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The bacterial chaperonin GroEL-GroES promotes protein folding through ATP-regulated cycles of substrate protein binding, encapsulation, and release. Here, we have used cryoEM to determine structures of GroEL, GroEL-ADP.BeF3, and GroEL-ADP.AlF3-GroES all complexed with the model substrate Rubisco. Our structures provide a series of snapshots that show how the conformation and interactions of non-native Rubisco change as it proceeds through the GroEL-GroES reaction cycle. We observe specific charged and hydrophobic GroEL residues forming strong initial contacts with non-native Rubisco. Binding of ATP or ADP.BeF3 to GroEL-Rubisco results in the formation of an intermediate GroEL complex displaying striking asymmetry in the ATP/ADP.BeF3-bound ring. In this ring, four GroEL subunits bind Rubisco and the other three are in the GroES-accepting conformation, explaining how GroEL can recruit GroES without releasing bound substrate. Our cryoEM structures of stalled GroEL-ADP.AlF3-Rubisco-GroES complexes show Rubisco folding intermediates interacting with GroEL-GroES via different sets of residues.

Volume electron microscopy (vEM) is a group of techniques that reveal the 3D ultrastructure of cells and tissues through continuous depths of at least 1 micrometer. A burgeoning grassroots community effort is fast building the profile and revealing the impact of vEM technology in the life sciences and clinical research.

Public participation in research, also known as citizen science, is being increasingly adopted for the analysis of biological volumetric data. Researchers working in this domain are applying online citizen science as a scalable distributed data analysis approach, with recent research demonstrating that non-experts can productively contribute to tasks such as the segmentation of organelles in volume electron microscopy data. This, alongside the growing challenge to rapidly process the large amounts of biological volumetric data now routinely produced, means there is increasing interest within the research community to apply online citizen science for the analysis of data in this context. Here, we synthesise core methodological principles and practices for applying citizen science for analysis of biological volumetric data. We collate and share the knowledge and experience of multiple research teams who have applied online citizen science for the analysis of volumetric biological data using the Zooniverse platform (www.zooniverse.org). We hope this provides inspiration and practical guidance regarding how contributor effort via online citizen science may be usefully applied in this domain.

Serial focussed ion beam scanning electron microscopy (FIB/SEM) enables imaging and assessment of subcellular structures on the mesoscale (10 nm to 10 µm). When applied to vitrified samples, serial FIB/SEM is also a means to target specific structures in cells and tissues while maintaining constituents’ hydration shells for in situ structural biology downstream. However, the application of serial FIB/SEM imaging of non-stained cryogenic biological samples is limited due to low contrast, curtaining, and charging artefacts. We address these challenges using a cryogenic plasma FIB/SEM. We evaluated the choice of plasma ion source and imaging regimes to produce high-quality SEM images of a range of different biological samples. Using an automated workflow we produced three-dimensional volumes of bacteria, human cells, and tissue, and calculated estimates for their resolution, typically achieving 20–50 nm. Additionally, a tag-free localisation tool for regions of interest is needed to drive the application of in situ structural biology towards tissue. The combination of serial FIB/SEM with plasma-based ion sources promises a framework for targeting specific features in bulk-frozen samples (>100 µm) to produce lamellae for cryogenic electron tomography.

Cryo-electron microscopy (cryoEM) has been transformed over the last decade, with continual new hardware and software tools coming online, pushing the boundaries of what is possible and the nature and complexity of projects that can be undertaken. Here we discuss some recent trends and new tools which are creating opportunities to make more effective use of the resources available within facilities (both staff and equipment). We present approaches for the stratification of projects based on risk and known information about the projects, and the impacts this might have on the allocation of microscope time. We show that allocating different resources (microscope time) based on this information can lead to a significant increase in 'successful' use of the microscope, and reduce lead time by enabling projects to 'fail faster'. This model results in more efficient and sustainable cryoEM facility operation.

An emergent volume electron microscopy (vEM) technique called cryogenic serial plasma focused ion beam milling scanning electron microscopy (pFIB/SEM) can decipher complex biological structures by building a three-dimensional picture of biological samples at mesoscale resolution. This is achieved by collecting consecutive SEM images after successive rounds of FIB milling that expose a new surface after each milling step. Due to instrumental limitations, some image processing is necessary before 3D visualisation and analysis of the data is possible. SEM images are affected by noise, drift, and charging effects, that can make precise 3D reconstruction of biological features difficult. This paper presents Okapi-EM, an open-source Napari plugin(1)developed to process and analyse cryogenic serial FIB/SEM images. Okapi-EM enables automated image registration of slices, evaluation of image quality metrics specific to FIB-SEM imaging, and mitigation of charging artefacts. Implementation of Okapi-EM within the Napari framework ensures that the tools are both user- and developer-friendly, through provision of a graphical user interface and access to Python programming. Napari also hosts a variety of other image processing plugins so Okapi-EM tools can be integrated into and combined with other workflows. Okapi-EM can be downloaded freely athttps://github.com/rosalindfranklininstitute/okapi-em, or installed from Python package index (PyPI).Impact statementCryogenic serial pFIB/SEM is an emerging microscopy technique that is used to visualise 3D structures of biological features at mesoscale resolutions(2). This technique requires common post processing of data such as alignment and charge mitigation to enable robust segmentation and analysis. In addition, approaches are needed to quantify data quality to enable an assessment of features and tune data acquisition parameters to enable optimal image acquisition. This article presents Okapi-EM, a combination of software tools designed to facilitate these important initial steps in assessing and processing images from these experiments. These tools have been assembled as a plugin for a popular 3D biological image visualiser called Napari, making their usage user-friendly and readily accessible.

Electron cryo-tomography (cryo-ET) is an imaging technique for probing 3D structures with at the nanometre scale. This technique has been used extensively in the biomedical field to study the complex structures of proteins and other macromolecules. With the advancement in technology, microscopes are currently capable of producing images amounting to terabytes of data per day, posing great challenges for scientists as the speed of processing of the images cannot keep up with the ever-higher throughput of the microscopes. Therefore, automation is an essential and natural pathway on which image processing – from individual micrographs to full tomograms – is developing. In this paper, we present Ot2Rec, an open-source pipelining tool which aims to enable scientists to build their own processing workflows in a flexible and automatic manner. The basic building blocks of Ot2Rec are plugins which follow a unified API structure, making it simple for scientists to contribute to Ot2Rec by adding features which are not already available. In this paper, we also present three case studies of image processing using Ot2Rec, through which we demonstrate the speedup of using a semi-automatic workflow over a manual one, the possibility of writing and using custom (prototype) plugins, and the flexibility of Ot2Rec which enables the mix-and-match of plugins. We also demonstrate, in the supplementary information, a built-in reporting feature in Ot2Rec which aggregates the metadata from all process being run, and output them in the Jupyter Notebook and/or HTML formats for quick review of image processing quality. Ot2Rec can be found athttps://github.com/rosalindfranklininstitute/ot2rec.Impact StatementThe field of cryo electron tomography has grown substantially in recent years, bringing about new advances in hardware and software which enable visualisation of cell and tissue architecture and proteins found in their native context. These same advances have, in some ways, stratified the field into those with access and those without. On the software side, this has emphasised the need for open-source options that do not require high levels of computational literacy to access. Additionally, it has highlighted the need for ways to both mix-and-match software for easy prototyping and comparisons between parameters and methods. Ot2Rec addresses these needs through a simple, unified plugin structure allowing the addition of existing software or the development of new and does so in a way which democratises access.

Protein misfolding is common to neurodegenerative diseases (NDs) including Alzheimer’s disease (AD), which is characterized by self-assembly and accumulation of Amyloid-beta in the brain. Furthermore, impaired proteostatic mechanisms have been implicated in NDs. Lysosomes are a critical component of the proteostasis network required to degrade and recycle material from outside and within the cell. Using a model of AD neurodegeneration where toxic amyloid beta oligomers are applied exogenously to primary hippocampal neurons, we have previously established that oligomers are endocytosed and trafficked to lysosomes where they accumulate and prevent further endocytosis. Here, we use pioneering correlative cryo-structured illumination microscopy and cryo-soft X-ray tomography imaging techniques to reconstruct 3D cellular architecture in the native state. We demonstrate that lysosomes in oligomer treated neurons are less X-ray dense suggesting they contain less carbon-rich material than untreated cells. Furthermore, we observe an increase in carbon dense lipid vesicles in oligomer treated cells. This work provides unprecedented and critical visual information on the changes to neuronal architecture inflicted by amyloid beta oligomers using advanced methods in structural cell biology.

Serial focussed ion beam scanning electron microscopy (FIB/SEM) enables imaging and assessment of sub-cellular structures on the mesoscale (10 nm to 10 μm). When applied to vitrified samples, serial FIB/SEM is also a means to target specific structures in cells and tissues while maintaining constituents’ hydration shells for in-situ structural biology downstream. However, the application of serial FIB/SEM imaging of non-stained cryogenic biological samples is limited due to low contrast, curtaining and charging artefacts. We address these challenges using a cryogenic plasma FIB/SEM (cryo-pFIB/SEM). We evaluated the choice of plasma ion source and imaging regimes to produce high quality SEM images of a range of different biological samples. Using an automated workflow we produced three dimensional volumes of bacteria, human cells, and tissue, and calculated estimates for their resolution, typically achieving 20 to 50 nm. Additionally, a tag-free tool is needed to drive the application of in situ structural biology towards tissue. The combination of serial FIB/SEM with plasmabased ion sources promises a framework for targeting specific features in bulk-frozen samples (>100 μm) to produce lamella for cryogenic electron tomography.