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  • Abstract talk
  • IM4.005

Cryo-STEM imaging for single particle structure determination

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aurum

Session

Development of cryo-EM instrumentation and techniques

Topics

  • IM 4: Development of cryo-EM instrumentation and techniques
  • LS 1: High-resolution cryo-EM

Authors

Carsten Sachse (Jülich / DE; Düsseldorf / DE), Ivan Lazić (Eindhoven / NL), Maarten Wirix (Eindhoven / NL), Max Leo Leidl (Jülich / DE; Munich / DE), Daniel Mann (Jülich / DE), Maximilian Beckers (Jülich / DE; Heidelberg / DE), Evgeniya V. Pechnikova (Eindhoven / NL), Knut Müller-Caspary (Munich / DE), Felix de Haas (Eindhoven / NL)

Abstract

Abstract text (incl. figure legends and references)

In electron cryo-microscopy (cryo-EM), molecular images of vitrified biological samples are obtained by conventional transmission electron microcopy (CTEM) using large underfocuses and subsequently computationally combined into a high-resolution 3D structure. Although scanning transmission electron microscopy (STEM) has been successfully applied to organic and inorganic materials and achieved exceptionally high resolution, its capability to resolve biological structures with high resolution is still to be determined. Here, we apply STEM using the integrated differential phase contrast mode also known as iDPC-STEM (1) to two cryo-EM test specimens, keyhole limpet hemocyanin (KLH) (2) and tobacco mosaic virus (TMV) (3). The micrographs show complete contrast transfer to high resolution and enable the cryo-EM structure determination for KLH at 6.5 Å resolution, as well as for TMV at 3.5 Å resolution using single-particle reconstruction methods, which share identical features with maps obtained by CTEM of a previously acquired same-sized TMV data set. The estimated B-factor from iDPC-STEM of 93 Å2 indicates that the accomplished micrograph quality exceeds CTEM recordings from 2015 using 2nd generation direct electron detection devices. These data show that STEM imaging in general, and in particular the iDPC-STEM approach, can be applied to vitrified single-particle specimens to determine near-atomic resolution cryo-EM structures of biological macromolecules. Our proof-of-principle study introduces high-resolution STEM to biological cryo-EM (4).

I. Lazić, E. G. T. Bosch, S. Lazar, Phase contrast STEM for thin samples: Integrated differential phase contrast. Ultramicroscopy. 160, 265–280 (2016). C. Gatsogiannis, J. Markl, Keyhole Limpet Hemocyanin: 9-Å CryoEM Structure and Molecular Model of the KLH1 Didecamer Reveal the Interfaces and Intricate Topology of the 160 Functional Units. J. Mol. Biol. 385, 963–983 (2009). S. A. Fromm, T. A. M. Bharat, A. J. Jakobi, W. J. H. Hagen, C. Sachse, Seeing tobacco mosaic virus through direct electron detectors. J. Struct. Biol. 189, 87–97 (2015). I. Lazić, M. Wirix, M. L. Leidl, F. de Haas, M. Beckers, E. V. Pechnikova, K. Müller-Caspary, R. Egoavil, E. G. T. Bosch, C. Sachse, "Single-particle cryo-EM structures from iDPC-STEM at near-atomic resolution" (preprint, Biophysics, 2021), , doi:10.1101/2021.10.12.464113. Accepted at Nature Methods

Fig.1 Cryo-iDPC-STEM resolves biological test specimens up to near-atomic resolution.

(a) Optical path of STEM, including the convergence semi-angle (α), the average electron path (red line) and the center of mass (COM) of the intensity at the detector. (b) Raster geometry and beam dimensions over the field of view. (c) iDPC-STEM micrograph of keyhole limpet hemocyanin (left) and corresponding 6.5-Å resolution map with superimposed atomic model (right). Scale bar is 100 nm. (d) iDPC-STEM micrograph of tobacco mosaic virus (left) and corresponding 3.5-Å resolution density (based on the 0.143 Fourier shell correlation (FSC) cutoff criterion) with superimposed atomic model. Scale bar is 100 nm. ADF, annular dark field; BF, bright field; E, electric field; f, focal length of the lens; rp, position of the probe in real space; V, electrostatic potential field.

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