Novel computational methods for in vitro and in situ cryo-electron microscopy

Over the past decade, advances in microscope hardware and image data processing algorithms have made cryo-electron microscopy (cryo-EM) a dominant technique for protein structure determination. Near-atomic resolution can now be obtained for many challenging in vitro samples using single-particle analysis (SPA), while sub-tomogram averaging (STA) can obtain sub-nanometer resolution for large protein complexes in a crowded cellular environment. Reaching high resolution requires large amounts of im-age data. Modern transmission electron microscopes (TEMs) automate the acquisition process and can acquire thousands of micrographs or hundreds of tomographic tilt se-ries over several days without intervention. In a first step, the data must be pre-processed: Micrographs acquired as movies are cor-rected for stage and beam-induced motion. For tilt series, additional alignment of all micrographs in 3D is performed using gold- or patch-based fiducials. Parameters of the contrast-transfer function (CTF) are estimated to enable its reversal during SPA refine-ment. Finally, individual protein particles must be located and extracted from the aligned micrographs. Current pre-processing algorithms, especially those for particle picking, are not robust enough to enable fully unsupervised operation. Thus, pre-processing is start-ed after data collection, and takes several days due to the amount of supervision re-quired. Pre-processing the data in parallel to acquisition with more robust algorithms would save time and allow to discover bad samples and microscope settings early on. Warp is a new software for cryo-EM data pre-processing. It implements new algorithms for motion correction, CTF estimation, tomogram reconstruction, as well as deep learn-ing-based approaches to particle picking and image denoising. The algorithms are more accurate and robust, enabling unsupervised operation. Warp integrates all pre-processing steps into a pipeline that is executed on-the-fly during data collection. Inte-grated with SPA tools, the pipeline can produce 2D and 3D classes less than an hour into data collection for favorable samples. Here I describe the implementation of the new algorithms, and evaluate them on various movie and tilt series data sets. I show that un-supervised pre-processing of a tilted influenza hemagglutinin trimer sample with Warp and refinement in cryoSPARC can improve previously published resolution from 3.9 Å to 3.2 Å. Warp’s algorithms operate in a reference-free manner to improve the image resolution at the pre-processing stage when no high-resolution maps are available for the particles yet. Once 3D maps have been refined, they can be used to go back to the raw data and perform reference-based refinement of sample motion and CTF in movies and tilt series. M is a new tool I developed to solve this task in a multi-particle framework. Instead of following the SPA assumption that every particle is single and independent, M models all particles in a field of view as parts of a large, physically connected multi-particle system. This allows M to optimize hyper-parameters of the system, such as sample motion and deformation, or higher-order aberrations in the CTF. Because M models these effects accurately and optimizes all hyper-parameters simultaneously with particle alignments, it can surpass previous reference-based frame and tilt series alignment tools. Here I de-scribe the implementation of M, evaluate it on several data sets, and demonstrate that the new algorithms achieve equally high resolution with movie and tilt series data of the same sample. Most strikingly, the combination of Warp, RELION and M can resolve 70S ribosomes bound to an antibiotic at 3.5 Å inside vitrified Mycoplasma pneumoniae cells, marking a major advance in resolution for in situ imaging

Towards Visual Proteomics at High Resolution

Traditionally, structural biologists approach the complexity of cellular proteomes in a reductionist manner. Proteomes are fractionated, their molecular components purified and studied one-by-one using the experimental methods for structure determination at their disposal. Visual proteomics aims at obtaining a holistic picture of cellular proteomes by studying them in situ, ideally in unperturbed cellular environments. The method that enables doing this at highest resolution is cryo-electron tomography. It allows to visualize cellular landscapes with molecular resolution generating maps or atlases revealing the interaction networks which underlie cellular functions in health and in disease states. Current implementations of cryo ET do not yet realize the full potential of the method in terms of resolution and interpretability. To this end, further improvements in technology and methodology are needed. This review describes the state of the art as well as measures which we expect will help overcoming current limitations. (C) 2021 Published by Elsevier Ltd

The promise and the challenges of cryo-electron tomography

Structural biologists have traditionally approached cellular complexity in a reductionist manner in which the cellular molecular components are fractionated and purified before being studied individually. This 'divide and conquer' approach has been highly successful. However, awareness has grown in recent years that biological functions can rarely be attributed to individual macromolecules. Most cellular functions arise from their concerted action, and there is thus a need for methods enabling structural studies performed in situ, ideally in unperturbed cellular environments. Cryo-electron tomography (Cryo-ET) combines the power of 3D molecular-level imaging with the best structural preservation that is physically possible to achieve. Thus, it has a unique potential to reveal the supramolecular architecture or 'molecular sociology' of cells and to discover the unexpected. Here, we review state-of-the-art Cryo-ET workflows, provide examples of biological applications, and discuss what is needed to realize the full potential of Cryo-ET