Structure of theLysinibacillus sphaericusTpp49Aa1 pesticidal protein elucidated from natural crystals using MHz-SFX

Abstract

Tpp49Aa1 from Lysinibacillus sphaericus is a Toxin_10 family protein that must interact with Cry48Aa1, a 3-domain crystal protein, to produce potent mosquitocidal activity, specifically against Culex quinquefasciatus mosquitoes. We use Culex cell lines to demonstrate for the first time transient detrimental effects of individual toxin components and widen the known target range of the proteins. MHz serial femtosecond crystallography at a nano-focused X-ray free electron laser allowed rapid and high-quality data collection to determine the Tpp49Aa1 structure at 2.2 Å resolution from the merged X-ray diffraction data. The structure revealed the packing of Cry49Aa1 within the natural nanocrystals isolated from sporulated bacteria, as a homodimer with a large intermolecular interface. We then modelled the potential interaction between Tpp49Aa1 and Cry48Aa1. The structure sheds light on natural crystallisation and, along with cell-based assays broadens our understanding of this two-component system.

Diode pumps for future laser plasma accelerators: perspectives from research and industry

“…We next briefly note some details of how a plasma accelerator can be constructed, with special focus on the relevance to diode laser pumps. The construction is illustrated using information from the planned European research infrastructure, the EuPRAXIA facility 3 , that is currently in the preparatory phase and whose concept is described in some detail in [8]. The basic power source for driving particle acceleration in a plasma consists of an ultrashort pulse based on Chirped Pulse Amplification (CPA) [13], consisting of a series of stages, using an optical arrangement as shown schematically in Fig.…”

“…Essentially, lessons and techniques from high intensity laser research are moving step by step toward real industrial application, in a similar manner to the way that skills in the basic science of quantum optics are enabling stepby-step an entirely new industry of quantum photonics. Specific examples of the use of secondary-sources include the generation of highly intense X-rays for diagnostic studies in life and material sciences [2][3][4], the generation of neutron or positron beams for volumetric non-destructive inspection and material studies [5,6], the development of new forms of radiation therapy for cancer patients [6], and their use in basic studies in accelerator science, photon science, laser science and high energy physics. A recent overview is provided in [1,[7][8][9].…”

“…The social and economic importance of studies into secondary-source generation is underlined by the establishment of national excellence centers, such as the Extreme Photonics Application Center (EPAC) in the UK 2 , the development of multinational research centers, for example the EuPRAXIA facility 3 and the establishment of start-up companies specializing in the field, such as TAU-systems 4 . Rapid research progress is also reported in the field at large laser facilities world-wide, for example at members of the Helmholtz Association in Germany 5 .…”

Investigating ultrafast structural dynamics using high repetition rate x-ray FEL radiation at European XFEL

“…This capability of European XFEL offers unique access to problems directly relevant to the societal challenge areas Health and Environment & Sustainability. A recent experiment employing MHz SFX determined the Tpp49 Aa1 structure of a bacterial insecticide to a final resolution of 2.2 Å [28]. The nano-focus option at the SPB/SFX instrument, in combination with megahertz repetition rates, was used for rapid and high-quality data collection from natively grown nanocrystals paving the way for investigations of the structure and dynamics of bacterial insecticides.…”

An experimental study of liquid micro-jets produced with a gas dynamic virtual nozzle under the influence of an electric field

“…Serial femtosecond crystallography (SFX) ( Chapman et al, 2011 ) utilizes femtosecond X-ray pulses generated by an X-ray Free-Electron Laser (XFEL) to produce diffraction patterns of crystals and provide insight into their internal molecular structure. In a particular scheme of SFX ( Stagno et al, 2017 ; Gisriel et al, 2019 ; Williamson et al, 2022 ), protein microcrystals are transferred to the X-ray beam in a liquid suspension focused into a thin micro-jet. The transfer is achieved using gas dynamic virtual nozzles (GDVNs) ( Gañán-Calvo, 1998 ; DePonte et al, 2008 ), which employ forces exerted by a co-flowing and expanding gas that creates a virtual nozzle, accelerating the liquid and focusing the diameter of the jet.…”