Workflow for Single-Particle-Analysis (SPA)

In SPA, data obtained from high amounts of two-dimensional (2D) projection images, featuring matching copies of a protein or protein complex in various orientations, are combined to produce a high resolution three dimensional (3D) reconstruction of the protein or protein complex.  

For this workflow, the facility is equipped with a Thermo Scientific Vitrobot and Gatan Cp3 plunger to freeze the protein solutions rapidly within milliseconds to avoid the formation of ice crystals. 

Process of plunge freezing

Sample screening utilises a Thermo Scientific Talos Arctica 200 kV cryo-electron microscope which can load and examine up to twelve vitrified SPA samples in a short time. After finding optimal freezing conditions, high resolution datasets are obtained with one of our two Thermo Scientific Titan Krios 300 kV instruments.

Both are fitted with the latest direct electron detection camera (Gatan K3) and energy filter to obtain structures of proteins or protein complexes with a resolution of below 2 Å. To improve the point resolution of the microscope one of the Krios instruments has a CETCOR Cs-corrector (CEOS GmbH) installed to compensate for all lens aberrations (up to the 3rd order).

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