The Ribosome - Three-Dimensional Structure and Ligand-Binding Studies

To date, cryo-electron microscopy has become the most successful technique for exploring the structure of the ribosome and for studying binding positions of its various ligands, with the resolution slowly extending toward 10 Å. Obstacles in the attempts to improve resolution are the limited stability and coherence of the electron microscope, the statistics of data collection, and the conformational heterogeneity of the specimen. The last factor in this list proved to be the reason why it has been difficult to go past 18-20 Å with many specimens despite the use of state-of-the-art electron microscopes and inclusion of tens of thousand of projections.A breakthrough has been achieved with a protein synthesis initiation-like complex in which mRNA and fMet-tRNA is bound to the E. coli ribosome. The high occupancy and extraordinary conformational homogeneity of this specimen has enabled us to reach a resolution of 15 Å.

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High Resolution Structure of the Mature Capsid of Ralstonia Solanacearum Bacteriophage ϕRSA1 by Cryo-Electron Microscopy

International Journal of Molecular Sciences ◽

10.3390/ijms222011053 ◽

2021 ◽

Vol 22 (20) ◽

pp. 11053

Author(s):

Grégory Effantin ◽

Akiko Fujiwara ◽

Takeru Kawasaki ◽

Takashi Yamada ◽

Guy Schoehn

Keyword(s):

Electron Microscopy ◽

Ralstonia Solanacearum ◽

Three Dimensional ◽

Dimensional Structure ◽

Icosahedral Symmetry ◽

E Coli ◽

Covalent Crosslinking ◽

Cryo Electron Microscopy ◽

Capsid Shell ◽

High Resolution Structure

The ϕRSA1 bacteriophage has been isolated from Ralstonia solanacearum, a gram negative bacteria having a significant economic impact on many important crops. We solved the three-dimensional structure of the ϕRSA1 mature capsid to 3.9 Å resolution by cryo-electron microscopy. The capsid shell, that contains the 39 kbp of dsDNA genome, has an icosahedral symmetry characterized by an unusual triangulation number of T = 7, dextro. The ϕRSA1 capsid is composed solely of the polymerization of the major capsid protein, gp8, which exhibits the typical “Johnson” fold first characterized in E. coli bacteriophage HK97. As opposed to the latter, the ϕRSA1 mature capsid is not stabilized by covalent crosslinking between its subunits, nor by the addition of a decoration protein. We further describe the molecular interactions occurring between the subunits of the ϕRSA1 capsid and their relationships with the other known bacteriophages.

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Structural Role of rRNAs on the Ribosomal Subunits from E. Coli by Scanning Transmission Electron Microscopy

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100098629 ◽

1981 ◽

Vol 39 ◽

pp. 424-425

Author(s):

M. Boublik ◽

N. Robakis ◽

J.S. Wall

Keyword(s):

Three Dimensional ◽

Uranyl Acetate ◽

Dimensional Structure ◽

Electron Microscopic ◽

Ribosomal Subunits ◽

E Coli ◽

Freeze Dried ◽

16S Rna ◽

Scanning Transmission ◽

And Function

The three-dimensional structure and function of biological supramolecular complexes are, in general, determined and stabilized by conformation and interactions of their macromolecular components. In the case of ribosomes, it has been suggested that one of the functions of ribosomal RNAs is to act as a scaffold maintaining the shape of the ribosomal subunits. In order to investigate this question, we have conducted a comparative TEM and STEM study of the structure of the small 30S subunit of E. coli and its 16S RNA.The conventional electron microscopic imaging of nucleic acids is performed by spreading them in the presence of protein or detergent; the particles are contrasted by electron dense solution (uranyl acetate) or by shadowing with metal (tungsten). By using the STEM on freeze-dried specimens we have avoided the shearing forces of the spreading, and minimized both the collapse of rRNA due to air drying and the loss of resolution due to staining or shadowing. Figure 1, is a conventional (TEM) electron micrograph of 30S E. coli subunits contrasted with uranyl acetate.

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

Microscopy and Microanalysis ◽

10.1017/s1431927613013937 ◽

2013 ◽

Vol 20 (1) ◽

pp. 164-174 ◽

Cited By ~ 10

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.

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Adhesion, Invasion, and Agglutination Mediated by Two Trimeric Autotransporters in the Human Uropathogen Proteus mirabilis

Infection and Immunity ◽

10.1128/iai.00718-10 ◽

2010 ◽

Vol 78 (11) ◽

pp. 4882-4894 ◽

Cited By ~ 28

Author(s):

Praveen Alamuri ◽

Martin Löwer ◽

Jan A. Hiss ◽

Stephanie D. Himpsl ◽

Gisbert Schneider ◽

...

Keyword(s):

Proteus Mirabilis ◽

Three Dimensional ◽

Beta Sheets ◽

Surface Proteins ◽

Matrix Proteins ◽

Dimensional Structure ◽

Passenger Domain ◽

Cell Monolayers ◽

E Coli ◽

Surface Adhesins

ABSTRACT Fimbriae of the human uropathogen Proteus mirabilis are the only characterized surface proteins that contribute to its virulence by mediating adhesion and invasion of the uroepithelia. PMI2122 (AipA) and PMI2575 (TaaP) are annotated in the genome of strain HI4320 as trimeric autotransporters with “adhesin-like” and “agglutinating adhesin-like” properties, respectively. The C-terminal 62 amino acids (aa) in AipA and 76 aa in TaaP are homologous to the translocator domains of YadA from Yersinia enterocolitica and Hia from Haemophilus influenzae. Comparative protein modeling using the Hia three-dimensional structure as a template predicted that each of these domains would contain four antiparallel beta sheets and that they formed homotrimers. Recombinant AipA and TaaP were seen as ∼28 kDa and ∼78 kDa, respectively, in Escherichia coli, and each also formed high-molecular-weight homotrimers, thus supporting this model. E. coli synthesizing AipA or TaaP bound to extracellular matrix proteins with a 10- to 60-fold-higher level of affinity than the control strain. Inactivation of aipA in P. mirabilis strains significantly (P < 0.01) reduced the mutants' ability to adhere to or invade HEK293 cell monolayers, and the functions were restored upon complementation. A 51-aa-long invasin region in the AipA passenger domain was required for this function. E. coli expressing TaaP mediated autoagglutination, and a taaP mutant of P. mirabilis showed significantly (P < 0.05) more reduced aggregation than HI4320. Gly-247 in AipA and Gly-708 in TaaP were indispensable for trimerization and activity. AipA and TaaP individually offered advantages to P. mirabilis in a murine model. This is the first report characterizing trimeric autotransporters in P. mirabilis as afimbrial surface adhesins and autoagglutinins.

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RNA minihelices as model substrates for ATP/CTP:tRNA nucleotidyltransferase

Biochemical Journal ◽

10.1042/bj3270847 ◽

1997 ◽

Vol 327 (3) ◽

pp. 847-851 ◽

Cited By ~ 16

Author(s):

Zengji LI ◽

Yue SUN ◽

L. David THURLOW

Keyword(s):

Escherichia Coli ◽

Saccharomyces Cerevisiae ◽

Three Dimensional ◽

Dimensional Structure ◽

Single Base ◽

Three Dimensional Structure ◽

E Coli ◽

Base Identity

Twenty-one RNA minihelices, resembling the coaxially stacked acceptor- /T-stems and T-loop found along the top of a tRNA's three-dimensional structure, were synthesized and used as substrates for ATP/CTP:tRNA nucleotidyltransferases from Escherichia coli and Saccharomyces cerevisiae. The sequence of nucleotides in the loop varied at positions corresponding to residues 56, 57 and 58 in the T-loop of a tRNA. All minihelices were substrates for both enzymes, and the identity of bases in the loop affected the interaction. In general, RNAs with purines in the loop were better substrates than those with pyrimidines, although no single base identity absolutely determined the effectiveness of the RNA as substrate. RNAs lacking bases near the 5ʹ-end were good substrates for the E. coli enzyme, but were poor substrates for that from yeast. The apparent Km values for selected minihelices were 2-3 times that for natural tRNA, and values for apparent Vmax were lowered 5-10-fold.

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Chimeric Nectin1-Poliovirus Receptor Molecules Identify a Nectin1 Region Functional in Herpes Simplex Virus Entry

Journal of Virology ◽

10.1128/jvi.75.17.7987-7994.2001 ◽

2001 ◽

Vol 75 (17) ◽

pp. 7987-7994 ◽

Cited By ~ 27

Author(s):

Francesca Cocchi ◽

Marc Lopez ◽

Patrice Dubreuil ◽

Gabriella Campadelli Fiume ◽

Laura Menotti

Keyword(s):

Herpes Simplex Virus ◽

Herpes Simplex ◽

Virus Entry ◽

Three Dimensional ◽

Dimensional Structure ◽

Immunoglobulin Superfamily ◽

Binding Studies ◽

Entry Site ◽

Poliovirus Receptor ◽

Simplex Virus

ABSTRACT Human nectin1 (hNectin1), an adhesion molecule belonging to the nectin family of the immunoglobulin superfamily, mediates entry of herpes simplex virus (HSV) into cells. The hNectin1 domain that mediates virus entry into cells and also binds glycoprotein D (gD) has been localized to the first N-terminal V-type domain. The poliovirus receptor (PVR) is a structural homolog to nectins, but it cannot function as an HSV entry receptor. hNectin1-PVR chimeras were constructed to functionally locate the site on hNectin1 involved in HSV entry (HSV entry site). The epitope recognized by monoclonal antibody (MAb) R1.302, which is able to block HSV entry, was also located. The chimeric receptors were designed to preserve the overall structure of the V domain. The HSV entry activity mapped entirely to the hNectin1 portion located between residues 64 and 94 (64-94), likely to encode the C, C′, and C" β-strands and intervening loops. In turn, this site consisted of two portions: one with low-level basal activity for HSV entry (77-94), and one immediately upstream (residues 64 to 76) which greatly enhanced the HSV entry activity of the downstream region. The gD-binding site mapped substantially to the same site, whereas the MAb R1.302 epitope also required a further downstream portion (95-102). The involvement of the 64-76 portion is at difference with previous indirect mapping results that were based on competitive binding studies (C. Krummenacher et al., J. Virol. 74:10863–10872, 2000). The A, A′, B, D, E, F, and G β-strands and intervening loops did not appear to play any role in HSV entry. According to the predicted three-dimensional structure of PVR, the C C′ C" site is located peripherally in the V domain and very likely represents an accessible portion at the cell surface.

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