Structural determinants of intermediate filament mechanics

Intermediate filaments (IFs) are integral to the cell cytoskeleton, supporting cellular mechanical stability. Unlike other cytoskeletal components, the detailed structure of assembled IFs has yet to be resolved. This review highlights new insights, linking the complex IF hierarchical assembly to their mechanical properties and impact on cellular functions. While we focus on vimentin IFs, we draw comparisons to keratins, showcasing the distinctive structural and mechanical features that underlie their unique mechanical responses

Filament assembly of the C. elegans lamin in the absence of helix 1A

Lamins are the major constituent of the nuclear lamina, a protein meshwork underlying the inner nuclear membrane. Nuclear lamins are type V intermediate filaments that assemble into ~3.5 nm thick filaments. To date, only the conditions for the in vitro assembly of Caenorhabditis elegans lamin (Ce-lamin) are known. Here, we investigated the assembly of Ce-lamin filaments by cryo-electron microscopy and tomography. We show that Ce-lamin is composed of ~3.5 nm protofilaments that further interact in vitro and are often seen as 6-8 nm thick filaments. We show that the assembly of lamin filaments is undisturbed by the removal of flexible domains, that is, the intrinsically unstructured head and tail domains. In contrast, much of the coiled-coil domains are scaffold elements that are essential for filament assembly. Moreover, our results suggest that Ce-lamin helix 1A has a minor scaffolding role but is important to the lateral assembly regulation of lamin protofilaments. Keywords: C. elegans; cryo-electron tomography; intermediate filaments; lamin

Structure of hibernating ribosomes studied by cryoelectron tomography in vitro and in situ

CryoET shows the configuration of the ephemeral translationally inactive 100S ribosomal dimer

Vimentin filaments integrate low-complexity domains in a complex helical structure

Intermediate filaments (IFs) are integral components of the cytoskeleton. They provide cells with tissue-specific mechanical properties and are involved in numerous cellular processes. Due to their intricate architecture, a 3D structure of IFs has remained elusive. Here we use cryo-focused ion-beam milling, cryo-electron microscopy and tomography to obtain a 3D structure of vimentin IFs (VIFs). VIFs assemble into a modular, intertwined and flexible helical structure of 40 α-helices in cross-section, organized into five protofibrils. Surprisingly, the intrinsically disordered head domains form a fiber in the lumen of VIFs, while the intrinsically disordered tails form lateral connections between the protofibrils. Our findings demonstrate how protein domains of low sequence complexity can complement well-folded protein domains to construct a biopolymer with striking mechanical strength and stretchability

Vimentin filaments integrate low-complexity domains in a complex helical structure

Intermediate filaments (IFs) are integral components of the cytoskeleton. They provide cells with tissue-specific mechanical properties and are involved in numerous cellular processes. Due to their intricate architecture, a 3D structure of IFs has remained elusive. Here we use cryo-focused ion-beam milling, cryo-electron microscopy and tomography to obtain a 3D structure of vimentin IFs (VIFs). VIFs assemble into a modular, intertwined and flexible helical structure of 40 α-helices in cross-section, organized into five protofibrils. Surprisingly, the intrinsically disordered head domains form a fiber in the lumen of VIFs, while the intrinsically disordered tails form lateral connections between the protofibrils. Our findings demonstrate how protein domains of low sequence complexity can complement well-folded protein domains to construct a biopolymer with striking mechanical strength and stretchability

Marker-free image registration of electron tomography tilt-series

<p>Abstract</p> <p>Background</p> <p>Tilt series are commonly used in electron tomography as a means of collecting three-dimensional information from two-dimensional projections. A common problem encountered is the projection alignment prior to 3D reconstruction. Current alignment techniques usually employ gold particles or image derived markers to correctly align the images. When these markers are not present, correlation between adjacent views is used to align them. However, sequential pairwise correlation is prone to bias and the resulting alignment is not always optimal.</p> <p>Results</p> <p>In this paper we introduce an algorithm to find regions of the tilt series which can be tracked within a subseries of the tilt series. These regions act as landmarks allowing the determination of the alignment parameters. We show our results with synthetic data as well as experimental cryo electron tomography.</p> <p>Conclusion</p> <p>Our algorithm is able to correctly align a single-tilt tomographic series without the help of fiducial markers thanks to the detection of thousands of small image patches that can be tracked over a short number of images in the series.</p

Correction of transfer functions <i>CTF</i> and <i>MTF</i> in cryo-electron tomography

Die Strukturaufklärung von makromolekularen Komplexen und Membranproteinen in ihrer natürlichen Umgebung ist eine große Herausforderung der Strukturbiologie. Hervorragend zur Lösung dieser Aufgabe geeignet ist die Kryoelektronentomographie (KET), die es ermöglicht, subzelluläre Strukturen direkt in ihrer zellulären Umgebung abzubilden. Durch die zusätzliche Anwendung von Subtomogrammalignierung und -mittelung können Elektronendichten von Proteinkomplexen mit deutlich erhöhten Signal-Rausch-Verhältnissen rekonstruiert werden. Die gegenwärtig erreichbare Auflösung hängt jedoch deutlich von der Genauigkeit ab, mit der die Kontrasttransferfunktion (Contrast transfer function, CTF) des Mikroskops und die Modulationstransferfunktion (Modulation transfer function, MTF) des Detektors bestimmt und anschließend korrigiert werden können. Das vorrangige Ziel dieser Arbeit war die Entwicklung und Validierung einer Methode zur Korrektur dieser Transferfunktionen mit hoher Genauigkeit, um die Auflösungsbegrenzung der KET in Kombination mit Subtomogrammalignierung zu umgehen. Zur Bestimmung und Korrektur der CTF wurde ein neuer Ansatz entwickelt, der auf einem erweiterten Bildaufnahmeschema basiert. Dabei werden mit hoher Elektronendosis zwei zusätzliche Bilder aufgenommen, die diametral entlang der Kippachse räumlich entfernt von der Aufnahmestelle des Objekts, um Strahlenschaden zu vermeiden, platziert werden. Durch die hohe Elektronendosis wird das Signal dieser Bilder verstärkt, so dass die CTF extrahiert und der Defokus genau bestimmt werden kann. Diese Defokuswerte werden schließlich zur Interpolation des Defokus an der Aufnahmestelle und zur CTF Korrektur verwendet. Zusätzlich wurde die Korrektur der MTF in den Prozess der Subtomogrammalignierung eingebunden. Diese neue Methode verhindert, auf der Ebene der Subtomogramme, eine zu hohe Verstärkung des Rauschanteils. Zur Validierung der Methode wurde das Porin MspA von Mycobacterium smegmatis eingesetzt. Durch die Anwendung von KET und Subtomogrammalignierung auf MspA, rekonstituiert in Lipidvesikel, war es möglich, eine dreidimensionale Rekonstruktion des Porins in einer nahezu natürlichen Umgebung zu erhalten. Trotz seiner geringen Molekülmasse von 160 kDa und dem geringem Kontrastunterschied zwischen Protein und Lipidmembran wurde eine Auflösung von etwa 12 Å (Fourier-shell correlation nach dem 0.5 Kriterium) erzielt.Elucidating the structure of macromolecular complexes and membrane proteins in their native environment is a major challenge of structural biology. Cryo-electron tomography (CET) is singular suited to solve this task with its potential to image sub-cellular structures directly in their cellular environment. Additionally, sub-tomogram alignment and averaging can be applied, resulting in electron density maps of protein complexes with intelligibly increased signal to noise ratios. However, the currently achievable resolution depends clearly on the accuracy with which the contrast transfer function (CTF) of the microscope and the modulation transfer function (MTF) of the detector can be determined and subsequently be corrected for. The major objective of this work was the development and validation of a method to correct for these transfer functions with high precision in order to overcome the resolution restriction of CET in combination with sub-tomogram alignment. For CTF determination and correction a new approach was developed, based on an extended image acquisition scheme. It adds to the acquisition protocol two high-dose electron micrographs, which are placed diametrically oriented along the tilt-axis spatially separated from the exposure position of the object, in order to avoid beam damage. The high electron dose used for these micrographs generates an increased signal, suitable for CTF extraction and accurate defocus determination. Finally these defocus values are used to interpolate the defocus at the exposure position and correct for the CTF. Additionally, MTF correction was integrated into the sub-tomogram alignment process. This novel procedure avoids the over-amplification of noise on the sub-tomogram level. For validation of the method the porin MspA of Mycobacterium smegmatis was chosen for analysis. By applying CET and sub-tomogram alignment to MspA, reconstituted in lipid vesicles, it was possible to obtain a three-dimensional reconstruction of this porin in its close-to-native environment. Despite its low molecular mass of 160 kDa and the minute contrast difference between protein and lipid membrane a resolution of approximately 12 Å (Fourier-shell correlation using the 0.5 criterion) was achieved

Structural Analysis of Supramolecular Assemblies by Cryo-Electron Tomography

Structural analysis of macromolecular assemblies in their physiological environment is a challenging task that is instrumental in answering fundamental questions in cellular and molecular structural biology. The continuous development of computational and analytical tools for cryo-electron tomography (cryo-ET) enables the study of these assemblies at a resolution of a few nanometers. Through the implementation of thinning procedures, cryo-ET can now be applied to the reconstruction of macromolecular structures located inside thick regions of vitrified cells and tissues, thus becoming a central tool for structural determinations in various biological disciplines. Here, we focus on the successful in situ applications of cryo-ET to reveal structures of macromolecular complexes within eukaryotic cells

Adenoviral vector with shield and adapter increases tumor specificity and escapes liver and immune control

Most systemic viral gene therapies have been limited by sequestration and degradation of virions, innate and adaptive immunity, and silencing of therapeutic genes within the target cells. Here we engineer a high-affinity protein coat, shielding the most commonly used vector in clinical gene therapy, human adenovirus type 5. Using electron microscopy and crystallography we demonstrate a massive coverage of the virion surface through the hexon-shielding scFv fragment, trimerized to exploit the hexon symmetry and gain avidity. The shield reduces virion clearance in the liver. When the shielded particles are equipped with adaptor proteins, the virions deliver their payload genes into human cancer cells expressing HER2 or EGFR. The combination of shield and adapter also increases viral gene delivery to xenografted tumors in vivo, reduces liver off-targeting and immune neutralization. Our study highlights the power of protein engineering for viral vectors overcoming the challenges of local and systemic viral gene therapies

Insights into the gate of the nuclear pore complex

Nuclear pore complexes (NPCs) serve as the gateway of the cell nucleus. These macromolecular assemblies form selective aqueous translocation channels permitting the free diffusion of small molecules, as well as receptor-mediated transport of large cargoes. Over the past decade, major progress has been made in both the structural determination of individual nucleoporins and subcomplexes by X-ray crystallography and in the structural analysis of the entire NPC by cryo-electron tomography (cryo-ET). The metazoan NPC structure from Xenopus laevis oocytes was recently resolved up to 20 Å by combining cryo-ET with advanced image processing techniques, revealing for the first time the architecture of the central channel. Here, we discuss the structure of the Xenopus laevis NPC and consider future perspectives that will eventually allow reconstructing the scaffold and gate of the NPC with higher resolution and identifying its transport-relevant regions. This will eventually allow us to describe the structure of the NPC 'in action'