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

Abstract

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.

Fig. 1. Experimental setup of 4D-STEM.

a A vitrified life sciences sample is illuminated with a convergent electron beam. Electrons are recorded using a pixelated detector. The convergence semi angle (CSA) is in the range of a few mrad. The beam is focused to a plane above or below sample with a defocus in the micrometer range. Figure created in Blender, using PDB 8J5A as apoferritin model. b An example ptychographic reconstruction of apoferritin. Scale bar: 50 nm. c The sum of three 4D-STEM diffraction patterns. A typical diffraction pattern shows most of the electrons in the bright field (BF) disk of the radius corresponding to the CSA of 6.1 mrad, and some electrons are scattered to higher angles to the dark field (DF) region.

Fig. 2. The ptychography data collection workflow.

A python client running on the SerialEM Server controls the electron microscope via the SerialEM software, and coordinates the TVIPS USG scan generator, the Dectris ELA camera recording, and the multi-GPU processing of recorded diffraction patterns. Computers: gray. Software: white. Microscope: dark gray. Reconstructed images: white. Dotted line: The parallax reconstruction is used to provide on-the-fly feedback about the data collection and serve as aberration estimate.

Fig. 3. Cryo-electron ptychography reconstructions of proteins.

a Apoferritin. b Bacteriophage Phi92 tail protein. c Tobacco mosaic virus (TMV). From left to right: Ptychographic image reconstruction (Scale bars are 30 nm); The same image Fourier transformed, showing circular amplitude oscillations (Scale bars are 0.5 nm⁻¹); The 3D reconstruction of the protein; A cross-section of the 3D reconstruction for a and a bottom view of the 3D reconstructions for b and c.