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Free keywords:
TRANSMISSION ELECTRON-MICROSCOPY; BEAM-INDUCED MOTION; MACROMOLECULAR
COMPLEXES; CRYOELECTRON MICROSCOPY; BIOLOGICAL SPECIMENS;
RADIATION-DAMAGE; IN-SITU; ORGANIZATION; LIMITATIONS; MICROGRAPHSLife Sciences & Biomedicine - Other Topics;
Abstract:
We present an approach to study macromolecular assemblies by detecting component proteins' characteristic high-resolution projection patterns, calculated from their known 3D structures, in single electron cryo-micrographs. Our method detects single apoferritin molecules in vitreous ice with high specificity and determines their orientation and location precisely. Simulations show that high spatial-frequency information and in the presence of protein background a whitening filter are essential for optimal detection, in particular for images taken far from focus. Experimentally, we could detect small viral RNA polymerase molecules, distributed randomly among binding locations, inside rotavirus particles. Based on the currently attainable image quality, we estimate a threshold for detection that is 150 kDa in ice and 300 kDa in 100 nm thick samples of dense biological material.
