Preparing the best sample of tick-borne encephalitis virus for single-particle imaging at EuXFEL

What is it about?

In our work, an inactivated enveloped virus was tested for the first time as an object for single-particle imaging (SPI) at the European X-ray free electron laser (XFEL). Single-particle imaging using XFELs is a method of determining spatial structure of biological objects under physiological conditions. For this structural method, approximately one millilitre of highly purified homogenous virus suspension at a high concentration is needed. The Sofjin vaccine strain of tick-borne encephalitis virus (TBEV) was chosen as a model. Combination of purification techniques accompanied by standard biochemical methods for estimation of infectivity and concentration together with a quality control pipeline mainly based on transmission electron microscopy and X-ray small-angle scattering allowed us to prepare the best inactivated TBEV sample. Finally, the protocol based on stepwise sucrose gradient ultracentrifugation yielded the most consistent, homogeneous, pure, and concentrated suspension of viral particles. This protocol was then used for sample preparation for high resolution cryo-electron microscopy (cryo-EM) reconstruction and further for sample preparation scale-up for SPI experiments at the European XFEL. We demonstrate a diffraction pattern obtained with a single-pulse X-ray from TBEV particles and a possibility of collecting single-particle diffraction data for the first time using the SPB/SFX instrument of the European XFEL. We were able to solve a high-resolution cryo-EM TBEV structure with the best resolution to date (3.0 Å), further confirming the high quality of the sample obtained by the optimized protocol.

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Photo by National Institute of Allergy and Infectious Diseases on Unsplash

Photo by National Institute of Allergy and Infectious Diseases on Unsplash

Why is it important?

The SPI at XFELs should eventually allow structural definition of different conformations of viral particles and proteins at physiological temperature in solution. These data are of fundamental importance for structure-based design of novel vaccines and antivirals. Biological single-particle analysis at XFELs is still in its early days and requires developing sample preparation methods, especially for such diverse objects as viruses. Certain success has been achieved earlier with non-enveloped viruses that form size-uniform, well-structured particles. Tick-borne encephalitis (TBE) is a severe viral disease that has substantial epidemiological importance. TBEV is a typical member of the flavivirus family that forms roughly spherical enveloped virions approximately 50 nm in diameter. Virions of flaviviruses are heterogeneous depending on their maturation state: each sample may simultaneously contain mature, immature, half-mature, and damaged particles. This diversity limits the choice of methods for determining the structure of flavivirus virions. This is the first time such an object has been studied by SPI at an XFEL, and the possibility of collecting single-particle diffraction data from iTBEV was successfully demonstrated.

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