Low-energy electron holographic imaging of individual tobacco mosaic virions

Modern structural biology relies on NMR, X-ray crystallography and cryo-electron microscopy for gaining information on biomolecules at nanometer, sub-nanometer or atomic resolution. All these methods, however, require averaging over a vast ensemble of entities and hence knowledge on the conformational landscape of an individual particle is lost. Unfortunately, there are now strong indications that even X-ray free electron lasers will not be able to image individual molecules but will require nanocrystal samples. Here, we show that non-destructive structural biology of single particles has now become possible by means of low-energy electron holography. As an example, individual tobacco mosaic virions deposited on ultraclean freestanding graphene are imaged at one nanometer resolution revealing structural details arising from the helical arrangement of the outer protein shell of the virus. Since low-energy electron holography is a lens-less technique and since electrons with a deBroglie wavelength of approximately 1 Angstrom do not impose radiation damage to biomolecules, the method has the potential for Angstrom resolution imaging of single biomolecules

Imaging and 3D reconstruction of membrane protein complexes by cryo-electron microscopy and single particle analysis

Cryo-electron microscopy (cryo-EM) in combination with single particle image processing and volume reconstruction is a powerful technology to obtain medium-resolution structures of large protein complexes, which are extremely difficult to crystallize and not amenable to NMR studies due to size limitation. Depending on the stability and stiffness as well as on the symmetry of the complex, three-dimensional reconstructions at a resolution of 10-30 ˚ can be achieved. In this range of resolution, we may not be able to answer A chemical questions at the level of atomic interactions, but we can gain detailed insight into the macromolecular architecture of large multi-subunit complexes and their mechanisms of action. In this thesis, several prevalently large membrane protein complexes of great physiological importance were examined by various electron microscopy techniques and single particle image analysis. The core part of my work consists in the imaging of a mammalian V-ATPase, frozen-hydrated in amorphous ice and of the completion of the first volume reconstruction of this type of enzyme, derived from cryo-EM images. This ubiquitous rotary motor is essential in every eukaryotic cell and is of high medical importance due to its implication in various diseases such as osteoporosis, skeletal cancer and kidney disorders. My contribution to the second and third paper concerns the volume reconstruction of two bacterial outer membrane pore complexes from cryo-EM images recorded by my colleague Mohamed Chami. PulD from Klebsiella oxytoca constitutes a massive translocating pore capable of transporting a fully folded cell surface protein PulA through the membrane. It is part of the Type II secretion system, which is common for Gram-negative bacteria. The second volume regards ClyA, a pore-forming heamolytic toxin of virulent Escherichia coli and Salmonella enterica strains that kill target cells by inserting pores into their membranes. To the last two papers, I contributed with cryo-negative stain imaging of the cell division protein DivIVA from Bacillus subtilis and with image processing of the micrographs displaying the siderophore receptor FrpB from Neisseria meningitidis

High-resolution cryo-electron microscopy on macromolecular complexes and cell organelles

Cryo-electron microscopy techniques and computational 3-D reconstruction of macromolecular assemblies are tightly linked tools in modern structural biology. This symbiosis has produced vast amounts of detailed information on the structure and function of biological macromolecules. Typically, one of two fundamentally different strategies is used depending on the specimens and their environment. A: 3-D reconstruction based on repetitive and structurally identical unit cells that allow for averaging, and B: tomographic 3-D reconstructions where tilt-series between approximately ±60 and ±70° at small angular increments are collected from highly complex and flexible structures that are beyond averaging procedures, at least during the first round of 3-D reconstruction. Strategies of group A are averaging-based procedures and collect large number of 2-D projections at different angles that are computationally aligned, averaged together, and back-projected in 3-D space to reach a most complete 3-D dataset with high resolution, today often down to atomic detail. Evidently, success relies on structurally repetitive particles and an aligning procedure that unambiguously determines the angular relationship of all 2-D projections with respect to each other. The alignment procedure of small particles may rely on their packing into a regular array such as a 2-D crystal, an icosahedral (viral) particle, or a helical assembly. Critically important for cryo-methods, each particle will only be exposed once to the electron beam, making these procedures optimal for highest-resolution studies where beam-induced damage is a significant concern. In contrast, tomographic 3-D reconstruction procedures (group B) do not rely on averaging, but collect an entire dataset from the very same structure of interest. Data acquisition requires collecting a large series of tilted projections at angular increments of 1–2° or less and a tilt range of ±60° or more. Accordingly, tomographic data collection exposes its specimens to a large electron dose, which is particularly problematic for frozen-hydrated samples. Currently, cryo-electron tomography is a rapidly emerging technology, on one end driven by the newest developments of hardware such as super-stabile microscopy stages as well as the latest generation of direct electron detectors and cameras. On the other end, success also strongly depends on new software developments on all kinds of fronts such as tilt-series alignment and back-projection procedures that are all adapted to the very low-dose and therefore very noisy primary data. Here, we will review the status quo of cryo-electron microscopy and discuss the future of cellular cryo-electron tomography from data collection to data analysis, CTF-correction of tilt-series, post-tomographic sub-volume averaging, and 3-D particle classification. We will also discuss the pros and cons of plunge freezing of cellular specimens to vitrified sectioning procedures and their suitability for post-tomographic volume averaging despite multiple artifacts that may distort specimens to some degree

3D reconstruction and comparison of shapes of DNA minicircles observed by cryo-electron microscopy

We use cryo-electron microscopy to compare 3D shapes of 158 bp long DNA minicircles that differ only in the sequence within an 18 bp block containing either a TATA box or a catabolite activator protein binding site. We present a sorting algorithm that correlates the reconstructed shapes and groups them into distinct categories. We conclude that the presence of the TATA box sequence, which is believed to be easily bent, does not significantly affect the observed shapes

The promise and the challenges of cryo-electron tomography

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

High-resolution cryo-electron microscopy study of structure anddynamics of yeast fatty acid synthase by single particle analysis

This thesis presents a 5.9 Å map of yeast FAS obtained by cryo-electron microscopy using single particle analysis (SPA). The EM-map has been analyzed both by quantitative and qualitative analysis to aid in understanding of the structure and dynamics of yeast FAS. This study approaches the factors limiting the resolution in EM (>20 Å) and further discusses the possibilities of achieving higher-resolutions (<10 Å) in cryo-EM by single particle analysis. Here, SPA is highlighted as a powerful tool for understanding the structure and dynamics of macro-molecular complexes at near native conditions. Though SPA has been used over the last four decades, the low-resolution range (20-30 Å) of the method has limited its use in structural biology. Over the last decade, sub nanometer resolution (<10 Å) structures solved by SPA have been reported --both in studies involving symmetric particles, such as GroEL (D7) and asymmetric particles, such as ribosomes (C1). Recently, near-atomic resolution in the range of 3.8-4.2 Å has been achieved in cases of highly symmetric icosahedral viral capsid structures as well. The yeast FAS structure (D3) presented here is one of two low symmetry structures submitted to the EM-database in a resolution range of 5-6 Å; the other being GroEL (D7). Fatty acid synthase (FAS) is the key enzyme for the biosynthesis of fatty acids in living organisms. There are two types of FAS, namely the type II FAS system in prokaryotes, consisting of a set of individual enzymes, and type I FAS found in eukaryotes as a multienzyme complex. Yeast fatty acid synthase (FAS) is a 2.6 MDa barrel-shaped multienzyme complex, which carries out cyclic synthesis of fatty acids. By electron cryomicroscopy of single particles we obtained a 3D map of yeast FAS at 5.9 Å resolution. Compared to the crystal structures of fungal FAS, the EM map reveals major differences and new features that indicate a considerably different arrangement of the complex in solution, as well as a high degree of variance inside the barrel. Distinct density regions in the reaction chambers next to each of the catalytic domains fit well with the substratebinding acyl carrier protein (ACP) domain. In each case, this resulted in the expected distance of ~18 Å from the ACP substrate binding site to the active site of the catalytic domains. The multiple, partially occupied positions of the ACP within the reaction chamber provide direct insight into the proposed substrate-shuttling mechanism of fatty acid synthesis in this large cellular machine.Die Fettsäure Synthase (FAS) ist das Schlüsselenzym bei der Biosynthese von Fettsäuren in lebenden Organismen. Zwei Arten der FAS sind bekannt. Der Typ-II FAS der Prokarioten ist aus verschiedenen Einzelenzymen aufgebaut, während der Typ-I FAS der Eukarioten aus einem Multi-Enzym Komplex besteht (Smith et al., 2003). Der Typ-I FAS Komplex der Säugetiere ist ein a2-Homodimer (Brink et al., 2002), der Typ-I FAS Komplex der Bäckerhefe wird dagegen aus einem a6b6 Heterododecamer aufgebaut, der eine molekulare Masse von 2,6 MDa besitzt (Brink et al., 2002). Obwohl Pilz- und Säugertyp FAS deutlich unterschiedliche Strukturen aufweisen, sind alle, zur Synthese von Fettsäuren notwendigen Enzyme (Fig. 1), in beiden FAS-Komplexen konserviert. Früheren elektronenmikrokopischen Untersuchungen (Kolodziej et al., 1996) zeigten die FAS als eine 260 Å x 230 Å Fass-ähnliche Struktur mit D3 Symmetrie. Die dreidimensionalen Röntgenkristallstrukturen der Thermomyces lanuginosus (Jenni et al., 2007) und S. cerevisiae FAS bei einer Auflösung von 3,1 Å beziehungsweise 4,0 Å (Leibundgut et al., 2007; Lomakin et al., 2007; Johansson et al., 2008) weisen kaum Unterschiede auf. Die sechs a-Untereinheiten der FAS bauen ein äquatoriales Rad auf, das die fassartige Struktur in zwei separate Kuppeln aufteilt. Die Kuppeln wiederum bestehen aus jeweils drei b-Untereinheiten. Die a- und b-Untereinheiten bilden pro Kuppel drei Reaktionsräume mit acht Reaktionszentren. Für die Reaktionszentren stellt die a-Untereinheit die Phosphopentetheinyl-Transferase (PPT), das Acyl-Carrier-Protein (ACP), die Ketoacyl-Synthase (KS), die Ketoacyl-Reduktase (KR) und einen Teil der Malonyl-Palmitoyl-Reduktase (MPT)-Domäne bereit. Die Acetyl-Transferase (AT), die Enoyl-Reduktase (ER), die Dehydratase (DH) sowie der Hauptteil der MPT wird von der b-Untereinheit eingebracht. In der Pilztyp FAS wird das ACP durch zwei flexible Verbindungsstücke gehalten, die es mit der MPT-Domäne und dem Zentrum des äquatorialen Rads verbindet. Die Verbindungsdomänen definieren den Reaktionsradius des ACP, der weitgehend mit dem Reaktionsraum übereinstimmt (Leibundgut et al., 2007). Die Reaktionsbereiche aller katalytischen Domänen, ausgenommen die der PPT, weisen in Richtung Reaktionsraum im Inneren der FAS (Leibundgut et al., 2007; Lomakin et al., 2007; Johansson et al., 2008). Zusätzlich zu den Reaktionsdomänen, existieren weitere sechs Strukturdomänen, zwei in der a-Untereinheit (SD1-2a) und vier in der b-Untereinheit (SD1-4b). Kürzlich veröffentliche Studien konnten zeigen, dass jeder Reaktionsraum unabhängig von einander arbeitet (Singh et al., 2008). Das ACP gehört zu der Klasse der universal vorkommenden, hochkonservierten Carrier-Proteine, die Acyl-Zwischenprodukte über den ~18 Å langen Phosphopantetheinarm binden und in sehr unterschiedlichen metabolischen Reaktionen, einschließlich denen der Biosynthese von Polyketiden oder Fettsäuren (Byers and Gong, 2007), activ sind. Die ACP werden in zwei Klassen, Typ-I und Typ-II, eingeteilt, die beide a-helicale Strukturen mit einer konservierten, zentralen vier-Helix Anordnung besitzen. Die vier- Helix Anordnung stellt die Bindestelle für die Acylketten-Zwischenprodukte zur Verfügung. ACP-abhängige Enzymreaktionen sind essentiell für jede Zelle. Daher ist die Pilztyp FAS eine wichtiges Angriffstelle medikamentöser Behandlung (Byers und Gong, 2007). ..

Compressed sensing electron tomography of needle-shaped biological specimens--Potential for improved reconstruction fidelity with reduced dose.

Electron tomography is an invaluable method for 3D cellular imaging. The technique is, however, limited by the specimen geometry, with a loss of resolution due to a restricted tilt range, an increase in specimen thickness with tilt, and a resultant need for subjective and time-consuming manual segmentation. Here we show that 3D reconstructions of needle-shaped biological samples exhibit isotropic resolution, facilitating improved automated segmentation and feature detection. By using scanning transmission electron tomography, with small probe convergence angles, high spatial resolution is maintained over large depths of field and across the tilt range. Moreover, the application of compressed sensing methods to the needle data demonstrates how high fidelity reconstructions may be achieved with far fewer images (and thus greatly reduced dose) than needed by conventional methods. These findings open the door to high fidelity electron tomography over critically relevant length-scales, filling an important gap between existing 3D cellular imaging techniques.The research leading to these results has received funding from the European Union Seventh Framework Programme under Grant Agreement 312483 - ESTEEM2 (Integrated Infrastructure Initiative–I3), as well as from the European Research Council under the European Union’s Seventh Framework Programme (FP/2007-2013)/ERC grant agreement 291522 - 3DIMAGE. B.W. and E.S. acknowledge financial support from the Deutsche Forschungsgemeinschaft (DFG) within the framework of the SPP 1570 as well as through the Cluster of Excellence “Engineering of Advanced Materials” at the Friedrich-Alexander-Universität ErlangenNürnberg. G.D. and C.D. acknowledge funding from the ERC under grant number 259619 PHOTO EM. B.W. acknowledges the Research Training Group “Disperse Systems for Electronic Applications” (DFG GEPRIS GRK 1161). R.L. acknowledges a Junior Research Fellowship from Clare College.This is the final version of the article. It first appeared from Elsevier via http://dx.doi.org/10.1016/j.ultramic.2015.10.02

Structural studies on viral receptor-binding proteins

Structure. Almost everything around us has it. But why? What is it needed for? Since the early days of human enquiry people have tried to understand how things come together and stay assembled. As scientific discovery advanced, researchers from all fields of science have been at some point or another puzzled by problems related to structure. Among them, biologists discovered that there is more to the natural world surrounding us than meets the eye. Structural biology studies are often hypothesis-driven and require flexible methods to address specific questions on the relationship between structure and biological function. My thesis discusses three cases in which structure is fundamental to function, and presents three different approaches to solving the three-dimensional structure of entire viruses or virus proteins, going from relatively well-ordered systems to increasing heterogeneous ones. The first study is the characterization of African horsesickness virus, a double-stranded RNA icosahedrally-symmetric virus causing a severe disease in horses. I used electron cryo-microscopy and icosahedral reconstruction to determine the virion structures of two serotypes to 11 and 14 Å resolution. The three-dimensional structure allowed us to map two domains of the receptor-binding protein VP2, an important step for the informed design of new subunit vaccines for African horsesickness virus. The second study is a description of the spike complex of bacteriophage PRD1, a membrane-containing virus. Here, the major problem was to determine the organization of flexible, low abundance proteins involved in cell recognition and attachment. This sort of heterogeneity in biological systems is key to their function, but very unfavourable for structural analysis. We used a combination of different mutants, electron cryo-microscopy three-dimensional image reconstruction and atomic modeling to address the symmetry mismatch between the icosahedrally-symmetric capsid and the spike complex situated at the five-fold vertices and determined the architecture of the spike complex formed by protein P5 and the receptor-binding protein P2. The third study is a comparative biological and structural study of seven recently isolated pleomorphic viruses, which infect extremely halophilic archaea. I established the pleomorphic nature of this novel virion type by electron cryo-tomography. Detailed analysis using subtomographic processing showed the radial distribution of the membrane and the spike protein VP4, and led to an average structure of VP4.Ei saatavill