystals is an effective strategy for producing composite materials with tunable mechanical, magnetic and optical properties. The type of guest materials that can be occluded can be limited, however, as incorporation is a complex process depending on many factors including binding of the additive to the crystal surface, the rate of crystal growth and the stability of the additives in the crystallisation solution. In particular, the size of occluded guests has been restricted to a few angstroms, as for single molecules, to a few hundred nanometers, as for polymer vesicles and particles. Here, we present a synthetic approach for occluding micrometer-scale objects, including high-complexity unicellular organisms and synthetic hollow calcite spheres within calcite single crystals. Both of these objects can transport functional additives, including organic molecules and nanoparticles that would not otherwise occlude within calcite. Therefore, this method constitutes a generic approach using calcite as a delivery system for active compounds, while providing them with effective protection against environmental factors that could cause degradation.	Dec 2021
Krios I-Titan Krios I at Diamond	Three-dimensional architecture and surface functionality of coccolith base plates B. Marzec , J. M. Walker , M. Panagopoulou , Y. Jhons , D. Clare , A. Wheeler , M. P. Shaver , F. Nudelman Journal Of Structural Biology DOI: 10.1016/j.jsb.2019.08.007 Diamond Proposal Number(s): [16989] Open Access Show Abstract ▼ Abstract: Coccolithophores are marine phytoplankton that are among the most prolific calcifiers widespread in Earth’s oceans, playing a crucial role in the carbon cycle and in the transport of organic matter to the deep sea. These organisms produce highly complex mineralized scales that are composed of hierarchical assemblies of nano-crystals of calcium carbonate in the form of calcite. Coccolith formation in vivo occurs within compartmentalized mineralisation vesicles derived from the Golgi body, which contain coccolith-associated polysaccharides (‘CAPs’) providing polymorph selection and mediating crystal growth kinetics, and oval organic mineralisation templates, also known as base plates, which promote heterogenous nucleation and further mechanical interlocking of calcite single crystals. Although the function of coccolith base plates in controlling crystal nucleation have been widely studied, their 3D spatial organization and the chemical functional groups present on the crystal nucleation sites, which are two crucial features impacting biomineralization, remain unsolved. Utilising cryo-electron tomography we show that base plates derived from an exemplary coccolithophore Pleurochrysis carterae (Pcar) in their native hydrated state have a complex 3-layered structure. We further demonstrate, for the first time, the edge and rim of the base plate – where the crystals nucleate - are rich in primary amine functionalities that provide binding targets for negatively charged complexes composed of synthetic macromolecules and Ca2+ ions. Our results indicate that electrostatic interactions between the negatively charged biogenic CAPs and the positively charged rim of the base plate are sufficient to mediate the transport of Ca2+ cations to the mineralization sites.	Aug 2019

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