Cryogenic electron ptychographic single particle analysis with wide bandwidth information transfer

Pei, Xudong; Zhou, Liqi; Boyce, Mark; Kim, Judy; Liberti, Emanuela; Hu, Yiming; Sasaki, Takeo; Nellist, Peter D.; Zhang, Peijun; Stuart, D.I.; Kirkland, Angus I.; Wang, Peng

doi:10.1038/s41467-023-38268-0

Cited by 17 publications

(4 citation statements)

References 70 publications

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“…Moreover, our reference images are formed by amplitude contrast which is less efficient than phase contrast for SPA samples, which are generally thin and composed of light elements. STEM SPA resolution can potentially be improved by phase retrieval methods [23,24]. Practical considerations constrain the combination of these methods with EELS but hold promise for the future [25][26][27][28].…”

Section: Discussionmentioning

confidence: 99%

Localising elements in single-particle reconstructions by REEL-EM: Reconstructed Electron Energy-Loss - Elemental Mapping

Pfeil-Gardiner,

Rosa,

Riedel

et al. 2024

Preprint

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For structures determined by single particle cryo-EM, no technique currently exists for mapping elements to defined locations, leading to errors in the assignment of metals and other ions, cofactors, substrates, inhibitors, and lipids that play essential roles in activity and regulation. Elemental mapping in the electron microscope is well established for dose-tolerant samples but is challenging for biological samples, especially in a cryo-preserved state. Here, we combine electron energy-loss spectroscopy (EELS) with single-particle image processing to allow elemental mapping in cryo-preserved macromolecular complexes. Proof-of-principle data show that our method, REEL-EM, allows 3D reconstruction of EELS data, such that a high total electron dose is accumulated across many copies of a complex. Working with two test samples, we demonstrate that we can reliably localise abundant elements. We discuss the current limitations of the method and potential future developments.

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Section: Discussionmentioning

confidence: 99%

Localising elements in single-particle reconstructions by REEL-EM: Reconstructed Electron Energy-Loss - Elemental Mapping

Pfeil-Gardiner,

Rosa,

Riedel

et al. 2024

Preprint

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“…Data processing workflows have to be established that can deal with the very low signal-to-noise ratio in the low-dose datasets and with the enormous quantity of recorded data, reaching terabytes in size for one sample. Nevertheless, significant progress in life sciences 4D-STEM has recently been demonstrated by the application of 4D-STEM ptychography to frozen-hydrated viruses 16,31 and virus-like particles 32 .…”

Section: Mainmentioning

confidence: 99%

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

Küçükoğlu,

Mohammed,

Guerrero-Ferreira

et al. 2024

Preprint

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Cryo-transmission electron microscopy (cryo-EM) of frozen hydrated specimens is an efficient method for the structural analysis of purified biological molecules. However, for particles smaller than 50kDa, single particle cryo-EM often fails due to the limited imaging contrast of individual micrographs. 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 resolution down to tens of picometers for thin specimens imaged at room temperature.Here we applied ptychographic data analysis to frozen hydrated single protein particles, reaching sub-nanometer resolution 3D reconstructions. We employed low-dose cryo-EM with an aberration-corrected, convergent electron beam to collect 4D-STEM data for our reconstructions. The high speed of the electron detector allowed us to record large datasets of electron diffraction patterns with substantial overlaps between the interaction volumes of adjacent scan position, from which the scattering potentials of the samples were iteratively reconstructed. The reconstructed micrographs show strong contrast enabling the reconstruction of the structure of apoferritin protein at up to 5.8 Å resolution. Ptychography is a promising tool to study the structure of smaller frozen hydrated protein particles, and, once combined with electron tomography tilt series, bears the potential to provide unique insight into the ultrastructure of vitrified biological tissue.

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“…iDPC-STEM has generated the first SPA map of macromolecules embedded in vitrified ice by a STEM technique at near-atomic resolution 23 . Initial attempts at low-dose ptychography have been performed on single particle VLPs at nanometer resolution with a limited number of particles 24,25 . More recent demonstrations have shown that SPA of thin sections with ptychography can resolve protein structures at a sub-nanometer level 26 , including a 5.8Å SPA map of apoferritin reconstructed from ∼11,000 particles.…”

Section: Mainmentioning

confidence: 99%

Dose-Efficient Cryo-Electron Microscopy for Thick Samples using Tilt-Corrected Scanning Transmission Electron Microscopy, Demonstrated on Cells and Single Particles

Yu,

Spoth,

Colletta

et al. 2024

Preprint

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Cryo-EM is a powerful tool in structural biology, providing insights through techniques like single-particle analysis (SPA) and cryogenic electron tomography (cryo-ET). In thick specimens, challenges arise as an exponentially larger fraction of the transmitted electrons lose energy from inelastic scattering and can no longer be properly focused as a result of chromatic aberrations in the post-specimen optics. Rather than filtering out the inelastic scattering at the price of reducing potential signal, as is done in energy-filtered transmission electron microscopy (EFTEM), we show how a dose-efficient and unfiltered image can be rapidly obtained using tilt-corrected bright-field scanning-TEM (tcBF-STEM) data collected on a pixelated detector. Enhanced contrast and a 3-5x improvement in collection efficiency are observed for 2D images of intact bacterial cells and large organelles using tcBF-STEM compared to EFTEM for thicknesses beyond 500 nm. As a proof of concept for the technique's performance in structural determination, we present an SPA map at a 7 angstrom nominal resolution for a highly symmetric virus-like particle (VLP) with 789 particles. These findings suggest applications for tcBF-STEM in cryo-EM of thicker cellular volumes where current approaches struggle.

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Cryogenic electron ptychographic single particle analysis with wide bandwidth information transfer

Cited by 17 publications

References 70 publications

Localising elements in single-particle reconstructions by REEL-EM: Reconstructed Electron Energy-Loss - Elemental Mapping

Localising elements in single-particle reconstructions by REEL-EM: Reconstructed Electron Energy-Loss - Elemental Mapping

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

Dose-Efficient Cryo-Electron Microscopy for Thick Samples using Tilt-Corrected Scanning Transmission Electron Microscopy, Demonstrated on Cells and Single Particles

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