scholarly journals Ex vivo mammalian prions are formed of paired double helical prion protein fibrils

Open Biology ◽  
2016 ◽  
Vol 6 (5) ◽  
pp. 160035 ◽  
Author(s):  
Cassandra Terry ◽  
Adam Wenborn ◽  
Nathalie Gros ◽  
Jessica Sells ◽  
Susan Joiner ◽  
...  
Keyword(s):  
Prion Protein ◽  
Ex Vivo ◽  
Electron Tomography ◽  
Three Dimensional ◽  
High Titre ◽  
Dimensional Structure ◽  
Structural Basis ◽  
Physical Relationship ◽  
Prion Infectivity ◽  
Mammalian Prions

Mammalian prions are hypothesized to be fibrillar or amyloid forms of prion protein (PrP), but structures observed to date have not been definitively correlated with infectivity and the three-dimensional structure of infectious prions has remained obscure. Recently, we developed novel methods to obtain exceptionally pure preparations of prions from mouse brain and showed that pathogenic PrP in these high-titre preparations is assembled into rod-like assemblies. Here, we have used precise cell culture-based prion infectivity assays to define the physical relationship between the PrP rods and prion infectivity and have used electron tomography to define their architecture. We show that infectious PrP rods isolated from multiple prion strains have a common hierarchical assembly comprising twisted pairs of short fibres with repeating substructure. The architecture of the PrP rods provides a new structural basis for understanding prion infectivity and can explain the inability to systematically generate high-titre synthetic prions from recombinant PrP.

scholarly journals Structural insights into the cause of human RSPH4A primary ciliary dyskinesia

2021 ◽  
pp. mbc.E20-12-0806
Author(s):  
Yanhe Zhao ◽  
Justine Pinskey ◽  
Jianfeng Lin ◽  
Weining Yin ◽  
Patrick R. Sears ◽  
...  
Keyword(s):  
Primary Ciliary Dyskinesia ◽  
Electron Tomography ◽  
Three Dimensional ◽  
Dimensional Structure ◽  
Structural Basis ◽  
Radial Spoke ◽  
Radial Spokes ◽  
Cilia And Flagella ◽  
Tract Infections ◽  
Ciliary Dyskinesia

Cilia and flagella are evolutionarily conserved eukaryotic organelles involved in cell motility and signaling. In humans, mutations in Radial Spoke Head Protein 4 homolog A ( RSPH4A) can lead to primary ciliary dyskinesia (PCD), a life-shortening disease characterized by chronic respiratory tract infections, abnormal organ positioning, and infertility. Despite its importance for human health, the location of RSPH4A in human cilia has not been resolved, and the structural basis of RSPH4A-/- PCD remains elusive. Here, we present the native, three-dimensional structure of RSPH4A-/- human respiratory cilia using samples collected non-invasively from a PCD patient. Using cryo-electron tomography and subtomogram averaging, we compared the structures of control and RSPH4A-/- cilia, revealing primary defects in two of the three radial spokes (RSs) within the axonemal repeat and secondary (heterogeneous) defects in the central pair complex. Similar to RSPH1-/- cilia, the radial spoke heads of RS1 and RS2, but not RS3, were missing in RSPH4A-/- cilia. However, RSPH4A-/- cilia also exhibited defects within the arch domains adjacent to the RS1 and RS2 heads, which were not observed with RSPH1 loss. Our results provide insight into the underlying structural basis for RSPH4A-/- PCD and highlight the benefits of applying cryo-ET directly to patient samples for molecular structure determination. [Media: see text]

scholarly journals A systematic investigation of production of synthetic prions from recombinant prion protein

Open Biology ◽  
2015 ◽  
Vol 5 (12) ◽  
pp. 150165 ◽  
Author(s):  
Christian Schmidt ◽  
Jeremie Fizet ◽  
Francesca Properzi ◽  
Mark Batchelor ◽  
Malin K. Sandberg ◽  
...  
Keyword(s):  
Prion Protein ◽  
Ex Vivo ◽  
Biological Properties ◽  
Systematic Investigation ◽  
High Titre ◽  
Cellular Prion Protein ◽  
Structural Basis ◽  
Recombinant Mouse ◽  
Infectious Titre ◽  
Recombinant Prion Protein

According to the protein-only hypothesis, infectious mammalian prions, which exist as distinct strains with discrete biological properties, consist of multichain assemblies of misfolded cellular prion protein (PrP). A critical test would be to produce prion strains synthetically from defined components. Crucially, high-titre ‘synthetic' prions could then be used to determine the structural basis of infectivity and strain diversity at the atomic level. While there have been multiple reports of production of prions from bacterially expressed recombinant PrP using various methods, systematic production of high-titre material in a form suitable for structural analysis remains a key goal. Here, we report a novel high-throughput strategy for exploring a matrix of conditions, additives and potential cofactors that might generate high-titre prions from recombinant mouse PrP, with screening for infectivity using a sensitive automated cell-based bioassay. Overall, approximately 20 000 unique conditions were examined. While some resulted in apparently infected cell cultures, this was transient and not reproducible. We also adapted published methods that reported production of synthetic prions from recombinant hamster PrP, but again did not find evidence of significant infectious titre when using recombinant mouse PrP as substrate. Collectively, our findings are consistent with the formation of prion infectivity from recombinant mouse PrP being a rare stochastic event and we conclude that systematic generation of prions from recombinant PrP may only become possible once the detailed structure of authentic ex vivo prions is solved.

scholarly journals Capturing Enveloped Viruses on Affinity Grids for Downstream Cryo-Electron Microscopy Applications

2013 ◽  
Vol 20 (1) ◽  
pp. 164-174 ◽  
Author(s):  
Gabriella Kiss ◽  
Xuemin Chen ◽  
Melinda A. Brindley ◽  
Patricia Campbell ◽  
Claudio L. Afonso ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Measles Virus ◽  
Influenza A ◽  
Electron Tomography ◽  
Three Dimensional ◽  
Nitrilotriacetic Acid ◽  
Dimensional Structure ◽  
Enveloped Viruses ◽  
Cryo Electron Microscopy ◽  
Human Immunodeficiency Virus Virus

AbstractElectron microscopy (EM), cryo-electron microscopy (cryo-EM), and cryo-electron tomography (cryo-ET) are essential techniques used for characterizing basic virus morphology and determining the three-dimensional structure of viruses. Enveloped viruses, which contain an outer lipoprotein coat, constitute the largest group of pathogenic viruses to humans. The purification of enveloped viruses from cell culture presents certain challenges. Specifically, the inclusion of host-membrane-derived vesicles, the complete destruction of the viruses, and the disruption of the internal architecture of individual virus particles. Here, we present a strategy for capturing enveloped viruses on affinity grids (AG) for use in both conventional EM and cryo-EM/ET applications. We examined the utility of AG for the selective capture of human immunodeficiency virus virus-like particles, influenza A, and measles virus. We applied nickel-nitrilotriacetic acid lipid layers in combination with molecular adaptors to selectively adhere the viruses to the AG surface. This further development of the AG method may prove essential for the gentle and selective purification of enveloped viruses directly onto EM grids for ultrastructural analyses.

scholarly journals High-resolution three-dimensional probes of biomaterials and their interfaces

2012 ◽  
Vol 370 (1963) ◽  
pp. 1337-1351 ◽  
Author(s):  
Kathryn Grandfield ◽  
Anders Palmquist ◽  
Håkan Engqvist
Keyword(s):  
High Resolution ◽  
Electron Tomography ◽  
Three Dimensional ◽  
Controlled Drug Release ◽  
Biological Tissues ◽  
Dimensional Structure ◽  
Bone Interface ◽  
Biomaterial Interfaces ◽  
Titania Coating

Interfacial relationships between biomaterials and tissues strongly influence the success of implant materials and their long-term functionality. Owing to the inhomogeneity of biological tissues at an interface, in particular bone tissue, two-dimensional images often lack detail on the interfacial morphological complexity. Furthermore, the increasing use of nanotechnology in the design and production of biomaterials demands characterization techniques on a similar length scale. Electron tomography (ET) can meet these challenges by enabling high-resolution three-dimensional imaging of biomaterial interfaces. In this article, we review the fundamentals of ET and highlight its recent applications in probing the three-dimensional structure of bioceramics and their interfaces, with particular focus on the hydroxyapatite–bone interface, titanium dioxide–bone interface and a mesoporous titania coating for controlled drug release.

scholarly journals A guided approach for subtomogram averaging of challenging macromolecular assemblies

2020 ◽  
Author(s):  
Danielle Grotjahn ◽  
Saikat Chowdhury ◽  
Gabriel C. Lander
Keyword(s):  
Electron Tomography ◽  
A Priori ◽  
Three Dimensional ◽  
Structural Heterogeneity ◽  
Dimensional Structure ◽  
Diverse Range ◽  
Macromolecular Assemblies ◽  
Cryo Electron Tomography ◽  
Subtomogram Averaging

AbstractCryo-electron tomography is a powerful biophysical technique enabling three-dimensional visualization of complex biological systems. Macromolecular targets of interest identified within cryo-tomograms can be computationally extracted, aligned, and averaged to produce a better-resolved structure through a process called subtomogram averaging (STA). However, accurate alignment of macromolecular machines that exhibit extreme structural heterogeneity and conformational flexibility remains a significant challenge with conventional STA approaches. To expand the applicability of STA to a broader range of pleomorphic complexes, we developed a user-guided, focused refinement approach that can be incorporated into the standard STA workflow to facilitate the robust alignment of particularly challenging samples. We demonstrate that it is possible to align visually recognizable portions of multi-subunit complexes by providing a priori information regarding their relative orientations within cryo-tomograms, and describe how this strategy was applied to successfully elucidate the first three-dimensional structure of the dynein-dynactin motor protein complex bound to microtubules. Our approach expands the application of STA for solving a more diverse range of heterogeneous biological structures, and establishes a conceptual framework for the development of automated strategies to deconvolve the complexity of crowded cellular environments and improve in situ structure determination technologies.

scholarly journals Cryo-Electron Tomography Reveals the Comparative Three-Dimensional Architecture of Prochlorococcus, a Globally Important Marine Cyanobacterium

2007 ◽  
Vol 189 (12) ◽  
pp. 4485-4493 ◽  
Author(s):  
Claire S. Ting ◽  
Chyongere Hsieh ◽  
Sesh Sundararaman ◽  
Carmen Mannella ◽  
Michael Marko
Keyword(s):  
Cell Wall ◽  
Electron Tomography ◽  
Three Dimensional ◽  
Membrane System ◽  
Niche Differentiation ◽  
Dimensional Structure ◽  
Comparative Genomic ◽  
Photosynthetic Membrane ◽  
Peptidoglycan Biosynthesis ◽  
Cryo Electron Tomography

ABSTRACT In an age of comparative microbial genomics, knowledge of the near-native architecture of microorganisms is essential for achieving an integrative understanding of physiology and function. We characterized and compared the three-dimensional architecture of the ecologically important cyanobacterium Prochlorococcus in a near-native state using cryo-electron tomography and found that closely related strains have diverged substantially in cellular organization and structure. By visualizing native, hydrated structures within cells, we discovered that the MED4 strain, which possesses one of the smallest genomes (1.66 Mbp) of any known photosynthetic organism, has evolved a comparatively streamlined cellular architecture. This strain possesses a smaller cell volume, an attenuated cell wall, and less extensive intracytoplasmic (photosynthetic) membrane system compared to the more deeply branched MIT9313 strain. Comparative genomic analyses indicate that differences have evolved in key structural genes, including those encoding enzymes involved in cell wall peptidoglycan biosynthesis. Although both strains possess carboxysomes that are polygonal and cluster in the central cytoplasm, the carboxysomes of MED4 are smaller. A streamlined cellular structure could be advantageous to microorganisms thriving in the low-nutrient conditions characteristic of large regions of the open ocean and thus have consequences for ecological niche differentiation. Through cryo-electron tomography we visualized, for the first time, the three-dimensional structure of the extensive network of photosynthetic lamellae within Prochlorococcus and the potential pathways for intracellular and intermembrane movement of molecules. Comparative information on the near-native structure of microorganisms is an important and necessary component of exploring microbial diversity and understanding its consequences for function and ecology.

scholarly journals Structural Basis for Env Incorporation into HIV-1 Particles

Proceedings ◽  
2020 ◽  
Vol 50 (1) ◽  
pp. 114
Author(s):  
R. Elliot Murphy ◽  
Alexandra B. Samal ◽  
Gunnar Eastep ◽  
Ruba H. Ghanam ◽  
Peter E. Prevelige ◽  
...  
Keyword(s):  
Structural Characterization ◽  
Late Phase ◽  
Three Dimensional ◽  
Dimensional Structure ◽  
Structural Basis ◽  
Membrane Interactions ◽  
Env Protein ◽  
Gag Assembly ◽  
Hiv 1

During the late phase of the HIV-1 replication cycle, the Gag polyproteins are transported to the plasma membrane (PM) for assembly. Gag targeting and assembly on the PM is dependent on interactions between its matrix (MA) domain and phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2). Subsequent to Gag assembly, the envelope (Env) protein is recruited to the PM for incorporation into virus particles. Evidence suggests that the incorporation of the Env protein is mediated by interactions between the MA domain of Gag and the cytoplasmic tail of the gp41 subunit of Env (gp41CT), a mechanism that remains to be elucidated. Trimerization of the MA domain of Gag appears to be an obligatory step for this interaction. The interplay between gp41CT, the MA trimer, and the membrane has yet to be determined. Our lab has pioneered methods and approaches to investigate, at the molecular level, how the retroviral MA domains of Gag interact with membranes, a key requirement for understanding the Gag assembly and Env incorporation. Herein, we devised innovative approaches that will enable the structural characterization of the gp41CT–MA–membrane interactions. We employed structural biology (NMR and cryo-electron microscopy, biophysical methods, and biochemical tools to generate a macromolecular picture of how the MA domain of Gag binds to the membrane and how it interacts with gp41CT. To this end, we: (i) determined the three-dimensional structure of HIV-1 gp41CT and characterized its interaction with the membrane, (ii) engineered trimeric constructs of gp41CT and the MA to recapitulate the native and functional states of the proteins, and (iii) utilized membrane nanodisc technology to anchor the MA and gp41CT proteins. Our studies will allow for a detailed structural characterization of the gp41CT–MA–membrane interactions, which will advance our knowledge of HIV-1 Gag assembly and Env incorporation.

Rational humanization of the powerful antithrombotic anti-GPIbα antibody: 6B4

2006 ◽  
Vol 96 (11) ◽  
pp. 671-684 ◽  
Author(s):  
Alexandre Fontayne ◽  
Karen Vanhoorelbeke ◽  
Inge Pareyn ◽  
Isabel Van Rompaey ◽  
Muriel Meiring ◽  
...  
Keyword(s):  
Ex Vivo ◽  
Three Dimensional ◽  
Vector System ◽  
Dimensional Structure ◽  
Fab Fragment ◽  
Variable Regions ◽  
Significant Prolongation ◽  
Von Willebrand ◽  
First Time

SummaryFab-fragments of the monoclonal antibody 6B4, raised against human glycoprotein Ibα (GPIbα), have a powerful antithrombotic effect in baboons by blocking the GPIbα binding site for von Willebrand factor (VWF), without significant prolongation of the skin bleeding time. In order to bring this antibody to the clinic,we here humanized for the first time an anti-human GPIbα by variable-domain resurfacing guided by computer modeling. First, the genes coding for the variable regions of the heavy and light chains of 6B4 were cloned and sequenced. Based on this,a three-dimensional structure of the Fv-fragment was constructed by using homology-based modeling, and with this and comparison with antibodies with known structure,”murine” putative immunogenic residues which are exposed, were changed for “human-like” residues. The humanized Fab-fragment, h6B4-Fab, was constructed in the pKaneo vector system, expressed and purified and showed in vitro an unaltered, even slightly higher binding affinity for its antigen than the murine form as determined by different ELISA set-ups and surface plasmon resonance. Finally, injection of doses of 0.1 to 1.5 mg/kg of h6B4-Fab in baboons showed that both pharmacokinetics and ex-vivo bio-activity of the molecule were to a large extent preserved.In conclusion, the method used here to humanize 6B4 by resurfacing resulted in a fully active derivative, which is now ready for further development.

scholarly journals Proposed three-dimensional structure for the cellular prion protein.

1994 ◽  
Vol 91 (15) ◽  
pp. 7139-7143 ◽  
Author(s):  
Z. Huang ◽  
J. M. Gabriel ◽  
M. A. Baldwin ◽  
R. J. Fletterick ◽  
S. B. Prusiner ◽  
...  
Keyword(s):  
Prion Protein ◽  
Three Dimensional ◽  
Cellular Prion Protein ◽  
Dimensional Structure ◽  
Three Dimensional Structure

scholarly journals Crystallization and preliminary crystallographic analysis of human muscle phosphofructokinase, the main regulator of glycolysis

2014 ◽  
Vol 70 (5) ◽  
pp. 578-582 ◽  
Author(s):  
Marco Kloos ◽  
Antje Brüser ◽  
Jürgen Kirchberger ◽  
Torsten Schöneberg ◽  
Norbert Sträter
Keyword(s):  
Three Dimensional ◽  
Crystallographic Analysis ◽  
Human Muscle ◽  
Yeast Cells ◽  
Dimensional Structure ◽  
Structural Basis ◽  
Diffusion Method ◽  
Rabbit Muscle ◽  
Main Regulator ◽  
Purification Protocol

Whereas the three-dimensional structure and the structural basis of the allosteric regulation of prokaryotic 6-phosphofructokinases (Pfks) have been studied in great detail, knowledge of the molecular basis of the allosteric behaviour of the far more complex mammalian Pfks is still very limited. The human muscle isozyme was expressed heterologously in yeast cells and purified using a five-step purification protocol. Protein crystals suitable for diffraction experiments were obtained by the vapour-diffusion method. The crystals belonged to space groupP6222 and diffracted to 6.0 Å resolution. The 3.2 Å resolution structure of rabbit muscle Pfk (rmPfk) was placed into the asymmetric unit and optimized by rigid-body and groupB-factor refinement. Interestingly, the tetrameric enzyme dissociated into a dimer, similar to the situation observed in the structure of rmPfk.

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