scholarly journals A microtubule RELION-based pipeline for cryo-EM image processing

2019 ◽  
Author(s):  
Alexander D. Cook ◽  
Szymon W. Manka ◽  
Su Wang ◽  
Carolyn A. Moores ◽  
Joseph Atherton
Keyword(s):  
Electron Microscopy ◽  
Image Processing ◽  
Structural Similarity ◽  
Geometric Lattice ◽  
Contrast Transfer Function ◽  
Biologically Relevant ◽  
Cryo Electron Microscopy

AbstractMicrotubules are polar filaments built from αβ-tubulin heterodimers that exhibit a range of architectures in vitro and in vivo. Tubulin heterodimers are arranged helically in the microtubule wall but many physiologically relevant architectures exhibit a break in helical symmetry known as the seam. Noisy 2D cryo-electron microscopy projection images of pseudo-helical microtubules therefore depict distinct but highly similar views owing to the high structural similarity of α- and β-tubulin. The determination of the αβ-tubulin register and seam location during image processing is essential for alignment accuracy that enables determination of biologically relevant structures. Here we present a pipeline designed for image processing and high-resolution reconstruction of cryo-electron microscopy microtubule datasets, based in the popular and user-friendly RELION image-processing package, Microtubule RELION-based Pipeline (MiRP). The pipeline uses a combination of supervised classification and prior knowledge about geometric lattice constraints in microtubules to accurately determine microtubule architecture and seam location. The presented method is fast and semi-automated, producing near-atomic resolution reconstructions with test datasets that contain a range of microtubule architectures and binding proteins.AbbreviationsMiRP, Microtubule RELION-based Pipeline; cryo-EM, cryo-electron microscopy; MT, microtubule; CTF, contrast transfer function; PF, protofilament.

scholarly journals Structural basis of prokaryotic ubiquitin-like protein engagement and translocation by the mycobacterial Mpa-proteasome complex

2022 ◽  
Vol 13 (1) ◽  
Author(s):  
Mikhail Kavalchuk ◽  
Ahmad Jomaa ◽  
Andreas U. Müller ◽  
Eilika Weber-Ban
Keyword(s):  
Electron Microscopy ◽  
Mycobacterium Tuberculosis ◽  
Early Stage ◽  
Structural Basis ◽  
Human Pathogen ◽  
Core Particle ◽  
Cryo Electron Microscopy ◽  
Complementary Interactions

AbstractProteasomes are present in eukaryotes, archaea and Actinobacteria, including the human pathogen Mycobacterium tuberculosis, where proteasomal degradation supports persistence inside the host. In mycobacteria and other members of Actinobacteria, prokaryotic ubiquitin-like protein (Pup) serves as a degradation tag post-translationally conjugated to target proteins for their recruitment to the mycobacterial proteasome ATPase (Mpa). Here, we use single-particle cryo-electron microscopy to determine the structure of Mpa in complex with the 20S core particle at an early stage of pupylated substrate recruitment, shedding light on the mechanism of substrate translocation. Two conformational states of Mpa show how substrate is translocated stepwise towards the degradation chamber of the proteasome core particle. We also demonstrate, in vitro and in vivo, the importance of a structural feature in Mpa that allows formation of alternating charge-complementary interactions with the proteasome resulting in radial, rail-guided movements during the ATPase conformational cycle.

scholarly journals The functions of SARS-CoV-2 neutralizing and infection-enhancing antibodies in vitro and in mice and nonhuman primates

2021 ◽  
Author(s):  
Dapeng Li ◽  
Robert J Edwards ◽  
Kartik Manne ◽  
David R. Martinez ◽  
Alexandra Schäfer ◽  
...  
Keyword(s):  
Neutralizing Antibodies ◽  
Nonhuman Primates ◽  
Safety Concern ◽  
Lung Pathology ◽  
Receptor Binding Domain ◽  
Biologically Relevant ◽  
Cryo Electron Microscopy ◽  
History Of

SummarySARS-CoV-2 neutralizing antibodies (NAbs) protect against COVID-19, making them a focus of vaccine design. A safety concern regarding SARS-CoV-2 antibodies is whether they mediate disease enhancement. Here, we isolated potent NAbs against the receptor-binding domain (RBD) and the N-terminal domain (NTD) of SARS-CoV-2 spike protein from individuals with acute or convalescent SARS-CoV-2 or a history of SARS-CoV-1 infection. Cryo-electron microscopy of RBD and NTD antibodies demonstrated function-specific modes of antibody binding. Select RBD NAbs also demonstrated Fc receptor-γ (FcγR)-mediated enhancement of virus infection in vitro, while five non-neutralizing NTD antibodies mediated FcγR-independent in vitro infection enhancement. However, both in vitro neutralizing and infection-enhancing RBD or infection-enhancing NTD antibodies protected from SARS-CoV-2 challenge in non-human primates and mice. One of 30 monkeys infused with enhancing antibodies had lung pathology and bronchoalveolar lavage cytokine evidence suggestive of enhanced disease. Thus, these in vitro assessments of enhanced antibody-mediated infection do not necessarily indicate biologically relevant in vivo infection enhancement.

scholarly journals Supramolecular assembly of the E. coli LdcI upon acid stress

2020 ◽  
Author(s):  
Matthew Jessop ◽  
Clarissa Liesche ◽  
Jan Felix ◽  
Ambroise Desfosses ◽  
Megghane Baulard ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Acid Stress ◽  
Super Resolution ◽  
Low Ph ◽  
Lysine Decarboxylase ◽  
E Coli ◽  
Cryo Electron Microscopy ◽  
Supramolecular Organisation

AbstractPathogenic and commensal bacteria often have to resist the harsh acidity of the host stomach. The inducible lysine decarboxylase LdcI buffers the cytosol and the local extracellular environment to ensure enterobacterial survival at low pH. Here, we investigate the acid-stress response regulation of E. coli LdcI by combining biochemical and biophysical characterisation with negative stain and cryo-electron microscopy, and wide-field and super-resolution fluorescence imaging. Due to deleterious effects of fluorescent protein fusions on native LdcI decamers, we opt for three-dimensional localisation of nanobody-labelled endogenous wild-type LdcI in acid-stressed E. coli cells, and show that it organises into distinct patches at the cell periphery. Consistent with recent hypotheses that in vivo clustering of metabolic enzymes often reflects their polymerisation as a means of stimulus-induced regulation, we show that LdcI assembles into filaments in vitro at physiologically relevant low pH. We solve the structures of these filaments and of the LdcI decamer formed at neutral pH by cryo-electron microscopy, and reveal the molecular determinants of LdcI polymerisation, confirmed by mutational analysis. Finally, we propose a model for LdcI function inside the enterobacterial cell, providing a structural and mechanistic basis for further investigation of the role of its supramolecular organisation in the acid stress response.Significance statementBacteria possess a sophisticated arsenal of defence mechanisms that allow them to survive in adverse conditions. Adaptation to acid stress and hypoxia is crucial for the enterobacterial transmission in the gastrointestinal tract of their human host. When subjected to low pH, E. coli and many other enterobacteria activate a proton-consuming resistance system based on the acid-stress inducible lysine decarboxylase LdcI. Here we develop generally-applicable tools to uncover the spatial localisation of LdcI inside the cell by super-resolution fluorescence microscopy, and investigate the in vitro supramolecular organisation of this enzyme by cryo-EM. We build on these results to propose a mechanistic model for LdcI function and offer tools for further in vivo investigations.

scholarly journals What macromolecular crystallogenesis tells us – what is needed in the future

IUCrJ ◽  
2017 ◽  
Vol 4 (4) ◽  
pp. 340-349 ◽  
Author(s):  
Richard Giegé
Keyword(s):  
Self Assembly ◽  
Inorganic Materials ◽  
Biological Processes ◽  
X Ray ◽  
Biologically Relevant ◽  
Cellular Processes ◽  
The Future

Crystallogenesis is a longstanding topic that has transformed into a discipline that is mainly focused on the preparation of crystals for practising crystallographers. Although the idiosyncratic features of proteins have to be taken into account, the crystallization of proteins is governed by the same physics as the crystallization of inorganic materials. At present, a diversified panel of crystallization methods adapted to proteins has been validated, and although only a few methods are in current practice, the success rate of crystallization has increased constantly, leading to the determination of ∼105X-ray structures. These structures reveal a huge repertoire of protein folds, but they only cover a restricted part of macromolecular diversity across the tree of life. In the future, crystals representative of missing structures or that will better document the structural dynamics and functional steps underlying biological processes need to be grown. For the pertinent choice of biologically relevant targets, computer-guided analysis of structural databases is needed. From another perspective, crystallization is a self-assembly process that can occur in the bulk of crowded fluids, with crystals being supramolecular assemblies. Life also uses self-assembly and supramolecular processes leading to transient, or less often stable, complexes. An integrated view of supramolecularity implies that proteins crystallizing eitherin vitroorin vivoor participating in cellular processes share common attributes, notably determinants and antideterminants that favour or disfavour their correct or incorrect associations. As a result, underin vivoconditions proteins show a balance between features that favour or disfavour association. If this balance is broken, disorders/diseases occur. Understanding crystallization underin vivoconditions is a challenge for the future. In this quest, the analysis of packing contacts and contacts within oligomers will be crucial in order to decipher the rules governing protein self-assembly and will guide the engineering of novel biomaterials. In a wider perspective, understanding such contacts will open the route towards supramolecular biology and generalized crystallogenesis.

scholarly journals Mapping of TNTs using Correlative Cryo-Electron Microscopy Reveals a Novel Structure

2018 ◽  
Author(s):  
Anna Sartori-Rupp ◽  
Diégo Cordero Cervantes ◽  
Anna Pepe ◽  
Elise Delage ◽  
Karine Gousset ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Tunneling Nanotubes ◽  
Cryo Electron Microscopy ◽  
Novel Structure ◽  
Organelle Transfer ◽  
Structural Identity ◽  
Membrane Bound ◽  
Multicellular Organisms

AbstractThe harmonious orchestration of intercellular communication is essential for multicellular organisms. One mechanism by which cells communicate is through long, actin-rich membranous protrusions, called tunneling nanotubes, that allow for the intercellular transport of various cargoes, including viruses, organelles, and proteins between the cytoplasm of distant cells in vitro and in vivo. Over the last decade, studies have focused on their functional role but information regarding their structure and the differences with other cellular protrusions such as filopodia, is still lacking. Here, we report the structural characterization of tunneling nanotubes using correlative light- and cryo-electron microscopy approaches. We demonstrate their structural identity compared to filopodia by showing that they are comprised of a bundle of functional individual Tunneling Nanotubes containing membrane-bound compartments and allowing organelle transfer.

Specific Labeling of Protein Domains with Antibody Fragments

1981 ◽  
Vol 39 ◽  
pp. 416-417
Author(s):  
U. Aebi ◽  
L.E. Buhle ◽  
W.E. Fowler
Keyword(s):  
Image Processing ◽  
Specific Protein ◽  
Antibody Fragments ◽  
Supramolecular Organization ◽  
Two Dimensional ◽  
Ordered Structures ◽  
Virus Capsids ◽  
Processing Techniques

Many important supramolecular structures such as filaments, microtubules, virus capsids and certain membrane proteins and bacterial cell walls exist as ordered polymers or two-dimensional crystalline arrays in vivo. In several instances it has been possible to induce soluble proteins to form ordered polymers or two-dimensional crystalline arrays in vitro. In both cases a combination of electron microscopy of negatively stained specimens with analog or digital image processing techniques has proven extremely useful for elucidating the molecular and supramolecular organization of the constituent proteins. However from the reconstructed stain exclusion patterns it is often difficult to identify distinct stain excluding regions with specific protein subunits. To this end it has been demonstrated that in some cases this ambiguity can be resolved by a combination of stoichiometric labeling of the ordered structures with subunit-specific antibody fragments (e.g. Fab) and image processing of the electron micrographs recorded from labeled and unlabeled structures.

New challenges to image processing posed by cryo-Electron microscopy of single particles

1991 ◽  
Vol 49 ◽  
pp. 422-423
Author(s):  
Joachim Frank
Keyword(s):  
Electron Microscopy ◽  
Phase Contrast ◽  
Particle Orientation ◽  
Single Particles ◽  
Contrast Transfer Function ◽  
Virtual Absence ◽  
Cryo Electron Microscopy ◽  
Spatial Frequencies ◽  
New Challenges ◽  
Particle Images

Compared with images of negatively stained single particle specimens, those obtained by cryo-electron microscopy have the following new features: (a) higher “signal” variability due to a higher variability of particle orientation; (b) reduced signal/noise ratio (S/N); (c) virtual absence of low-spatial-frequency information related to elastic scattering, due to the properties of the phase contrast transfer function (PCTF); and (d) reduced resolution due to the efforts of the microscopist to boost the PCTF at low spatial frequencies, in his attempt to obtain recognizable particle images.

Enhanced information from biological materials through technological improvements in cryo-electron microscopy

1992 ◽  
Vol 50 (1) ◽  
pp. 788-789
Author(s):  
Marc J.C. de Jong ◽  
Wim M. Busing ◽  
Max T. Otten
Keyword(s):  
Electron Microscopy ◽  
Electron Beam ◽  
Biological Materials ◽  
Electron Crystallography ◽  
Cryo Electron Microscopy ◽  
Transmission Electron ◽  
The Many ◽  
Technological Improvements ◽  
Beam Damage

Biological materials damage rapidly in the electron beam, limiting the amount of information that can be obtained in the transmission electron microscope. The discovery that observation at cryo temperatures strongly reduces beam damage (in addition to making it unnecessaiy to use chemical fixatives, dehydration agents and stains, which introduce artefacts) has given an important step forward to preserving the ‘live’ situation and makes it possible to study the relation between function, chemical composition and morphology.Among the many cryo-applications, the most challenging is perhaps the determination of the atomic structure. Henderson and co-workers were able to determine the structure of the purple membrane by electron crystallography, providing an understanding of the membrane's working as a proton pump. As far as understood at present, the main stumbling block in achieving high resolution appears to be a random movement of atoms or molecules in the specimen within a fraction of a second after exposure to the electron beam, which destroys the highest-resolution detail sought.

A Molecular Approach to Immunoscintigraphy: A Study of the T-Antigen Conformation on the Surface of Tumors

1987 ◽  
Vol 26 (01) ◽  
pp. 1-6 ◽  
Author(s):  
S. Selvaraj ◽  
M. R. Suresh ◽  
G. McLean ◽  
D. Willans ◽  
C. Turner ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Tumor Cell ◽  
T Antigen ◽  
Human Carcinomas ◽  
Animal Tumor ◽  
Synthetic Antigens

The role of glycoconjugates in tumor cell differentiation has been well documented. We have examined the expression of the two anomers of the Thomsen-Friedenreich antigen on the surface of human, canine and murine tumor cell membranes both in vitro and in vivo. This has been accomplished through the synthesis of the disaccharide terminal residues in both a and ß configuration. Both entities were used to generate murine monoclonal antibodies which recognized the carbohydrate determinants. The determination of fine specificities of these antibodies was effected by means of cellular uptake, immunohistopathology and immunoscintigraphy. Examination of pathological specimens of human and canine tumor tissue indicated that the expressed antigen was in the β configuration. More than 89% of all human carcinomas tested expressed the antigen in the above anomeric form. The combination of synthetic antigens and monoclonal antibodies raised specifically against them provide us with invaluable tools for the study of tumor marker expression in humans and their respective animal tumor models.

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