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Hagen, W. J.

Publications and source records attributed to Hagen, W. J..

2 recordsLinked to original sources

Light 'em up: efficient screening of gold grids in cryo-EM

Transmission electron cryo-microscopy (cryo-EM) allows for obtaining 3D structural information by imaging macromolecules embedded in thin layers of amorphous ice. To obtain high-resolution structural information, samples need to be thin to minimize inelastic scattering which blurs images. During data collection sessions, time spent on finding areas on the cryo-EM grid with optimal ice thickness should be minimized as imaging time on high-end Transmission Electron Microscope TEM systems is costly. Recently, grids covered with thin gold films have become popular due to their stability and reduced beam-induced motion of the sample. Gold foil grids have substantially different densities between the gold foil and ice, effectively resulting in the loss of dynamic range between thin and thick regions of ice, making it challenging to find areas with suitable ice thickness efficiently during grid screening and thus increase expensive imaging time. Here, an energy filter-based plasmon imaging is presented as a fast and easy method for grid screening of the gold grids.

molecular biology↗

Exploring high-resolution cryo-ET and subtomogram averaging capabilities of contemporary DEDs

The potential of energy filtering and direct electron detection for cryo-electron microscopy (cryo-EM) image processing has been well documented for single particle analysis (SPA). Here, we assess the performance of recently introduced hardware for cryo-electron tomography (cryo-ET) and subtomogram averaging (STA), an increasingly popular structural determination method for complex 3D specimens. We acquired cryo-ET datasets of EIAV virus-like particles (VLPs) on two contemporary cryo-EM systems equipped with different energy filters and direct electron detectors (DED), specifically a Krios G4, equipped with a cold field emission gun (CFEG), Thermo Fisher Scientific Selectris X energy filter, and a Falcon 4 DED; and a Krios G3i, with a Schottky field emission gun (XFEG), a Gatan Bioquantum energy filter, and a K3 DED. We performed constrained cross-correlation-based STA on equally sized datasets acquired on the respective systems. The resulting EIAV CA hexamer reconstructions show that both systems perform comparably in the 4-6 [A] resolution range. In addition, by employing a recently introduced multiparticle refinement approach, we obtained a reconstruction of the EIAV CA hexamer at 2.9 [A]. Our results demonstrate the potential of the new generation of energy filters and DEDs for STA, and the effects of using different processing pipelines on their STA outcomes.

molecular biology↗