Cryo-Electron Microscopy – a Primer for the Non-Microscopist Jacqueline L
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Electron Microscopy Workflows: from Single Particle Analysis to Volume Imaging
Electron microscopy workflows: from single particle analysis to volume imaging Kristian Wadel History 1949 – World’s first production TEM introduced FEI Company: global reach, many applications FEI Life Science segmentation The importance of scale Small organisms and tissues: millimeters Cell: ≈300 µm3 3 Tomogram: ≈0.4 µm Top image: Courtesy of D. McCarthy, University College London Middle and bottom: Courtesy of J. Mahamid, J. Plitzko and W. Baumeister, MPI for Biochemistry Organism Tissue Glomerulus Cell Synapse Vesicle Lipid bi-layer (~10 mm) (~1 mm) (~100 μm) (~10 μm) (~500nm) (~30nm) (4.3nm) Resolution - Z Resolution limit resin embedded specimen 5 STEM Tomo TEM Tomo DualBeam FIB tomography 10 Serial Blockface SEM 50 Serial Section SEM Serial Section TEM Superresolution LM 100 500 Light Microscopy 10.000.000 1.000.000 100.000 10.000 1.000 100 10 1 Volume size (µm3) Structure size (nm) FEI Life Science portfolio TEM Tecnai Talos Talos Artica Titan Halo Titan Krios SEM SDB Inspect Quanta NovaNano Teneo Verios Scios Helios CLEM Software CorrSight iCorr Living bio. sample Chemical Cryo- Cryo fixation fixation protection Preembedd. Rehydration labelling Freeze- Dehydration Freezing Freeze drying CEMOVIS substitution Humbel, UNIL Lausanne Resin Freeze embedding fracture Cryo- Critical point sectioning drying Adapted from slideAdapted from of Bruno Thawed Whole mount Frozen Resin section section replica hydrated TEM applications Cell & Tissue Classical 2D Tomography Correlative Microscopy Biology Negative Staining Immuno-labeling Cryo-TEM -
Structure of the Immature HIV-1 Capsid in Intact Virus Particles at 8.8 Ĺ
LETTER doi:10.1038/nature13838 Structure of the immature HIV-1 capsid in intact virus particles at 8.8 A˚ resolution Florian K. M. Schur1,2, Wim J. H. Hagen1, Michaela Rumlova´3,4, Toma´ˇs Ruml5, Barbara Mu¨ller2,6, Hans-Georg Kra¨usslich2,6 & John A. G. Briggs1,2 Human immunodeficiency virus type 1 (HIV-1) assembly proceeds form, while the N-terminal CA domain (CA-NTD) adopts a presum- in two stages. First, the 55 kilodalton viral Gag polyprotein assem- ably non-physiological form7. bles into a hexameric protein lattice at the plasma membrane of the Subtomogram averaging has been used to solve low-resolution struc- infected cell, inducing budding and release of an immature particle. tures of biological molecules within pleiotropic native environments8, Second, Gag is cleaved by the viral protease, leading to internal rear- including structures of the immature HIV-1 Gag lattice within virus rangement of the virus into the mature, infectious form1. Immature particles at ,20 A˚ resolution4,9. Higher-resolution structures have not and mature HIV-1 particles are heterogeneous in size and morpho- yet been obtained for any component of such heterogeneous viruses logy, preventing high-resolution analysis of their protein arrangement in situ. in situ by conventional structural biology methods. Here we apply Recently, we showed that optimized subtomogram averaging methods cryo-electron tomography and sub-tomogram averaging methods can recover structural data at below 10 A˚ resolution10. Their application to resolve the structure of the capsid lattice within intact immature to immature HIV-1 might enable determination of a subnanometre- HIV-1 particles at subnanometre resolution, allowing unambiguous resolution structure of Gag within intact virus. -
Electron Tomography—A Tool for Ultrastructural 3D Visualization in Cell Biology and Histology
main topic Wien Med Wochenschr (2018) 168:322–329 https://doi.org/10.1007/s10354-018-0646-y Electron tomography—a tool for ultrastructural 3D visualization in cell biology and histology Josef Neumüller Received: 16 March 2018 / Accepted: 22 June 2018 / Published online: 6 August 2018 © The Author(s) 2018 Summary Electron tomography (ET) was developed lischen Grundlagen, Möglichkeiten und Grenzen. Die to overcome some of the problems associated re- Entwicklung innovativer Verfahren wird besprochen, constructing three-dimensional (3D) images from 2D relevante Untersuchungen aus dem Institut der Au- election microscopy data from ultrathin slices. Virtual toren und in Zusammenarbeit mit anderen werden sections of semithin sample are obtained by incre- dargestellt. Durch die ET erschließt sich die dritte mental rotation of the target and this information is Dimension auf der ultrastrukturellen Ebene, sie stellt used to assemble a 3D image. Herein, we provide einen Meilenstein in der strukturellen Molekularbio- an instruction to ET including the physical principle, logie dar. possibilities, and limitations. We review the develop- ment of innovative methods and highlight important Schlüsselwörter Elektronentomografie · 3D-Darstel- investigations performed inourdepartmentandwith lung · Ultrastruktur · Zellbiologie · Histologie our collaborators. ET has opened up the third di- mension at the ultrastructural level and represents a Introduction milestone in structural molecular biology. When inspecting the organization of organelles of Keywords Electron tomography · 3D visualization · a particular cell or the cellular arrangement in sev- Ultrastructure · Cell biology · Histology eral tissues at the ultrastructural level, we usually start from two-dimensional images. We then inter- Elektronentomografie – Ein Verfahren zur 3D- polate a three-dimensional(3D)imagefromsections Visualisierung von ultrastrukturellen Details in at different heights of the same site of an electron Zellbiologie und Histologie microscopic (EM) preparation. -
Cryo-Electron Tomography of Bacterial Viruses
Virology 435 (2013) 179–186 Contents lists available at SciVerse ScienceDirect Virology journal homepage: www.elsevier.com/locate/yviro Review Cryo-electron tomography of bacterial viruses Ricardo C. Guerrero-Ferreira, Elizabeth R. Wright n Division of Pediatric Infectious Diseases, Emory University School of Medicine, Children’s Healthcare of Atlanta, Atlanta, GA 30322, USA article info abstract Bacteriophage particles contain both simple and complex macromolecular assemblages and machines Keywords: that enable them to regulate the infection process under diverse environmental conditions with a broad Bacteriophage range of bacterial hosts. Recent developments in cryo-electron tomography (cryo-ET) make it possible Cryo-electron microscopy to observe the interactions of bacteriophages with their host cells under native-state conditions at Cryo-EM unprecedented resolution and in three-dimensions. This review describes the application of cryo-ET to Cryo-electron tomography studies of bacteriophage attachment, genome ejection, assembly and egress. Current topics of Cryo-ET investigation and future directions in the field are also discussed. Sub-tomogram averaging & 2012 Elsevier Inc. All rights reserved. Contents Introduction. ........................................................................................................179 Biological electron microscopy and the development of cryo-electron microscopy . .................................................180 Imaging whole virus (isolated particles) ...................................................................................182 -
Electron Crystallography of Ultrathin 3D Protein Crystals: Atomic Model with Charges
Electron crystallography of ultrathin 3D protein crystals: Atomic model with charges Koji Yonekura (米倉 功治)a,b, Kazuyuki Kato (加藤 一幸)c, Mitsuo Ogasawara (小笠原 光雄)b, Masahiro Tomita (富田 正弘)b,d, and Chikashi Toyoshima (豊島 近)b,1 aBiostructural Mechanism Laboratory, RIKEN SPring-8 Center, 1-1-1 Kouto, Sayo, Hyogo 679-5148, Japan; bInstitute of Molecular and Cellular Biosciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo, 113-0032, Japan; cHitachi High-Tech Fielding Corporation, 4-28-8 Yotsuya, Shinjuku-ku, Tokyo, 160-0004, Japan; and dHitachi High-Technologies Corporation, 1-24-14 Nishi-Shinbashi, Minato-ku, Tokyo, 105-8717, Japan Contributed by Chikashi Toyoshima, January 23, 2015 (sent for review August 28, 2014) Membrane proteins and macromolecular complexes often yield F and G). These features of Coulomb potential maps result from crystals too small or too thin for even the modern synchrotron the fact that atomic scattering factors for electrons vary consid- X-ray beam. Electron crystallography could provide a powerful erably over a range of spatial frequency depending on the means for structure determination with such undersized crystals, charged state (Fig. 1A) and can become close to zero or even − as protein atoms diffract electrons four to five orders of magni- negative (e.g., for O , Fig. 1A). An advantageous consequence is tude more strongly than they do X-rays. Furthermore, as electron that it is possible to determine experimentally the charged states crystallography yields Coulomb potential maps rather than elec- of protein residues and metals. As proteins use metals of different tron density maps, it could provide a unique method to visualize ionic states for many purposes, notably for catalysis and electron the charged states of amino acid residues and metals. -
Charting the Native Architecture of Thylakoid Membranes with Single-Molecule Precision
bioRxiv preprint doi: https://doi.org/10.1101/759001; this version posted September 5, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. Charting the native architecture of thylakoid membranes with single-molecule precision Wojciech Wietrzynski1*, Miroslava Schaffer1*, Dimitry Tegunov2*, Sahradha Albert1, Atsuko Kanazawa3, Jürgen M. Plitzko1, Wolfgang Baumeister1, Benjamin D. Engel1† Thylakoid membranes scaffold an assortment of large protein complexes that work together to harness the energy of light to produce oxygen, NADPH, and ATP. It has been a longstanding challenge to visualize how the intricate thylakoid network organizes these protein complexes to finely tune the photosynthetic reactions. Using cryo-electron tomogra- phy to analyze membrane surface topology, we have mapped the native molecular landscape of thylakoid membranes within green algae cells. Our tomograms provide insights into the molecular forces that drive thylakoid stacking and reveal that photosystems I and II are strictly segregated at the borders between appressed and non-appressed mem- brane domains. This new approach to charting thylakoid topology lays the foundation for dissecting photosynthetic regulation at the level of single protein complexes within the cell. Membranes orchestrate cellular life. In addition to compart- thylakoids (7, 8, 15). However, the platinum replicas produced mentalizing the cell into organelles, membranes can organize by this technique have limited resolution and only provide ac- their embedded proteins into specialized domains, concentrat- cess to random fracture planes through the membranes. More ing molecular partners together to drive biological processes (1, recently, atomic force microscopy (AFM) has been used to map 2). -
The Subcellular Proteome of a Planctomycetes Bacterium Shows That Newly Evolved Proteins Have Distinct Fractionation Patterns
fmicb-12-643045 May 3, 2021 Time: 15:59 # 1 ORIGINAL RESEARCH published: 04 May 2021 doi: 10.3389/fmicb.2021.643045 The Subcellular Proteome of a Planctomycetes Bacterium Shows That Newly Evolved Proteins Have Distinct Fractionation Patterns Christian Seeger1†, Karl Dyrhage1†, Mayank Mahajan1, Anna Odelgard1, Sara Bergström Lind2‡ and Siv G. E. Andersson1* 1 Science for Life Laboratory, Molecular Evolution, Department of Cell and Molecular Biology, Biomedical Centre, Uppsala University, Uppsala, Sweden, 2 Department of Chemistry-BMC, Analytical Chemistry, Uppsala University, Uppsala, Sweden Edited by: Anthony Poole, The Planctomycetes bacteria have unique cell architectures with heavily invaginated University of Auckland, New Zealand membranes as confirmed by three-dimensional models reconstructed from FIB-SEM Reviewed by: Christian Jogler, images of Tuwongella immobilis and Gemmata obscuriglobus. The subcellular proteome Radboud University Nijmegen, of T. immobilis was examined by differential solubilization followed by LC-MS/MS Netherlands Damien Paul Devos, analysis, which identified 1569 proteins in total. The Tris-soluble fraction contained Andalusian Center for Development mostly cytoplasmic proteins, while inner and outer membrane proteins were found in the Biology (CABD), Spain Triton X-100 and SDS-soluble fractions, respectively. For comparisons, the subcellular *Correspondence: proteome of Escherichia coli was also examined using the same methodology. A notable Siv G. E. Andersson [email protected] difference in the overall fractionation pattern of the two species was a fivefold higher †These authors have contributed number of predicted cytoplasmic proteins in the SDS-soluble fraction in T. immobilis. equally to this work and share first One category of such proteins is represented by innovations in the Planctomycetes authorship lineage, including unique sets of serine/threonine kinases and extracytoplasmic sigma ‡ Present address: Sara Bergström Lind, factors with WD40 repeat domains for which no homologs are present in E. -
Electron Tomography Analysis of 3D Order and Interfacial Structure in Nano-Precipitates
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology 1380 Electron tomography analysis of 3D order and interfacial structure in nano-precipitates LING XIE ACTA UNIVERSITATIS UPSALIENSIS ISSN 1651-6214 ISBN 978-91-554-9590-9 UPPSALA urn:nbn:se:uu:diva-284102 2016 Dissertation presented at Uppsala University to be publicly examined in Polhemssalen, Ångströmlaboratoriet, Lägerhyddsvägen 1, Uppsala, Tuesday, 14 June 2016 at 13:00 for the degree of Doctor of Philosophy. The examination will be conducted in English. Faculty examiner: Professor Christian Kübel (Electron Microscopy and Spectroscopy Laboratory, Karlsruhe Institute of Technology). Abstract Xie, L. 2016. Electron tomography analysis of 3D order and interfacial structure in nano-precipitates. Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology 1380. 84 pp. Uppsala: Acta Universitatis Upsaliensis. ISBN 978-91-554-9590-9. Structural characterization is essential to understand the formation mechanisms of the nanostructures in thin absorber layers in third generation solar cells and amyloid protein aggregates. Since to the dimension of the precipitated structures is in nanometer scale, electron tomography technique in transmission electron microscopy (TEM) has been applied as a major tool to analyze the 3D order and distribution of precipitates using the electron tomography technique. Silicon rich silicon carbide (SRSC) films were deposited by plasma enhanced chemical vapor deposition (PECVD) technique and annealed in the nitrogen atmosphere for 1 hour at 1100 °C. The spectrum-imaging (SI) technique in Energy filtered TEM (EFTEM) imaging mode was used to develop electron tomography. From the reconstructed sub-volumes, the complex, three dimensional interfacial nanostructure between the precipitated NPs and their parental matrix was observed and explained in terms of thermodynamic concepts. -
Recent Advances in Electron Crystallography
pISSN 2287-5123·eISSN 2287-4445 https://doi.org/10.9729/AM.2017.47.3.160 Review Article Recent Advances in Electron Crystallography Jeong Min Chung†, Sangmin Lee†, Hyun Suk Jung* Department of Biochemistry, College of Natural Sciences, Kangwon National University, Chuncheon 24341, Korea Electron crystallography has been used as the one of powerful tool for studying the structure of biological macromolecules at high resolution which is sufficient to provide †These authors contributed equally details of intramolecular and intermolecular interactions at near-atomic level. Previously to this work. it commonly uses two-dimensional crystals that are periodic arrangement of biological molecules, however recent studies reported a novel technical approach to electron *Correspondence to: crystallography of three-dimensional crystals, called micro electron-diffraction (MicroED) Jung HS, which involves placing the irregular and small sized protein crystals in a transmission Tel: +82-33-250-8513 electron microscope to determine the atomic structure. In here, we review the advances in Fax: +82-33-259-9363 electron crystallography techniques with several recent studies. Furthermore, we discuss E-mail: [email protected] the future direction of this structural approach. Received August 7, 2017 Revised September 6, 2017 Key Words: Electron crystallography, Protein structure, Transmission electron microscopy, Accepted September 8, 2017 Micro-electron diffraction, Structural biology INTRODUCTION crystals found during the screening process (Bill et al., 2011). Since early 1940s, electron diffraction has been used to solve The ultimate goal of structural biology is to understand the crystallographic problems (Bendersky & Gayle, 2001). the protein function and its physiological mechanisms by The basic principle of electron crystallography is similar determining the three-dimensional (3D) structure. -
Electron Crystallography of Aquaporins
Portland State University PDXScholar Chemistry Faculty Publications and Presentations Chemistry 7-2008 Electron Crystallography of Aquaporins Simeon Andrews University of Washington Tacoma Steve Reichow [email protected] Tamir Gonen Howard Hughes Medical Institute Follow this and additional works at: https://pdxscholar.library.pdx.edu/chem_fac Part of the Biochemistry, Biophysics, and Structural Biology Commons, and the Chemistry Commons Let us know how access to this document benefits ou.y Citation Details Andrews, S., Reichow, S. L., & Gonen, T. (2008). Electron crystallography of aquaporins. IUBMB life, 60(7), 430-436. This Post-Print is brought to you for free and open access. It has been accepted for inclusion in Chemistry Faculty Publications and Presentations by an authorized administrator of PDXScholar. Please contact us if we can make this document more accessible: [email protected]. NIH Public Access Author Manuscript IUBMB Life. Author manuscript; available in PMC 2009 June 4. NIH-PA Author ManuscriptPublished NIH-PA Author Manuscript in final edited NIH-PA Author Manuscript form as: IUBMB Life. 2008 July ; 60(7): 430±436. doi:10.1002/iub.53. Electron Crystallography of Aquaporins Simeon Andrews, Steve L. Reichow, and Tamir Gonen Department of Biochemistry, University of Washington, Seattle, WA, USA Summary Aquaporins are a family of ubiquitous membrane proteins that form a pore for the permeation of water. Both electron and X-ray crystallography played major roles in determining the atomic structures of a number of aquaporins. This review focuses on electron crystallography, and its contribution to the field of aquaporin biology. We briefly discuss electron crystallography and the two-dimensional crystallization process. -
Electron Crystallography: Imaging and Single-Crystal Diffraction from Powders
feature articles Acta Crystallographica Section A Foundations of Electron crystallography: imaging and single-crystal Crystallography diffraction from powders ISSN 0108-7673 Xiaodong Zoua,b* and Sven Hovmo¨llera Received 28 September 2007 Accepted 16 November 2007 aStructural Chemistry, Stockholm University, SE-106 91 Stockholm, Sweden, and bBerzelii Centre EXSELENT on Porous Materials, Stockholm University, SE-106 91 Stockholm, Sweden. Correspondence e-mail: [email protected] The study of crystals at atomic level by electrons – electron crystallography – is an important complement to X-ray crystallography. There are two main advantages of structure determinations by electron crystallography compared to X-ray diffraction: (i) crystals millions of times smaller than those needed for X-ray diffraction can be studied and (ii) the phases of the crystallographic structure factors, which are lost in X-ray diffraction, are present in transmission- electron-microscopy (TEM) images. In this paper, some recent developments of electron crystallography and its applications, mainly on inorganic crystals, are shown. Crystal structures can be solved to atomic resolution in two dimensions as well as in three dimensions from both TEM images and electron diffraction. Different techniques developed for electron crystallography, including three- dimensional reconstruction, the electron precession technique and ultrafast electron crystallography, are reviewed. Examples of electron-crystallography # 2008 International Union of Crystallography applications are given. There is in principle no limitation to the complexity of Printed in Singapore – all rights reserved the structures that can be solved by electron crystallography. 1. Introduction Henderson, 1975) using Fourier-transform-based image processing to get both the crystallographic structure-factor Electron diffraction (ED) of crystals was discovered in 1927, amplitudes and phases from the TEM images and retrieve the only 15 years after the discovery of X-ray diffraction. -
Pulsed EPR Determination of Water Accessibility to Spin-Labeled Amino Acid Residues in Lhciib
1124 Biophysical Journal Volume 96 February 2009 1124–1141 Pulsed EPR Determination of Water Accessibility to Spin-Labeled Amino Acid Residues in LHCIIb A. Volkov,† C. Dockter,‡ T. Bund,‡ H. Paulsen,‡ and G. Jeschke§* †Max-Planck Institute for Polymer Research, Mainz, Germany; ‡Institute of General Botany, Johannes Gutenberg University, Mainz, Germany; and §Laboratory of Physical Chemistry, Swiss Federal Institute of Technology, Zu¨rich, Switzerland ABSTRACT Membrane proteins reside in a structured environment in which some of their residues are accessible to water, some are in contact with alkyl chains of lipid molecules, and some are buried in the protein. Water accessibility of residues may change during folding or function-related structural dynamics. Several techniques based on the combination of pulsed elec- tron paramagnetic resonance (EPR) with site-directed spin labeling can be used to quantify such water accessibility. Accessibility parameters for different residues in major plant light-harvesting complex IIb are determined by electron spin echo envelope modulation spectroscopy in the presence of deuterated water, deuterium contrast in transversal relaxation rates, analysis of longitudinal relaxation rates, and line shape analysis of electron-spin-echo-detected EPR spectra as well as by the conventional techniques of measuring the maximum hyperfine splitting and progressive saturation in continuous-wave EPR. Systematic comparison of these parameters allows for a more detailed characterization of the environment of the spin-labeled residues. These techniques are applicable independently of protein size and require ~10–20 nmol of singly spin-labeled protein per sample. For a residue close to the N-terminus, in a domain unresolved in the existing x-ray structures of light-harvesting complex IIb, all methods indicate high water accessibility.