Selected reference guide

Find structure and function with cryo-electron tomography

Revolutionizing our understanding of biological systems

As the world leader in serving science, our innovative microscopy and application expertise helps customers find meaningful answers to the questions that accelerate breakthrough discoveries, increase productivity, and ultimately change the world.

Thermo Fisher Scientific’s Materials & Structural Analysis division develops high-end electron microscopes. The main locations and development centers for electron microscopy are located in the cities of Eindhoven (NL), Brno (CZ) and Hillsboro, Oregon (US). At these sites, R&D engineers cover all disciplines necessary to develop an electron microscope, including physics, mechatronics, electronics and software. By continually expanding capabilities and driving innovation, Thermo Fisher Scientific is uniquely positioned to take cryo-EM technology to new heights, enabling our customers to explore biological complexity and make new discoveries for many years to come.

About Thermo Fisher Scientific

COVER IMAGE: Cryo-electron tomography of Chlamydomonas golgi. Data courtesy of Dr. Benjamin Engel, formerly MPI Biochemistry, now Helmholtz Zentrum München. Data visualization with Thermo Scientific™ Amira™ Software.

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The 2017 Nobel Prize in Chemistry recognized the pioneering work of three scientists, Jacques Dubochet, Joachim Frank, and Richard Henderson, whose breakthrough developments in cryo-electron microscopy (cryo-EM) have helped to broaden the use of this technology within the structural biology community.

Introduction

The winners worked with Thermo Scientific™ instruments which allow scientists around the globe to routinely produce highly resolved, three-dimensional images of protein structures. It has taken decades of dedicated work to develop the technological advancements for the structural biology community – hardware, automation, software and detectors – that have made modern cryo-EM possible.

In recent years, single-particle cryo-EM has emerged as a mainstream structural biology technique which can determine the 3D structure of proteins and protein complexes at near-atomic resolution. However, single-particle cryo-EM is limited to highly purified and isolated proteins that are averaged to determine their 3D structure and lacks a connection to the cellular context. Here, cryo-electron tomography fills the gap by visualizing proteins within their functional cellular environments. This allows for observation of their relationships and interactions with other cellular components and holds great promise for cell biology, where the ultimate goal is to understand every molecule in the cell – its structure, function, location, and interactions.

Video: Cryo-EM: Advancing research for more users than ever before

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Cryo-electron microscopy (cryo-ET) combines the best possible structural preservation with nanometer-scale resolution. The method acquires 3D snapshots of the cellular interior and visualizes protein complexes within their crowded physiological environments. Such high-resolution 3D images of the interior of cells provide new insights into cellular function and shed light on the arrangement and structure of native protein complexes. One key aspect is that we not only have structural information, but we can link this information to the spatial arrangement within the cell. The technique can bridge the gap between light microscopy and atomic-resolution techniques like single-particle cryo-electron microscopy.

Cryo-electron tomography

Structure size

Cells Viruses Proteins Atoms

Light microscopy

Conventional SEM / TEM

Cryo-light microscopy

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NMR

X-ray crystallography (XRD)

Super res

1 mm 100 µm 100 nm 1 nm 1 Å10 µm 10 nm1 µm

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Video: How are cells prepared for cryo-ET? Visit the virtual cryo-EM lab

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In scientific literature

Cryo-tomography articles by application, based on published peer-reviewed

articles using Thermo Scientific instruments.

Neuroscience12%

Virology10%

Image processing/

Methods27% Microbiology

16%

Cell biology34%

Soft matter1%

Cryo-ET has provided first insights into the cellular mechanisms underlying neurodegenerative diseases such as Huntington’s, frontotemporal dementia (FTD), and amyotrophic lateral sclerosis (ALS).

Introduction

Top 3 reasons cryo-ET is used to image cells1. Maintains both molecular and

structural integrity through the vitrification process

2. Enables the study of proteins at work, thus revealing their functional interactions

3. Provides label-free, fixation-free, 3D nanometer-resolution imaging of cells’ inner workings

Since 2015, 32% of cryo-ET articles have appeared in Cell, Science, Nature, or PNAS, and 90% of these articles used Thermo Scientific instruments.

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Cryo-electron tomography benefits cellular ultrastructure imaging because it can obtain nanometer-scale information about macromolecular complexes within frozen samples. Cryo-ET was selected by Nature Methods as a “Method to Watch” in 2016 and 2019. In 2015 single-particle cryo-EM won “Method of the Year.” Since then, methods continue to improve for focused ion beam milling of cell and tissue samples for cryo-ET and data/imaging processing by subtomogram averaging continues to get faster.

Recommended reviewsMethod to watch

Turk, M., Baumeister, W. The promise and the challenges of cryo-electron tomography. FEBS Letters 2020. doi: 10.1002/1873-3468.13948

Wang, Z, Grange M, Wagner T, et al. The molecular basis for sarcomere organization in vertebrate skeletal muscle. doi: 10.1016/j.cell.2021.02.047

Strack, R. 2020. Structures in situ. Nature Methods 17, 21. doi: 10.1038/s41592-019-0704-4

Doerr, A. 2017. Cryo-electron tomography. Nature Methods 14: 34. doi: 10.1038/nmeth.4115

Four cryo-ET experts discuss recent advancements to encourage cell biologists to try cryo-ET, including the technology used for sample prep and correlated light-electron microscopy as well as new cryo-electron microscopy (cryo-EM) access and training centers being set up through Common Fund grants from the National Institutes of Health. Experts interviewed include E. Villa (UCSD), S. Subramanian (UBC), G. Jensen (Caltech), and W. Baumeister (MPI Martensreid).

Marx V., 2018. Calling all cell biologists to try cryo-ET. Nature Methods 15: 575–578. doi: 10.1038/s41592-018-0079-y

It is very easy to answer many of these fundamental biological questions; you just look at the thing!

–Richard Feynman

Hurtley, Science, 15 Nov 2019.

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Cell BiologyThe interior of eukaryotic cells is mysterious. This review discusses some of the key ideas, strategies, auxiliary techniques, and innovations that an aspiring structural cell biologist will consider when asking bold questions.

Ng, C. T. & Gan, L. 2020. Investigating eukaryotic cells with cryo-ET. MBoC 31, 87–100. doi: 10.1091/mbc.E18-05-0329

Imaging and methods In this special issue, Schur reviews the current status of methodology as well as overviews in vitro and in situ applications of cryo-ET and subtomogram averaging. Nievergelt et al. provides a more in depth look at cryo-EM technology and correlative microscopy workflows to provide localization and complement dynamic information.

Schur, F. K. 2019. Toward high-resolution in situ structural biology with cryo-electron tomography and subtomogram averaging. Current Opinion in Structural Biology, 58, 1-9. doi: 10.1016/j.sbi.2019.03.018

Nievergelt, A. P.; Viar, G. A. & Pigino, G. 2019. Towards a mechanistic understanding of cellular processes by cryoEM. Current Opinion in Structural Biology, 58, 149-158. doi: 10.1016/j.sbi.2019.06.008

Neuroscience Iadanza, M. G.; Jackson, M. P.; Hewitt, E. W.; Ranson, N. A. & Radford, S. E. 2018. A new era for understanding amyloid structures and disease. Nature Reviews Molecular Cell Biology, 19, 755-773. doi: 10.1038/s41580-018-0060-8

Microbiology Oikonomou C.M., Chang Y., Jensen G.J. 2016. Cryo-Electron Tomography: can it reveal the molecular sociology of cells in atomic detail? Trends in Cell Biology 26(11): 825-837. doi: 10.1016/j.tcb.2016.08.006

Virology Authors discuss recent viral assembly analyses by cryo-EM and cryo-ET showing how natural assembly mechanisms are used to encapsulate heterologous cargos including chemicals, enzymes, and/or nucleic acids for a variety of nanotechnological applications.

Luque, D., Castón, J. R. 2020. Cryo-electron microscopy for the study of virus assembly. Nature Chemical Biology, 16, 231-239. doi: 10.1038/s41589-020-0477-1

SARS-CoV-2 The molecular architecture of the authentic SARS-CoV-2 virus is unveiled by cryo-electron tomography and subtomogram averaging. H. Yao et al. Molecular Architecture of the SARS-CoV-2 Virus. Cell 183, 730 (2020) doi: 10.1016/j.cell.2020.09.018

Cryo-tomography solves the puzzle how new positive stranded (+)RNA genomes inside viral replication compartments can enter the cytoplasm: Wolff G, Limpens R, Zevenhoven-Dobbe J, et al. A molecular pore spans the double membrane of the coronavirus replication organelle Science 369, 1395 (2020) doi: 10.1126/science.abd3629

…the future of structural biology is all cryo-tomography all the time.

–Grant Jensen

Ask bold questionsReview articles to feed the curious mind

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Cell biology

Cell biologists now have access to the inner workings of cells through 3D renderings and at unprecedented nanoscale resolution using cryo-electron tomography. Cryo-ET avoids the alterations caused by conventional chemical fixation and dehydration resulting in the imaging of cellular morphology and proteins under fully hydrated, near-native conditions. Results from this technique are having profound effects on our understanding of cell biology.

Recommended review: Chakraborty, S., Jasnin, M., & Baumeister, W. (2020). Three-dimensional organization of the cytoskeleton: a cryo-electron tomography perspective. Protein Science, online 26 Mar 2020. doi: 10.1002/pro.3858

A detailed view of protein quality control in the cellCryo-electron tomography provides a close-up view of the endoplasmic reticulum-associated degradation machinery at distinct microsites near the ER, giving rise to new questions: How do these sites form? Are these sites examples of liquid-liquid phase separation? Do other sites within the cell exhibit this type of compartmentalization? How conserved are these sites across species?

Albert, S., Wietrzynski, W., Lee, C.-W., Schaffer, M., Beck, F., Schuller, J. M., Salomé, P. A., Plitzko, J. M., Baumeister, W., & Engel, B. D. (2020). Direct visualization of degradation microcompartments at the ER membrane. Proceedings of the National Academy of Sciences, 117(2), 1069 LP – 1080. doi: 10.1073/pnas.1905641117

3D rendering of a degradation microcompartment within a cell. ER membrane (white), proteasomes (red), cytosolic ribosomes (light blue), membrane-bound ribosomes (dark blue), cdc48 (yellow). Data courtesy of Dr. Benjamin Engel, formerly with Max Plank Institute for Biochemistry, currently at Helmholtz Zentrum München. Data visualization with Thermo Scientific Amira™ Software.

In situ cryo-electron tomogram of the native Chlamydomonas Golgi. Image courtesy of Ben Engel from Bykov et al. Elife, 6, E32493, 2017.

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See inside the cell to discover your answer

Cryo-ET provides the first view of a cell’s nucleus in its natural, undisturbed environmentCryo-electron tomography reveals the molecular organization of various components of the HeLa cell in their natural environment. This technique shows that protein filaments make the nucleus the stiffest organelle around.

Source: Sarah Everts, C&EN (29 Feb 2016 Vol. 94 Issue 9)

Mahamid, J., Pfeffer, S., Schaffer, M., Villa, E., Danev, R., Kuhn Cuellar, L., Förster, F., Hyman, A. A., Plitzko, J. M., & Baumeister, W. (2016). Visualizing the molecular sociology at the HeLa cell nuclear periphery. Science, 351(6276), 969 LP – 972. doi: 10.1126/science.aad8857

Discovery of new endoplasmic reticulum subcompartmentCryo-ET has sparked new interest in the endoplasmic reticulum (ER) via the discovery of ER subcompartments known as ribosome-associated vesicles (RSVs). Scientists now seek to answer how RSVs form and what role they play in cells.

Carter, S. D., Hampton, C. M., Langlois, R., Melero, R., Farino, Z. J., Calderon, M. J., Li, W., Wallace, C. T., Tran, N. H., Grassucci, R. A., Siegmund, S. E., Pemberton, J., Morgenstern, T. J., Eisenman, L., Aguilar, J. I., Greenberg, N. L., Levy, E. S., Yi, E., Mitchell, W. G., … Freyberg, Z. (2020). Ribosome-associated vesicles: A dynamic subcompartment of the endoplasmic reticulum in secretory cells. Science Advances, 6(14), eaay9572. doi: 10.1126/sciadv.aay9572

Data courtesy of Dr. J. Mahamid, formerly MPI Biochemistry, now EMBL. 3D rendering of a ribosome-associated vesicle (RAV): RAV membrane (green), ribosome 40S subunit (yellow), ribosome 60S subunit (blue)

Image © 2020 CC BY 4.0

Video: 3D nanometer resolution of cell inner workings

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Neuroscience

How do the building blocks of a synapse work together? In order to understand synaptic mechanisms and their components it is necessary to visualize them. Understanding subcellular mechanisms and components also is essential to understanding larger life processes.

Recommended review: Iadanza, M. G.; Jackson, M. P.; Hewitt, E. W.; Ranson, N. A. & Radford, S. E. 2018 A new era for understanding amyloid structures and disease. Nature Reviews Molecular Cell Biology, 19, 755-773. doi: 10.1038/s41580-018-0060-8

In situ analysis of ultrastructural organization underlying distinct synaptic functions The image on the right shows a 3D view of an excitatory synapse between cultured hippocampal neurons revealed by cryo-ET. All essential structural synaptic elements are rendered to facilitate visualization. Post-synaptic structures like PSD filaments and glutamate receptors are clearly depicted as well as pre-synaptic structures such as vesicles or adhesion proteins. Cryo-electron tomography gives a unique insight into the structure of synapses and will lead to a better understanding of the interplay of its components.

Tao C.-L., Liu Y.-T., Sun R., Zhang B., Qi L., Shivakoti S., Tian C.-L., Zhang P., Lau P.M., Zhou Z.H., et al. 2018. Differentiation and characterization of excitatory and inhibitory synapses by cryoelectron tomography and correlative microscopy. JNeurosci, 1548-17. doi: 10.1523/JNEUROSCI.1548-17.2017

Liu, Y.-T., Tao, C.-L., Zhang, X., et al. 2020. Mesophasic organization of GABAA receptors in hippocampal inhibitory synapse. bioRxiv, 2020.01.06.895425; doi: 10.1101/2020.01.06.895425

Synapse ultrastructureA combination of crystallography, cryo-EM, and cryo-ET was used to validate the zipper model of cluster protocadherin assembly between membranes.

Brasch, J.; Goodman, K. M.; Noble, A. J.; Rapp, M.; Mannepalli, S.; Bahna, F.; Dandey, V. P.; Bepler, T.; Berger, B.; Maniatis, T.; Potter, C. S.; Carragher, B.; Honig, B. & Shapiro, L. 2019. Visualization of clustered protocadherin neuronal self-recognition complexes. Nature, 569, 280-283. doi: 10.1038/s41586-019-1089-3

Synapse transmissionCorrelative light and electron microscopy (CLEM) workflow summarized and was used to find transfected neurite varicosities for cryo-ET.

Li, X., Radhakrishnan, A., Grushin, K., Kasula, R., Chaudhuri, A., Gomathinayagam, S., Krishnakumar, S. S., Liu, J. & Rothman, J. E. 2019. Symmetrical organization of proteins under docked synaptic vesicles. FEBS Letters, Wiley, 2019, 593, 144-153. doi:10.1002/1873-3468.13316

Video: Anthony Fitzpatrick: Revealing Alzheimer’s. Courtesy of Columbia University’s Zuckerman Institute

Image courtesy of G. Bi, USTC and H. Zhou, UCLA. Visualization by Thermo Scientific Amira™ Software.

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Protein aggregates and neurodegeneration

Image © 2018 Elsevier Inc.

Protein aggregates caught stallingProtein aggregates involved in motor neuron disease (or ALS) have been captured stalling the molecular machines needed for normal protein degradation.

Protein aggregates are a hallmark of neurodegeneration. High-resolution snapshots of the structure of one such aggregate offer an unprecedented view of how these proteins disrupt crucial cellular functions.

Details of contrasting mechanisms of Poly(GA) and Poly(Q) aggregates are detailed in the publications of Guo et al. (Cell, 2018) and Bauerlein (Cell, 2017), respectively, and highlighted in the next section.

Image © 2018 Elsevier Inc. Image © 2017 Elsevier Inc.

Are ALS Dipeptide Repeat Ribbons Entangling Proteasomes?In neurons, ALS/FTD poly-Gly-Ala peptides aggregate into a dense network of twisted ribbons.The ribbons sequester a large fraction of the cells’ proteasomes. Many trapped proteasomes are frozen in a catalytic transition state.

Source: Daisuke Ito, ALZ Forum, 02 Feb 2018

Guo Q., Lehmer, C., Martínez-Sánchez A., Rudack T., Beck F., Hartmann, H., Pérez-Berlanga M., Frottin F., Hipp M.S., Hartl F.U., et al. 2018. In Situ Structure of Neuronal C9orf72 Poly-GA Aggregates Reveals Proteasome Recruitment. Cell, 172, 696-705.e12. doi: 10.1016/j.cell.2017.12.030

Weeds in the brainA common feature of neurodegenerative diseases such as Alzheimer’s, Parkinson’s, and Huntington’s is the accumulation of toxic protein deposits in the nerve cells of patients. Once these aggregates appear, they begin to proliferate like weeds. If and how these deposits damage nerve cells and lead to their demise remains largely unexplained. A detailed insight into the three-dimensional structure of the protein aggregates should help researchers to solve this puzzle.

Source: MPI of Biochemistry, 07 Sept 2017

Bäuerlein F.J.B., Saha I., Mishra A., Kalemanov M., Martínez-Sánchez A., Klein R., Dudanova I., Hipp M.S., Hartl F.U., Baumeister W., et al. 2017. In Situ Architecture and Cellular Interactions of PolyQ Inclusions. Cell, 171, 179-187.e10. doi: 10.1016/j.cell.2017.08.009

Neuroscience

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Understanding Parkinson’s disease

LRRK2 structure solved in situ The top contributor to familial Parkinson’s disease—the second most common neurodegenerative disease—is mutations in leucine-rich repeat kinase 2 (LRRK2), whose large and difficult structure has finally been solved, paving the way for targeted therapies. Lead author Dr. Watanbe says she is hopeful that “with this structure in hand, pharmaceutical companies can design a drug that can inhibit LRRK2 activity,” which could provide a widely applicable cure for Parkinson’s disease. Image by Reika Watanabe and Guillaume Castillon. Ref: Biophysical Society 2019

Watanabe, R.; Buschauer, R.; Böhning, J.; Audagnotto, M.; Lasker, K.; Lu, T. W.; Boassa, D.; Taylor, S. & Villa, E. The in situ structure of Parkinson’s disease-linked LRRK2. bioRxiv, doi: 10.1101/837203

Deniston, C. K.; Salogiannis, J.; Mathea, S.; Snead, D. M.; Lahiri, I.; Donosa, O.; Watanabe, R.; et al. 2020 Parkinson’s Disease-linked LRRK2 structure and model for microtubule interaction. bioRxiv, doi: 10.1101/2020.01.06.895367

Neuroscience

Lewy bodies different than expected In a breakthrough for Parkinson’s disease research, University of Basel researchers have discovered unexpected structures within the postmortem human brain tissue. Using correlative light-electron microscopy and tomography, researchers showed that Lewy bodies and neurites are stuffed full of membranes, not protein filaments. Image by Henning Stahlberg.

Shahmoradian, S. H., Lewis, A. J., Genoud, C. et al. 2019. Lewy pathology in Parkinson’s disease consists of crowded organelles and lipid membranes. Nature Neuroscience, 22, 1099-1109. doi: 10.1038/s41593-019-0423-2

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Microbiology

Bacteria and archaea can no longer be viewed as mainly undifferentiated sacs of jumbled enzymes. Cryo-ET has led to a broadened view of the intricate processes within prokaryotic cells. Finer details reveal organized assemblies of macromolecular machines that are optimized to travel through and interact with complex and dynamic environments. These fascinating observations raise a number of unanswered questions for uncharted areas of discovery.

Recommended review: Wang, W., Briegel, A. 2020. Diversity of bacterial chemosensory arrays. Trends in Microbiology 28, 68-80. doi: 10.1016/j.tim.2019.08.002

Chaikeeratisak V., Nguyen K., Khanna K., Brilot A.F., Erb M.L., Coker J.K.C., Vavilina A., Newton G.L., Buschauer R., Pogliano K., Villa E., Agard D.A., Pogliano, J. 2017 Assembly of a nucleus-like structure during viral replication in bacteria. Science 355: 194-197. doi: 10.1126/science.aal2130

Chaikeeratisak V., Khanna K., Nguyen KT., Sugie J., Egan ME., Erb ML., Vavilina A., Nonejuie P., Nieweglowska E., Pogliano K., Agard DA., Villa E., Pogliano J. 2019. Viral capsid trafficking along treadmilling tubulin filaments in bacteria. Cell, 177: 1771-1780. doi: 10.1016/j.cell.2019.05.032

Bacteria…not so simpleFor the first time, biologists have documented how very large viruses reprogram the cellular machinery of bacteria during infection to more closely resemble an animal or human cell. A previous study found that several Pseudomonas phages assemble a bipolar nucleus-like spindle composed of a phage tubulin-like protein (PhuZ) that encloses phage DNA (image above). In a following study, using time-lapse light microscopy and cryo-electron tomography, Chaikeeratisak et al. observed that capsids traffic along a viral encoded tubulin filament and this

treadmilling of the filament provides the mechanism of capsid movement through the cell. The spindle rotates the phage nucleus, which the authors hypothesize distributes capsids for efficient DNA packaging. The discovery demonstrates that bacteria have more in common with sophisticated human cells than previously believed and are part of a renewed interest due to their potential of using them for phage therapy.

Microbiology

Cryo-electron tomography shows how the bacterial cell is reorganized to resemble a more complicated plant or animal cell with a blue nucleus like compartment and ribosomes, the smaller yellow structures.

The Atlas of Bacterial & Archaeal Cell Structure -an open access digital textbook offers a tour of microbial cells guided by cutting-edge 3D electron microscopy. Courtesy Catherine M. Oikonomou & Grant J. Jensen California Institute of Technology

Video: Viral Capsid Trafficking along Treadmilling Tubulin Filaments in Bacteria. Courtesy of the Villa Lab.

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Microbial pathogenesis

Bacteria assemble specialized type IV secretion systems (T4SSs) to inject molecular cargo into target cells. Using cryo-ET, Chang and Shaffer et al. report the first structure of a cancer-associated T4SS in vivo and describe unique membranous appendages that are produced when H. pylori encounters gastric epithelial cells.

Chang, Y.-W., Shaffer, C. L., Rettberg, L. A., Ghosal, D., & Jensen, G. J. (2018). In Vivo Structures of the Helicobacter pylori cag Type IV Secretion System. Cell Reports, 23(3), 673–681. doi.org: 10.1016/j.celrep.2018.03.085

Bdellovibrio bacteriovorus. Image © 2016 Elsevier Inc. Oikonomou C.M., 2016 Trends in Cell Biology 26(11): 825-837.

Video: Prof. Briegel and new discoveries in microbes with cryo-ETComplete structure of the Salmonella type III secretion machinery explains how bacteria deliver proteins into eukaryotic cells. Image © 2017 Elsevier Inc.

Bacterial delivery of proteinsSalmonella and many other bacterial pathogens use a nano syringe-like device to deliver toxic proteins into target human cells. “The device is like a stinger and injects ready-made bacterial proteins into mammalian cells to commandeer them for the benefit of the pathogen,” said Jorge Galan, the Lucille P. Markey Professor of Microbial Pathogenesis and co-senior author of the paper. Knowledge of the structure could help researchers devise new anti-infective strategies against a variety of bacterial pathogens such as Salmonella, Pseudomonas, Escherichia coli, Yersinia pestis, and Chlamydia. Source: Yale University Reference: Hu et al., Cell, 168, 1065, 2017.

Microbiology

Hu B., Lara-Tejero M., Kong Q., Galán J.E., and Liu J. 2017. In Situ Molecular Architecture of the Salmonella Type III Secretion Machine. Cell, 168, 1065-1074.e10. doi: 10.1016/j.cell.2017.02.022

VirologyViruses are infectious agents that come in a wide variety of shapes and sizes, with most having a diameter between ten and a few hundred nanometers. Cryo-EM has been used to study virus morphology for over 20 years, resolving the structures of viruses such as Ebola, HIV, and coronaviruses. The near-atomic resolution information provided by single-particle analysis cryo-EM is critical for a better understanding of the molecular mechanisms behind antibody-antigen interactions. Cryo-ET provides structural information with broader cellular context, such as viral assembly within bacteria. Additionally, cryo-ET provides 3D analysis of asymmetric or pleomorphic viral particles such as HIV, herpesvirus, and influenza.

Structural insights into HIV-1 capsid flexibilityViral capsids are protein structures that enclose the genetic material of viruses. Previous structural studies of the HIV-1 capsid have relied on recombinant, cross-linked, or mutant capsid proteins. Mattei et al. reported sub-nanometer resolution cryo–ET structures of the HIV-1 capsid from intact virions. These structures confirm the hollow cone shape of the capsid and allow for the specific placement of each individual capsid hexamer and pentamer within the lattice structure. The structures also reveal the flexible nature of the capsid, which likely helps it accommodate interactions with host cell factors.

Source: Mattei, et al. Science, 254, 1434, 2016

Recommended review: Luque, D., Castón, J. R. 2020. Cryo-electron microscopy for the study of virus assembly. Nature Chemical Biology, 16, 231-239. doi: 10.1038/s41589-020-0477-1 doi: 0.1038/s41589-020-0477-1

Insights into HIV in unprecedented resolution: The green and red CA protein structures form the conical protective envelope of the virus genome. Image by Simone Mattei, EMBL.

Ebola Sugita, Y.; Matsunami, H.; Kawaoka, Y.; Noda, T. & Wolf, M. Cryo-EM structure of the Ebola virus nucleoprotein–RNA complex at 3.6 Å. 2018 Nature, 563, 137-140. doi: 10.1038/s41586-018-0630-0

Wan, W. et al. Structure and assembly of the Ebola virus nucleocapsid. Nature, 551, 394–397 (2017). doi: 10.1038/nature24490

Influenza Gallagher J.R., Torian U., McCraw D.M., and Harris A.K. 2017 “Structural studies of influenza virus RNPs by electron microscopy indicate molecular contortions within NP supra-structures” J. Structural Biology, 197, 294–307. doi: 10.1016/j.jsb.2016.12.007

Arranz, R.; Coloma, R.; Chichon, F. J.; Conesa, J. J.; Carrascosa, J. L.; Valpuesta, J. M.; Ortin, J. & Martin-Benito, J. 2012. The Structure of Native Influenza Virion Ribonucleoproteins. Science, 338, 1634-1637 doi: 10.1126/science.1228172

Left: A 3D rendering of the cryo-EM reconstruction. The RNA and NP are colored in red and grey, respectively. A single NP molecule is highlighted in blue. Right: An atomic model of a single NP molecule with the RNA in the complex. The model is superposed with the cryo-EM map in a polygon mesh representation. Courtesy of Yukihiko Sugita, OIST.

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Molecular details of the intact virusThe molecular architecture of the authentic SARS-CoV-2 virus is unveiled by cryo-electron tomography and subtomogram averaging. Courtesy of Sai Li.

Yao, C. Molecular Architecture of the SARS-CoV-2 Virus, Cell, 183: 730- 739, 2020.

Global researchers have deposited over 1000 structures of SARS-CoV-2 biomolecules to the Worldwide Protein Data Bank (PDB) solved by cryo-EM or X-ray crystallography. Cryo-electron tomography (cryo-ET) and subtomogram averaging were used in studies to characterize the pleomorphic structure and the replication mechanism of SARS-CoV-2.

Zimmer, Carl. “The Coronavirus Unveiled,” New York Times 9 Oct 2020.© Tsinghua University School of Life Sciences

Cryo-Tomography against SARS-CoV-2

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Cryo-Tomography against SARS-CoV-2

Viral replication puzzle sovledCoronaviruses transform host cell membranes into peculiar double-membrane vesicles that have long been thought to accommodate viral genome replication. Cryo-tomography solves the puzzle how new positive stranded (+)RNA genomes inside viral replication compartments can enter the cytoplasm. A molecular pore (right) acts as a gateway to

the cytosol. This recently discovered coronavirus-specific structure likely plays a key role in coronavirus replication and thus constitutes a potential drug target.

Wolff, G. et al. A molecular pore spans the double membrane of the coronavirus replication organelle. Science 369, 1395, 2020.

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Virus induced changes in cellular membrane Characterizing the replication cycle inside the host cell is key to understanding virulence and discovering targets restricting virus replication. SARS-CoV-2 virion budding and assembly at the ERGIC membrane. A 3D volume rendering is shown with cellular and viral membranes in green and magenta respectively. Viral spike proteins are shown in yellow, and viral ribonucleoproteins in cyan.

Klein, S. et al. Nat Commun 11, 5885, 2002.

Cryo-Tomography against SARS-CoV-2

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Preparing for the next major virusHerpes simplex virus 1 glycoprotein BBen-Mordehai, T. Z.; et al. 2016. Two distinct trimeric conformations of natively membrane-anchored full-length herpes simplex virus 1 glycoprotein B. PNAS, 113, 4176-418. doi: 10.1073/pnas.1523234113

Meyer, N. L.; Hu, G.; Davulcu, O.; Xie, Q.; Noble, A. J.; Yoshioka, C.; Gingerich, D. S.; Trzynka, A.; David, L.; Stagg, S. M. & Chapman, M. S. 2019. Structure of the gene therapy vector, adeno-associated virus with its cell receptor, eLife, 8. doi: 10.7554/eLife.44707

Viral assembly in selected regions of virus-infected cellsThe Elizabeth Wright lab at the University of Wisconsin—Madison describes its protocol for correlated cryo-fluorescence light microscopy (cryo-fLM), cryo-EM, and cryo-ET, together known as cryo-CLEM, of virus-infected or transfected mammalian cells.

Hampton, C. M.; Strauss, J. D.; Ke, Z.; Dillard, R. S.; Hammonds, J. E.; Alonas, E.; Desai, T. M.; Marin, M.; Storms, R. E.; Leon, F. et al. 2017 Correlated fluorescence microscopy and cryo-electron tomography of virus-infected or transfected mammalian cells. Nature protocols, 12, 150. doi: 10.1038/nprot.2016.168

Cryo-ET of MeV-infected HeLa cell. Site of measles virus (MeV) assembly is indicated by the dashed white box where 3D cryo-ET tomogram was collected (inset). Asterisks indicate released MeV particles and red dashed lines indicate the cell membrane. Inset is a false-colored 3D segmented view of the cryo-ET dataset. Scale bar is 100 nm (inset) and 500 nm.

Measles and Rubella Ke, Z. et al. 2018. Promotion of virus assembly and organization by the measles virus matrix protein. Nat. Commun. 9, 1736. doi: 10.1038/s41467-018-04058-2

Mangala Prasad, V., Klose, T. & Rossmann, M. G.2017. Assembly, maturation and three-dimensional helical structure of the teratogenic rubella virus. PLoS Pathog. 13, e1006377. doi: 10.1371/journal.ppat.1006377

Video: Thermo Fisher’s COVID-19 Global Response

Image © 2018 CC BY 4.0

Find out more at thermofisher.com/emvirusanalysis

The Peijun Wang lab at the University of Pittsburg describes the AutoCLEM workflow that significantly accelerates the correlation efficiency between live cell fluorescence imaging and cryo-EM/ET structural analysis, as demonstrated by visualizing human immunodeficiency virus type 1 (HIV-1) interacting with host cells.

Fu, X.; Ning, J.; Zhong, Z.; Ambrose, Z.; Watkins, S. C. & Zhang, P. 2019 AutoCLEM: An Automated Workflow for Correlative Live-Cell Fluorescence Microscopy and Cryo-Electron Tomography. Scientific Reports, 9. doi: 10.1038/s41598-019-55766-8

Introduction Cell Biology Neuroscience Microbiology Virology Workflow AppendixReviews Virology

Introduction Cell Biology Neuroscience Microbiology Virology Workflow AppendixReviews

Cryo-electron tomography workflowCryo-ET provides label-free, fixation-free, 3D nanometer-scale resolution imaging of cells’ inner workings. Next to this, it allows 3D snapshots of the cellular interior and visualizes protein complexes within their physiological environments. Using a correlative light and electron microscopy approach allows for targeting of tagged proteins by fluorescence microscopy before subsequent higher resolution imaging with cryo-EM. Many cells are too thick for electrons, so the vitrified cells must be thinned with a cryo-focused ion beam microscope (cryo-FIB) prior to imaging in a transmission electron microscope.

Sample preparation by vitrificationCells prepared by routine culture methods are grown on carbon-coated gold electron microscopy (EM) grids and plunge-frozen in a cryogenically cooled fluid, typically liquid ethane. The water in the sample freezes so rapidly that it does not crystallize, thus avoiding the molecular-scale disruption (by formed ice crystals) that would occur with a normal slow freezing process.

Electron beam

Ion beam

-196ºC

Thinning

Fluorescence imaging

Cryo-electron tomography

Structural Analysis

Cryo-focused ion beam milling

Cryo-correlative Microscopy

Vitrification

Localization by fluorescenceUsing cryo-correlative microscopy the sample is transferred to a cryo-fluorescence light microscope, where structures of interest are identified. A dedicated cryo-LM stage keeps the sample in its vitrified state during cryo-fluorescence imaging.

Thinning by millingA dedicated cryo-FIB prepares a thin, uniform lamella at the vitreous temperature (approximately -170°C).

Imaging by TEMDuring cryo-ET, the sample is tilted in known increments about an axis. The individual projection images from the tomographic tilt series are then combined computationally in a procedure known as back-projection, which creates the 3D tomographic volume.

Reconstruction & VisualizationThe 3D tomogram featuring cellular structures can be segmented and colored in a variety of ways to enhance its display and presentation. From the tomogram small subsets of data containing the structures of interest can be computationally extracted and subjected to image processing methods.

Find out more at thermofisher.com/cryo-tomography

Workflow

Introduction Cell Biology Neuroscience Microbiology Virology Workflow AppendixReviews

How do I get started with cryo-electron tomography?

1. EM-learning.com is a free learning platform that features over 70 hours of theoretical lectures and practical videos. It was created by Thermo Fisher Scientific in collaboration with Professor Grant Jensen at Caltech and is intended for participants of all levels. Upon completion, you will have a fundamental base of knowledge in the field of cryo-electron microscopy. Learn more at www.em-learning.com

Workflow

What kind of samples can I image?Samples for cryo-ET imaging can be prepared from cells and tissues. Cells can be grown on gold grids which are used later as carriers in the microscope (a). Target regions can be selected and ‘lift out’ tools (b) are currently available

to automate the process on the new Thermo Fisher™ Aquilos™ 2 cryo-focused ion beam microscope (cryo-FIB). High pressure frozen tissues can also be prepared in the Aquilos 2 through the cryo-lift out technique (b).

2. Central cryo-electron microscopy core facilities are available worldwide. To find the closest facility near you, visit this online directory of cryo-EM core facilities or service labs.

3. Accelerate your cryo-electron tomography research. While this technology is transformative, implementing it can cause a lot of stress and introduce risk—especially if you are new to cryo-electron microscopy. You can rest assured that Thermo Scientific™ Accelerate and Advance for Cryo-Electron Tomography will help you every step of the way to successfully implement this workflow in your lab. We will train your users and partner with you from the warranty period and beyond, delivering intensive support with onsite and remotely support features. Learn more at thermofisher.com/cryo-tomo-service

Learn more with on-demand cryo-ET webinars

Video: Accelerate and Advance service packages for cryo-tomography

Cryo-electron tomography reveals a phase-separated protein degradation microcompartment at the ER membrane. Data courtesy of Dr. Benjamin Engel, formerly Max Plank Institute for Biochemistry, now Helmholtz Zentrum München. Data visualization with Amira Software.

New Features: Extended cryo-operation, automation for Cryo-Milling, Cryo Slice and View and Cryo-Lift Out

Cryo-electron tomography allows you to visualize and study proteins in their functional cellular environments at unprecedented resolution. This 3D imaging technique provides insights into complex supramolecular structures and assemblies that cannot be achieved by conventional purification and structural imaging methods.

The Thermo Scientific™ Aquilos™ 2 Cryo-FIB is a cryo-DualBeam™ (focused ion beam/scanning electron microscope) system dedicated to the preparation of frozen, thin lamella samples from

Find out more at thermofisher.com/aquilos2

Resolve protein structures inside cells biological specimens, now with automated workflow software. It maintains the vitrified cellular sample’s structural integrity and ensures its accessibility for tomographic imaging in a cryo-transmission electron microscope such as the newly released Thermo Scientific™ Krios™ G4 Cryo-TEM.

With the new Aquilos 2 Cryo-FIB, you can automate the fast production of multiple lamellas extracted from specific regions.

AppendixCell biologyAlbert, S., Schaffer, M., Beck, F., Mosalaganti, S., Asano, S., Thomas, H. F., Plitzko, J. M., Beck, M., Baumeister, W., & Engel, B. D. (2017). Proteasomes tether to two distinct sites at the nuclear pore complex. PNAS, 114(52), 13726 LP – 13731. doi: 10.1073/

pnas.1716305114

Albert, S., Wietrzynski, W., Lee, C.-W., Schaffer, M., Beck, F., Schuller, J. M., Salomé, P. A., Plitzko, J. M., Baumeister, W., & Engel, B. D. (2020). Direct visualization of degradation microcompartments at the ER membrane. PNAS, 117(2), 1069 LP – 1080. doi: 10.1073/pnas.1905641117

Allegretti, M., Zimmerli, C. E., Rantos, V., Wilfling, F., Ronchi, P., Fung, H. K. H., Lee, C.-W., Hagen, W., Turonova, B., Karius, K., Zhang, X., Müller, C., Schwab, Y., Mahamid, J., Pfander, B., Kosinski, J., & Beck, M. (2020). In cell architecture of the nuclear pore complex and snapshots of its turnover. BioRxiv, 2020.02.04.933820. doi: 10.1101/2020.02.04.933820

Anderson, K. L., Page, C., Swift, M. F., Suraneni, P., Janssen, M. E. W., Pollard, T. D., Li, R., Volkmann, N., & Hanein, D. (2017). Nano-scale actin-network characterization of fibroblast cells lacking functional Arp2/3 complex. J. Struct. Bio, 197(3), 312–321. doi: 10.1016/j.jsb.2016.12.010

Azubel, M., Carter, S. D., Weiszmann, J., Zhang, J., Jensen, G. J., Li, Y., & Kornberg, R. D. (2019). FGF21 trafficking in intact human cells revealed by cryo-electron tomography with gold nanoparticles. ELife, 8, e43146. doi: 10.7554/eLife.43146

Bharat, T. A. M., Hoffmann, P. C., & Kukulski, W. (2018). Correlative Microscopy of Vitreous Sections Provides Insights into BAR-Domain Organization In Situ. Structure, 26(6), 879-886.e3. doi: 10.1016/j.str.2018.03.015

Bykov, Y. S., Schaffer, M., Dodonova, S. O., Albert, S., Plitzko, J. M., Baumeister, W., Engel, B. D., & Briggs, J. A. G. (2017). The structure of the COPI coat determined within the cell. ELife, 6, e32493. doi: 10.7554/eLife.32493

Cornell, C. E., Mileant, A., Thakkar, N., Lee, K. K., & Keller, S. L. (2020). Direct Imaging of Liquid Domains in Membranes by Cryo Electron Tomography. BioRxiv, 2020.02.05.935684. doi: 10.1101/2020.02.05.935684

Craig, E. W., Mueller, D. M., Bigge, B. M., Schaffer, M., Engel, B. D., & Avasthi, P. (2019). The elusive

actin cytoskeleton of a green alga expressing both conventional and divergent actins. Molecular Biology of the Cell, 30(22), 2827–2837. doi: 10.1091/mbc.E19-03-0141

Faelber, K., Dietrich, L., Noel, J. K., Wollweber, F., Pfitzner, A.-K., Mühleip, A., Sánchez, R., Kudryashev, M., Chiaruttini, N., Lilie, H., Schlegel, J., Rosenbaum, E., Hessenberger, M., Matthaeus, C., Kunz, S., von der Malsburg, A., Noé, F., Roux, A., van der Laan, M., … Daumke, O. (2019). Structure and assembly of the mitochondrial membrane remodelling GTPase Mgm1. Nature, 571(7765), 429–433. doi: 10.1038/s41586-019-1372-3

Freeman Rosenzweig, E. S., Xu, B., Kuhn Cuellar, L., Martinez-Sanchez, A., Schaffer, M., Strauss, M., Cartwright, H. N., Ronceray, P., Plitzko, J. M., Förster, F., Wingreen, N. S., Engel, B. D., Mackinder, L. C. M., & Jonikas, M. C. (2017). The Eukaryotic CO2-Concentrating Organelle Is Liquid-like and Exhibits Dynamic Reorganization. Cell, 171(1), 148-162.e19. doi: 10.1016/j.cell.2017.08.008

Klein, S., Wimmer, B. H., Winter, S. L., Kolovou, A., Laketa, V., & Chlanda, P. (2020). Membrane architecture of pulmonary lamellar bodies revealed by post-correlation on-lamella cryo-CLEM. BioRxiv, 2020.02.27.966739. doi: 10.1101/2020.02.27.966739

Kovtun, O., Leneva, N., Bykov, Y. S., Ariotti, N., Teasdale, R. D., Schaffer, M., Engel, B. D., Owen, D. J., Briggs, J. A. G., & Collins, B. M. (2018). Structure of the membrane-assembled retromer coat determined by cryo-electron tomography. Nature, 561(7724), 561–564. doi: 10.1038/s41586-018-0526-z

Le Guennec, M., Klena, N., Gambarotto, D., Laporte, M. H., Tassin, A.-M., van den Hoek, H., Erdmann, P. S., Schaffer, M., Kovacik, L., Borgers, S., Goldie, K. N., Stahlberg, H., Bornens, M., Azimzadeh, J., Engel, B. D., Hamel, V., & Guichard, P. (2020). A helical inner scaffold provides a structural basis for centriole cohesion. Science Advances, 6(7), eaaz4137. doi: 10.1126/sciadv.aaz4137

Lin, J., & Nicastro, D. (2018). Asymmetric distribution and spatial switching of dynein activity generates ciliary motility. Science, 360(6387), eaar1968. doi: 10.1126/science.aar1968

McArthur, K., Whitehead, L. W., Heddleston, J. M., Li, L., Padman, B. S., Oorschot, V., Geoghegan, N. D., Chappaz, S., Davidson, S., San Chin, H., Lane, R. M., Dramicanin, M., Saunders, T. L., Sugiana, C., Lessene, R., Osellame, L. D., Chew, T.-L., Dewson, G., Lazarou, M., … Kile, B. T.

(2018). BAK/BAX macropores facilitate mitochondrial herniation and mtDNA efflux during apoptosis. Science, 359(6378), eaao6047. doi: 10.1126/science.aao6047

O’Reilly, F. J., Xue, L., Graziadei, A., Sinn, L., Lenz, S., Tegunov, D., Blötz, C., Hagen, W. J. H., Cramer, P., Stülke, J., Mahamid, J., & Rappsilber, J. (2020). In-cell architecture of an actively transcribing-translating expressome. BioRxiv, 2020.02.28.970111. doi: 10.1101/2020.02.28.970111

Paul, D. M., Mantell, J., Borucu, U., Coombs, J., Surridge, K. J., Squire, J., Verkade, P., & Dodding, M. P. (2019). In situ cryo-electron tomography reveals filamentous actin within the microtubule lumen. BioRxiv, 844043. doi: 10.1101/844043

Siegmund, S. E., Grassucci, R., Carter, S. D., Barca, E., Farino, Z. J., Juanola-Falgarona, M., Zhang, P., Tanji, K., Hirano, M., Schon, E. A., Frank, J., & Freyberg, Z. (2018). Three-Dimensional Analysis of Mitochondrial Crista Ultrastructure in a Patient with Leigh Syndrome by In Situ Cryoelectron Tomography. IScience, 6, 83–91. doi: 10.1016/j.isci.2018.07.014

Swulius, M. T., Nguyen, L. T., Ladinsky, M. S., Ortega, D. R., Aich, S., Mishra, M., & Jensen, G. J. (2018). Structure of the fission yeast actomyosin ring during constriction. PNAS, 115(7), E1455 LP-E1464. doi: 10.1073/pnas.1711218115

Turgay, Y., Eibauer, M., Goldman, A. E., Shimi, T., Khayat, M., Ben-Harush, K., Dubrovsky-Gaupp, A., Sapra, K. T., Goldman, R. D., & Medalia, O. (2017). The molecular architecture of lamins in somatic cells. Nature, 543(7644), 261–264. doi: 10.1038/nature21382

MicrobiologyBöck, D., Medeiros, J. M., Tsao, H.-F., Penz, T., Weiss, G. L., Aistleitner, K., Horn, M., & Pilhofer, M. (2017). In situ architecture, function, and evolution of a contractile injection system. Science, 357(6352), 713 LP – 717. doi: 10.1126/science.aan7904

Chang, Y.-W., Shaffer, C. L., Rettberg, L. A., Ghosal, D., & Jensen, G. J. (2018). In Vivo Structures of the Helicobacter pylori cag Type IV Secretion System. Cell Reports, 23(3), 673–681. doi: 10.1016/j.celrep.2018.03.085

Chetrit, D., Hu, B., Christie, P. J., Roy, C. R., & Liu, J. (2018). A unique cytoplasmic ATPase complex defines the Legionella pneumophila type IV secretion channel. Nature Microbiology, 3(6), 678–686. doi: 10.1038/s41564-018-0165-z

Ghosal, D., Jeong, K. C., Chang, Y.-W., Gyore, J., Teng, L., Gardner, A., Vogel, J. P., & Jensen, G. J. (2019). Molecular architecture, polar targeting and biogenesis of the Legionella Dot/Icm T4SS. Nature Microbiology, 4(7), 1173–1182. doi: 10.1038/s41564-019-0427-4

The journal for the rapid publication of short reports in the molecular biosciences

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ISSN 00145793 ISSN 00145793 Volume 593 Number 2 Volume 593 Number 2

January 2019January 2019

Symmetrical Organiza on of Proteins Under Docked Synap c Vesicles

Introduction Cell Biology Neuroscience Microbiology Virology Workflow AppendixReviews Appendix

Introduction Cell Biology Neuroscience Microbiology Virology Workflow AppendixReviews

Lopez-Garrido, J., Ojkic, N., Khanna, K., Wagner, F. R., Villa, E., Endres, R. G., & Pogliano, K. (2018). Chromosome Translocation Inflates Bacillus Forespores and Impacts Cellular Morphology. Cell, 172(4), 758-770.e14. doi: 10.1016/j.cell.2018.01.027

Park, D., Lara-Tejero, M., Waxham, M. N., Li, W., Hu, B., Galán, J. E., & Liu, J. (2018). Visualization of the type III secretion mediated Salmonella–host cell interface using cryo-electron tomography. ELife, 7, e39514. doi: 10.7554/eLife.39514

Rapisarda, C., Cherrak, Y., Kooger, R., Schmidt, V., Pellarin, R., Logger, L., Cascales, E., Pilhofer, M., Durand,

E., & Fronzes, R. (2019). In situ and high-resolution cryo-EM structure of a bacterial type VI secretion system membrane complex. The EMBO Journal, 38(10), e100886. doi: 10.15252/embj.2018100886

von Kügelgen, A., Tang, H., Hardy, G. G., Kureisaite-Ciziene, D., Brun, Y. V, Stansfeld, P. J., Robinson, C. V, & Bharat, T. A. M. (2020). In Situ Structure of an Intact Lipopolysaccharide-Bound Bacterial Surface Layer. Cell, 180(2), 348-358.e15. doi: 10.1016/j.cell.2019.12.006

Wang, C., Tu, J., Liu, J., & Molineux, I. J. (2019). Structural dynamics of bacteriophage P22 infection initiation revealed by cryo-electron tomography. Nature Microbiology, 4(6), 1049–1056. doi: 10.1038/s41564-019-0403-z

ReviewsAsano S., Engel B.D., Baumeister W. 2016 In Situ Cryo-Electron Tomography: A Post-Reductionist Approach to Structural Biology. J Mol Biol. 428(2 Pt A):332-343. doi: 10.1016/j.jmb.2015.09.030

Baker L.A., Grange M., Grunewald K. 2017 Electron cryotomography captures macromolecular complexes in native Environments. Curr Opin Struct Biol. 46: 149-156. doi: 10.1016/j.sbi.2017.08.005

Beck M., Baumeister W. 2016 Cryo-Electron Tomography: can it reveal themolecular sociology of cells in atomic detail? Trends in Cell Biology 26(11): 825-837. doi: 10.1016/j.tcb.2016.08.006

Burbaum L., Schaffer M., Engel B.D., Mahamid J., Albert S., Danev R., Baumeister W., Plitzko J.M. 2017 Charting molecular landscapes using cryo-electron tomography. Microscopy Today 25(3): 26-31. doi: 10.1017/S1551929517000384

Chlanda P., Krijnse Locker J. 2017 The sleeping beauty kissed awake: new methods in electron microscopy to study cellular membranes. Biochemical Journal 474: 1041-1053. doi: 10.1042/BCJ20160990

Collado J., Fernandez-Busnadiego R. 2017 Deciphering the molecular architecture of membrane contact sites by cryoelectron tomography. BBA – Molecular Cell Research 1864(9): 1507-1512. doi: 10.1016/j.bbamcr.2017.03.009

Dubrovsky A., Sorrentino S., Harapin J., Sapra K.T., Medalia O. 2015 Developments in cryo-electron tomography for in situ structural analysis. Archives of Biochemistry and Biophysics 581: 78-85. doi: 10.1016/j.abb.2015.04.006

Irobalieva R.N., Martins B., Medalia O. 2016 Cellular structural biology as revealed by cryo-electron tomography. J Cell Science 129: 469-476. doi: 10.1242/jcs.171967

Kizilyaprak C., Daraspe J., Humbel B.M. 2014 Focused ion beam scanning electron microscopy in biology. J Microscopy 254(3): 109-114. doi: 10.1111/jmi.12127

Medeiros J.M., Bock D., Weiss G.L., Kooger R., Wepf R.A., and Pilhofer M. 2018 Robust workflow and instrumentation for cryo-focused ion beam milling of samples for electron cryotomography. Ultramicroscopy 190, 1–11. doi: 10.1016/j.ultramic.2018.04.002

Lučić V., Rigort A., Baumeister W. 2013 Cryo-electron tomography: the challenge of doing structural biology in situ. J. Cell Biol. 202, 407–419. doi: 10.1083/jcb.201304193

Oikonomou C.M., Jensen G.J. 2016 A new view into the prokaryotic cell biology from electron cryotomography. Nature Reviews Microbiology 14: 205-220. doi: 10.1038/nrmicro.2016.7

Oikonomou C.M., Jensen G.J. 2017 Cellular electron cryotomography: towards structural biology in situ. Annual Review of Biochemistry 86: 873-896. doi: 10.1146/annurev-biochem-061516-044741

Orlov I., Myasnikov A.G., Andronov L., et al. 2017 The integrative role of cryo electron microscopy in molecular and cellular structural biology. Biol Cell 109: 81-93. doi: 10.1111/boc.201600042

Rigort A., Plitzko J.M. 2015 Cryo-focused-ion-beam applications in structural biology. Archives of Biochemistry and Biophysics 581: 122-130. doi: 10.1016/j.abb.2015.02.009

Narayan K., Subramaniam S. 2015 Focused ion beams in biology. Nature Methods 12(11): 1021-1031. doi: 10.1038/nmeth.3623

Villa E., Schaffer M., Plitzko J.M., Baumeister W. 2013 Opening windows into the cell: focused- ion-beam milling for cryo-electron tomography. Curr Opin Struct Biol. 23:771-777. doi: 10.1016/j.sbi.2013.08.006

Wagner J., Schaffer M., Fernandez-Busnadiego R. 2017 Cryo-electron tomography – the cell biology that came in from the cold. FEBS Letters 591: 2520-2533. doi: 10.1002/1873-3468.12757

NeuroscienceEditorial. Vaites L.P., Harper, J.W. 2018 Protein aggregates caught stalling. Nature 555, 449-451. doi: 10.1038/d41586-018-03000-2

Gruber A., Hornburg D., Antonin M., et al. 2018 Molecular and structural architecture of

polyQ aggregates in yeast. PNAS 115, 3446-E3453. doi: 10.1073/pnas.1717978115

Jasnin, M.; Beck, F.; Ecke, M.; Fukuda, Y.; Martinez-Sanchez, A.; Baumeister, W. & Gerisch, G. 2019 The Architecture of Traveling Actin Waves Revealed by Cryo-Electron Tomography. Structure, 27, 1211-1223.e5. doi: 10.1016/j.str.2019.05.009

Basnet, N., Nedozralova, H., Crevenna, A. H., Bodakuntla, S., Schlichthaerle, T., Taschner, M., Cardone, G., Janke, C., Jungmann, R., Magiera, M. M., Biertümpfel, C., & Mizuno, N. (2018). Direct induction of microtubule branching by microtubule nucleation factor SSNA1. Nature Cell Biology, 20(10), 1172–1180. doi: 10.1038/s41556-018-0199-8

Metlagel, Z., Krey, J. F., Song, J., Swift, M. F., Tivol, W. J., Dumont, R. A., Thai, J., Chang, A., Seifikar, H., Volkmann, N., Hanein, D., Barr-Gillespie, P. G., & Auer, M. (2019). Electron cryo-tomography of vestibular hair-cell stereocilia. Journal of Structural Biology, 206(2), 149–155. doi: 10.1016/j.jsb.2019.02.006

Schaefer, T., Riera-Tur, I., Hornburg, D., Mishra, A., Fernández-Mosquera, L., Raimundo, N., Mann, M., Baumeister, W., Klein, R., Meissner, F., Fernández-Busnadiego, R., & Dudanova, I. (2019). Amyloid-like aggregates cause lysosomal defects in neurons via gain-of-function toxicity. BioRxiv, 2019.12.16.877431. doi: 10.1101/2019.12.16.877431

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Appendix

Appendix

Introduction Cell Biology Neuroscience Microbiology Virology Workflow AppendixReviews

VirusBharat T.A.M., Kureisaite-Ciziene D., Hardy G.G., Yu E.W., Devant J.M., Hagen W.J.H., Brun Y.V., Briggs J.A.G., and Lowe J. 2017. Structure of the hexagonal surface layer on Caulobacter crescentus cells. Nature Microbiology 2, 17059. doi: 10.1038/nmicrobiol.2017.59

Murine lukemia virus. Qu, K. et al. 2018 Structure and architecture of immature and mature murine leukemia virus capsids. Proc. Natl. Acad. Sci. 115, E11751–E11760. doi: 10.1073/pnas.1811580115

Archael virus structuresHochstein, R. et al. 2018 Structural studies of Acidianus tailed spindle virus reveal a structural paradigm used in the assembly of spindle-shaped viruses. Proc. Natl. Acad. Sci. USA 115, 2120–2125. doi: 10.1073/pnas.1719180115

Hartman, R., Munson-McGee, J., Young, M. J. & Lawrence, C. M. 2019 Survey of high-resolution archaeal virus structures. Curr. Opin. Virol. 36, 74–83. doi: 10.1016/j.coviro.2019.05.008

WorkflowSchaffer M., Engel B.D., Laugks T., Mahamid J., Plitzko J.M., Baumeister W. 2015. Cryo-focused Ion Beam Sample Preparation for Imaging Vitreous Cells by Cryo-electron Tomography. Bio Protoc 5(17) pii: e1575. doi: 10.21769/BioProtoc.1575

Schaffer M., Mahamid J., Engel B.D., Laugks T., Beumeister W., Plitzko W. 2016. Optimized cryo-focused ion beam sample preparation aimed at in situ structural studies of membrane proteins. J Struct Biol. 197(2): 73-82. doi: 10.1016/j.jsb.2016.07.010

Arnold J., Mahamid J., Lucic V., de Marco A., Fernandez J.J., Laugks T., Mayer T., Hyman A., Baumeister W., Plizko J. 2016. Site-Specific Cryo-Focused Ion Beam Sample Preparation Guided by 3D Correlative Microscopy. Biophys J 1 110(4): 860-869. doi: 10.1016/j.bpj.2015.10.053

Castaño-Díez, D., & Zanetti, G. (2019). In situ structure determination by subtomogram averaging. Current Opinion in Structural Biology, 58, 68–75. doi: 10.1016/j.sbi.2019.05.011

Chen, M., Bell, J. M., Shi, X., Sun, S. Y., Wang, Z., & Ludtke, S. J. (2019). A complete data processing workflow for cryo-ET and subtomogram averaging. Nature Methods, 16(11), 1161–1168. doi: 10.1038/s41592-019-0591-8

Chreifi, G., Chen, S., Metskas, L. A., Kaplan, M., & Jensen, G. J. (2019). Rapid tilt-series acquisition for electron cryotomography. J. Struct. Bio, 205(2), 163–169. doi: 10.1016/j.jsb.2018.12.008

Danev, R., Iijima, H., Matsuzaki, M., & Motoki, S. (2019). Fast and accurate defocus for improved tunability of cryo-EM experiments. BioRxiv, 2019.12.12.874925. doi: 10.1101/2019.12.12.874925

Eisenstein, F., Danev, R., & Pilhofer, M. (2019). Improved applicability and robustness of fast cryo-electron tomography data acquisition. J. Struct. Bio, 208(2), 107–114. doi: 10.1016/j.jsb.2019.08.006

Gorelick, S., Buckley, G., Gervinskas, G., Johnson, T. K., Handley, A., Caggiano, M. P., Whisstock, J. C., Pocock, R., & de Marco, A. (2019). PIE-scope, integrated cryo-correlative light and FIB/SEM microscopy. ELife, 8, e45919. doi: 10.7554/eLife.45919

Grotjahn, D., Chowdhury, S., & Lander, G. C. (2020). A guided approach for subtomogram averaging of challenging macromolecular assemblies. BioRxiv, 2020.02.01.930297. doi: 10.1101/2020.02.01.930297

Himes, B. A., & Zhang, P. (2018). emClarity: software for high-resolution cryo-electron tomography and subtomogram averaging. Nature Methods, 15(11), 955–961. doi: 10.1038/s41592-018-0167-z

Lewis, A. J., Genoud, C., Pont, M., van de Berg, W. D. J., Frank, S., Stahlberg, H., Shahmoradian, S. H., & Al-Amoudi, A. (2019). Imaging of post-mortem human brain tissue using electron and X-ray microscopy. Current Opinion in Structural Biology, 58, 138–148. doi: 10.1016/j.sbi.2019.06.003

Liu, N., Zhang, J., Chen, Y., Liu, C., Zhang, X., Xu, K., Wen, J., Luo, Z., Chen, S., Gao, P., Jia, K., Liu, Z., Peng, H., & Wang, H.-W. (2019). Bioactive Functionalized Monolayer Graphene for High-Resolution Cryo-Electron Microscopy. Journal of the American Chemical Society, 141(9), 4016–4025. doi: 10.1021/jacs.8b13038

Ortega, D. R., Oikonomou, C. M., Ding, H. J., Rees-Lee, P., Alexandria, & Jensen, G. J. (2019). ETDB-Caltech: A blockchain-based distributed public database for electron tomography. PLOS ONE, 14(4), e0215531. doi: 10.1371/journal.pone.0215531

Schaffer, M., Pfeffer, S., Mahamid, J., Kleindiek, S., Laugks, T., Albert, S., Engel, B. D., Rummel, A., Smith, A. J., Baumeister, W., & Plitzko, J. M. (2019). A cryo-FIB lift-out technique enables molecular-resolution cryo-ET within native Caenorhabditis elegans tissue. Nature Methods, 16(8), 757–762. doi: 10.1038/s41592-019-0497-5

Tacke, S., Erdmann, P., Wang, Z., Klumpe, S., Grange, M., Kříž, R., Dolník, M., Mitchels, J., Plitzko, J., & Raunser, S. (2020). A streamlined workflow for automated cryo focused ion beam milling. BioRxiv, 2020.02.24.963033. doi: 10.1101/2020.02.24.963033

Tegunov, D., & Cramer, P. (2019). Real-time cryo-electron microscopy data preprocessing with Warp. Nature Methods, 16(11), 1146–1152. doi: 10.1038/s41592-019-0580-y

Toro-Nahuelpan, M., Zagoriy, I., Senger, F., Blanchoin, L., Théry, M., & Mahamid, J. (2020). Tailoring cryo-electron microscopy grids by photo-micropatterning for in-cell structural studies. Nature Methods, 17(1), 50–54. doi: 10.1038/s41592-019-0630-5

Turoňová, B., Hagen, W. J. H., Obr, M., Kräusslich, H.-G., & Beck, M. (2019). Benchmarking tomographic acquisition schemes for high-resolution structural biology. BioRxiv, 742254. doi: 10.1101/742254

Wang, H.-W., & Fan, X. (2019). Challenges and opportunities in cryo-EM with phase plate. Current Opinion in Structural Biology, 58, 175–182. doi: 10.1016/j.sbi.2019.06.013

Wolff, G., Limpens, R. W. A. L., Zheng, S., Snijder, E. J., Agard, D. A., Koster, A. J., & Bárcena, M. (2019). Mind the gap: Micro-expansion joints drastically decrease the bending of FIB-milled cryo-lamellae. J. Struct. Bio, 208(3), 107389. doi: 10.1016/j.jsb.2019.09.006

Xu, M., Singla, J., Tocheva, E. I., Chang, Y.-W., Stevens, R. C., Jensen, G. J., & Alber, F. (2019). De Novo Structural Pattern Mining in Cellular Electron Cryotomograms. Structure, 27(4), 679-691.e14. doi: 10.1016/j.str.2019.01.005

Zhang, J., Zhang, D., Sun, L., Ji, G., Huang, X., Niu, T., & Sun, F. (2019). VHUT-cryo-FIB, a method to fabricate frozen-hydrated lamella of tissue specimen for in situ cryo-electron tomography. BioRxiv, 727149. doi: 10.1101/727149

Zhang, P. (2019). Advances in cryo-electron tomography and subtomogram averaging and classification. Current Opinion in Structural Biology, 58, 249–258. doi: 10.1016/j.sbi.2019.05.021

Appendix

Appendix

3D visualization of a Chlamydomonas Golgi apparatus obtained by cryo-electron tomography. Data courtesy of Dr. B. Engel, Department of Molecular Structural Biology, Max Planck Institute of Biochemistry, Martinsried, Germany.

Understanding the structural basis for cellular processes is essential for understanding how cells function. Within the Aquilos Cryo-FIB, the new optional Thermo Scientific iFLM Correlative System combines light and electron microscopy into one system, eliminating extra sample transfer steps and enabling users to create a streamlined

Search less, discover moreiFLM™ Correlative System for Aquilos 2 Cryo-FIB

cryo-correlative solution for cryo-tomography. The iFLM Correlative System delivers the localization of fluorescent targets inside the chamber, allowing users to check the fluorescence signal inside milled lamellae and correlate two imaging modalities directly within one system.

Cryo-lamella prepared with the Aquilos 2 Cryo-FIB (left). Overlay of SEM image with fluorescence image obtained with the iFLM Correlative System (right).

10 µm

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1 µm

Then...Electron micrograph of 30S dynein from cilia isolated from Tetrahymena pyriformis. Gibbons & Rowe, 1965.

The structure and behavior of these flagella weren’t revealed until the advent of electron microscopy. Cryo-ET was used to image the active flagellum of swimming sea urchin sperm cells. Image © 2018 The Authors, some rights reserved; exclusive licensee AAAS. No claim to original U.S. Government Works.

Lin J., and Nicastro D. 2018. Asymmetric distribution and spatial switching of dynein activity generates ciliary motility. Science 360, eaar1968. doi: 10.1126/science.aar1968

...Now

Swimming sperm cell

Inhibited dynein motors

Active dynein motors

Introduction Cell Biology Neuroscience Microbiology Virology Workflow AppendixReviews