Low-dose cryo-electron ptychography of proteins at sub-nanometer resolution

Article https://doi.org/10.1038/s41467-024-52403-5 Low-dose cryo-electron ptychography of proteins at sub-nanometer resolution Received: 10 March 2024 Accepted: 5 September 2024 Berk Küçükoğlu1, Inayathulla Mohammed1, Ricardo C. Guerrero-Ferreira 1,9, Stephanie M. Ribet2, Georgios Varnavides 2,3, Max Leo Leidl 4,5, Kelvin Lau Sergey Nazarov7, Alexander Myasnikov 7, Massimo Kube1, Julika Radecke1, Carsten Sachse 4,8, Knut Müller-Caspary 5, Colin Ophus 2 & Henning Stahlberg 1 6 , Cryo-transmission electron microscopy (cryo-EM) of frozen hydrated specimens is an efficient method for the structural analysis of purified biological molecules. However, cryo-EM and cryo-electron tomography are limited by the low signal-to-noise ratio (SNR) of recorded images, making detection of smaller particles challenging. For dose-resilient samples often studied in the physical sciences, electron ptychography – a coherent diffractive imaging technique using 4D scanning transmission electron microscopy (4D-STEM) – has recently demonstrated excellent SNR and resolution down to tens of picometers for thin specimens imaged at room temperature. Here we apply 4D-STEM and ptychographic data analysis to frozen hydrated proteins, reaching sub-nanometer resolution 3D reconstructions. We employ low-dose cryo-EM with an aberration-corrected, convergent electron beam to collect 4D-STEM data for our reconstructions. The high frame rate of the electron detector allows us to record large datasets of electron diffraction patterns with substantial overlaps between the interaction volumes of adjacent scan positions, from which the scattering potentials of the samples are iteratively reconstructed. The reconstructed micrographs show strong SNR enabling the reconstruction of the structure of apoferritin protein at up to 5.8 Å resolution. We also show structural analysis of the Phi92 capsid and sheath, tobacco mosaic virus, and bacteriorhodopsin at slightly lower resolutions. 1234567890():,; 1234567890():,; Check for updates Cryo-transmission electron microscopy (cryo-EM) has revolutionized life sciences and pharmaceutical research in academia and industry. Cryo-EM has been shown to only require a few hours to determine the near-atomic resolution structure of proteins that have been frozen as single particles in a thin aqueous layer. The method generally requires that the proteins are available in sufficient concentration as homogeneous populations, adopt sufficiently stable conformations, and are embedded in random 1 Laboratory of Biological Electron Microscopy, Institute of Physics, School of Basic Sciences, EPFL, and Department of Fundamental Microbiology, Faculty of Biology and Medicine, UNIL, Rte. de la Sorge, 1015 Lausanne, Switzerland. 2National Center for Electron Microscopy (NCEM), Molecular Foundry, Lawrence Berkeley National Laboratory, Berkeley, CA 94720, USA. 3Miller Institute for Basic Research in Science, University of California, Berkeley, CA 94720, USA. 4 Ernst Ruska-Centre for Microscopy and Spectroscopy with Electrons (ER-C-3): Structural Biology, Jülich, Germany. 5Department of Chemistry and Centre for NanoScience, Ludwig-Maximilians-Universität München, Butenandstr. 11, 81377 München, Germany. 6Protein Production and Structure Core Facility (PTPSP), School of Life Sciences, EPFL, Rte Cantonale, 1015 Lausanne, Switzerland. 7Dubochet Center for Imaging Lausanne, EPFL and UNIL, EPFL VPA DCI-Lausanne, 1015 Lausanne, Switzerland. 8Department of Biology, Heinrich Heine University, Düsseldorf, Germany. 9Present address: Robert P. Apkarian Integrated e-mail: henning.stahlberg@epfl.ch Electron Microscopy Core, Emory University School of Medicine, 1521 Dickey Drive NE, Atlanta, GA 30322, USA. Nature Communications | (2024)15:8062 1 Article orientations in a thin ice layer without excessive adsorption to the air-water interface. Cryo-EM analysis of proteins suffers from the low signal-to-noise ratio (SNR) in the images, so that only larger protein particles typically bigger than 50 kDa, or larger details in cryo-electron tomography reconstructions of tissue slices can be analyzed at high resolution. The low SNR stems from the fact that for an electron beam, proteins are weak-phase objects and are highly fragile under the beam. Typically, the electron fluence must be limited to below 20 e–/Å2, if a protein is to be imaged at high resolution. Data can be recorded with slightly higher doses if fractionation of the electron dose is used, and recorded frames are resolution-weighted with dose-dependent frequency filters before averaging, to obtain images with higher differential contrast, i.e., signal-to-noise ratio (SNR)1. Cryo-EM furthermore suffers from particle movements under the electron beam, which can be corrected partly during image processing through motion correction in dosefractionated “movies“2–4. Finally, conventional cryo-EM suffers from the oscillating contrast transfer function (CTF), which is dampened towards higher resolution from the limited spatial and temporal coherence of the beam, the detector modulation transfer function (MTF), and non-corrected specimen movements, among others. Phase plates, such as Volta or laser phase plates5,6 partly improve the CTF for low-resolution components, but so far have not yet shown improvements in final resolution. An alternative data acquisition scheme is scanning transmission electron microscopy (STEM), which uses a focused electron probe that is scanned across the sample, while electron detectors record the number of electrons scattered to a certain angle covered by the detector pixel(s) as a function of the probe position7. Bright-field (BF) STEM images provide only weak phase contrast8, and dark-field (DF) STEM yields high mass-thickness contrast, yet at low dose efficiency, so that STEM was until recently of limited use in the life sciences. STEM Z-contrast imaging, employing annular detectors covering high scattering angles, is based on Rutherford scattering, where the detector signal is approximately proportional to the square of the atomic number and linear to the number of atoms within the interaction volume. This linearity of the high-angle annular DF (HAADF) STEM signal has been exploited for detecting heavier atoms in proteins by cryo-STEM with an ADF signal9. It has also been exploited for mass measurements of protein particles that had been freeze-dried on ultrathin carbon film supports10,11. Early attempts of high-resolution lowdose aberration-corrected HAADF STEM imaging showed only amplitude contrast and was found unsuitable for life sciences imaging12. However, cryo-STEM tomography, combining BF and DF STEM with sample tilt series to compute a 3D reconstruction of the vitrified specimen13, has shown promising results for thicker biological specimens up to 1 µm diameter. More recently, integrated differential phase contrast (iDPC) STEM was applied to cryo-EM specimens, reaching 3.5 Å resolution for protein 3D reconstructions fro (...truncated)