Structural Analysis of gp6 and gp6/gpl5/gpl6 Complex by Cryo-Electron Microscopy At 10 Å Resolution

2000 ◽  
Vol 6 (S2) ◽  
pp. 266-267
Author(s):  
E.V. Orlova ◽  
R. Lurz ◽  
A. Droege ◽  
B. Gowen ◽  
P. Tavares ◽  
...  
Keyword(s):  
Three Dimensional ◽  
Wild Type ◽  
Viral Dna ◽  
3D Reconstructions ◽  
Portal Protein ◽  
Double Stranded Dna ◽  
Cryo Electron Microscopy ◽  
Phage Tail ◽  
Phage Capsid ◽  
Icosahedral Head

The mechanism of encapsidation of DNA into a bacteriophage head is a most intriguing problem. The portal protein is essential for the assembly of tailed double-stranded DNA (dsDNA) bacteriophages. These turbine-like homo-oligomers consist of 12 or 13 subunits surrounding a central channel. Portal oligomers are located at the vertex of the icosahedral head that binds to the phage tail. It was shown that gp6 portal protein from bacteriophage SPP1 has 13 subunits, prior to their incorporation into the viral procapsid structure. After packaging of the viral DNA inside the phage capsid, additional proteins have been found attached to the portal oligomer: gpl5 and gp 16. A complex of gp6/gpl5/gpl7 forms the connector structure that provides the interface for attachment of the phage tail. We here present the three-dimensional (3D) reconstructions of the gp6 wild-type protein alone and of the gp6/gpl5/gpl6 complex - both at 10Å resolution - based on electron cryo-microscopy using the angular reconstitution single particle methodology.

scholarly journals Hypersusceptibility of Human Cytomegalovirus to Foscarnet Induced by Mutations in Helices K and P of the Viral DNA Polymerase

2020 ◽  
Vol 64 (4) ◽  
Author(s):  
Karima Zarrouk ◽  
Van Dung Pham ◽  
Jocelyne Piret ◽  
Rong Shi ◽  
Guy Boivin
Keyword(s):  
Dna Polymerase ◽  
Human Cytomegalovirus ◽  
Three Dimensional ◽  
Wild Type ◽  
Viral Dna ◽  
Recombinant Viruses ◽  
Closed Conformation ◽  
Three Dimensional Modeling ◽  
Dimensional Modeling ◽  
Modeling Analysis

ABSTRACT Herein, we phenotypically and enzymatically characterize the theoretical mutation Q579I in helix K and the already described clinical mutation K805Q in helix P of cytomegalovirus DNA polymerase for susceptibility to foscarnet. Q579I and K805Q recombinant viruses were hypersusceptible to foscarnet (respective mean 50% effective concentrations [EC50] of 0.12- and 0.19-fold that of the wild type). Three-dimensional modeling analysis suggested that both mutations favor the closed conformation of the enzyme to which foscarnet binds with a higher affinity.

Visualization of co-axially coiled dsDNA in bacteriophage T7 capsids by cryo-electron microscopy

1996 ◽  
Vol 54 ◽  
pp. 68-69
Author(s):  
Naiqian Cheng ◽  
Mario E. Cerritelli ◽  
Alan H. Rosenberg ◽  
Frank P. Booy ◽  
Alasdair C. Steven
Keyword(s):  
Electron Microscopy ◽  
Life Cycle ◽  
Bacteriophage T7 ◽  
Efficient Utilization ◽  
Viral Dna ◽  
Viral Capsids ◽  
Double Stranded Dna ◽  
Cryo Electron Microscopy ◽  
Invaluable Tool ◽  
Repulsive Forces

The packaging of viral DNA into a pre-formed procapsid structure and its subsequent release from the mature virion during infection, constitutes one of the basic phenomena of the viral life-cycle which still remains to be understood. The DNA must package at a high density into the head to allow efficient utilization of the space available, overcome the mutual electrostatic repulsive forces between the strands, and be readily available for release upon infection. Cryo-electron microscopy has proven to be an invaluable tool for visualizing the internal organization of the packaged DNA inside viral capsids. We report on the effectiveness of this technique in examining the packaging of the ∼40,000 bp double-stranded DNA genome inside the capsids of bacteriophage T7.

Cryo-Electron Microscopy of Aura Viruses

1998 ◽  
Vol 4 (S2) ◽  
pp. 946-947
Author(s):  
W. Zhang ◽  
N. H. Olson ◽  
B. R. McKinney ◽  
R. J. Kuhn ◽  
T. S. Baker
Keyword(s):  
Electron Microscopy ◽  
Three Dimensional ◽  
Single Copy ◽  
Wild Type Virus ◽  
Genomic Rna ◽  
Wild Type ◽  
Subgenomic Rna ◽  
Enveloped Viruses ◽  
Virus Particles ◽  
Cryo Electron Microscopy

Alphaviruses are a group of enveloped viruses in the Togaviridae family. Studies of several alphaviruses, including Ross River, Sindbis and Semliki Forest viruses, by cryo-electron microscopy (cryo-EM), three-dimensional (3D) image resconstruction and other techniques have illustrated that these spherical viruses have a T=4, multi-layered structure.Aura virus, which is closely related to Sindbis, was first isolated in South America. Unlike the other alphaviruses, both genomic RNA (12kb, 49S) and subgenomic RNA(4.2kb, 26S) are encapsidated efficiently and form mature virions. Studies on negatively-stained virus particles demonstrated that there are two major size classes. The first contains particles of ∼72nm diameter, which are most similar to wild type virus, whereas the second class includes particles of ∼62nm in diameter. The 72nm particles are believed to have one copy of genomic RNA or one to three copies of subgenomic RNA, and a T=4 structure. The 62nm particles probably only have a single copy of subgenomic RNA and are presumed to be T=3 structures.

Improved 3-D reconstruction using stereo computer graphics and multiple tilt EM images

1995 ◽  
Vol 53 ◽  
pp. 630-631
Author(s):  
Lee D. Peachey ◽  
Lou Fodor ◽  
John C. Haselgrove ◽  
Stanley M. Dunn ◽  
Junqing Huang
Keyword(s):  
Computer Graphics ◽  
High Performance ◽  
Transverse Section ◽  
Three Dimensional ◽  
Stereo Pair ◽  
3D Reconstructions ◽  
Video Cameras ◽  
Biological Objects ◽  
Microscope Images ◽  
High Performance Computer

Stereo pairs of electron microscope images provide valuable visual impressions of the three-dimensional nature of specimens, including biological objects. Beyond this one seeks quantitatively accurate models and measurements of the three dimensional positions and sizes of structures in the specimen. In our laboratory, we have sought to combine high resolution video cameras with high performance computer graphics systems to improve both the ease of building 3D reconstructions and the accuracy of 3D measurements, by using multiple tilt images of the same specimen tilted over a wider range of angles than can be viewed stereoscopically. Ultimately we also wish to automate the reconstruction and measurement process, and have initiated work in that direction.Figure 1 is a stereo pair of 400 kV images from a 1 micrometer thick transverse section of frog skeletal muscle stained with the Golgi stain. This stain selectively increases the density of the transverse tubular network in these muscle cells, and it is this network that we reconstruct in this example.

Two-Dimensional Crystallization of Tetanus Toxin

1985 ◽  
Vol 43 ◽  
pp. 528-529
Author(s):  
Jeffry A. Reidler ◽  
John P. Robinson
Keyword(s):  
Electron Microscopy ◽  
Tetanus Toxin ◽  
Structural Information ◽  
Three Dimensional ◽  
Two Dimensional ◽  
Membrane Interface ◽  
3D Reconstructions ◽  
Cellular Receptors ◽  
Computer Reconstruction ◽  
2D Crystals

We have prepared two-dimensional (2D) crystals of tetanus toxin using procedures developed by Uzgiris and Kornberg for the directed production of 2D crystals of monoclonal antibodies at an antigen-phospholipid monolayer interface. The tetanus toxin crystals were formed using a small mole fraction of the natural receptor, GT1, incorporated into phosphatidyl choline monolayers. The crystals formed at low concentration overnight. Two dimensional crystals of this type are particularly useful for structure determination using electron microscopy and computer image refinement. Three dimensional (3D) structural information can be derived from these crystals by computer reconstruction of photographs of toxin crystals taken at different tilt angles. Such 3D reconstructions may help elucidate the mechanism of entry of the enzymatic subunit of toxins into cells, particularly since these crystals form directly on a membrane interface at similar concentrations of ganglioside GT1 to the natural cellular receptors.

scholarly journals Structural Analysis of Jumbo Coliphage phAPEC6

2020 ◽  
Vol 21 (9) ◽  
pp. 3119 ◽  
Author(s):  
Jeroen Wagemans ◽  
Jessica Tsonos ◽  
Dominique Holtappels ◽  
Kiandro Fortuna ◽  
Jean-Pierre Hernalsteens ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Capsid Protein ◽  
Tem Analysis ◽  
Host Interaction ◽  
Cryo Electron Microscopy ◽  
Transmission Electron ◽  
Tail Sheath ◽  
Associated Proteins ◽  
Contractile Tail ◽  
Phage Capsid

The phAPEC6 genome encodes 551 predicted gene products, with the vast majority (83%) of unknown function. Of these, 62 have been identified as virion-associated proteins by mass spectrometry (ESI-MS/MS), including the major capsid protein (Gp225; present in 1620 copies), which shows a HK97 capsid protein-based fold. Cryo-electron microscopy experiments showed that the 350-kbp DNA molecule of Escherichia coli virus phAPEC6 is packaged in at least 15 concentric layers in the phage capsid. A capsid inner body rod is also present, measuring about 91 nm by 18 nm and oriented along the portal axis. In the phAPEC6 contractile tail, 25 hexameric stacked rings can be distinguished, built of the identified tail sheath protein (Gp277). Cryo-EM reconstruction reveals the base of the unique hairy fibers observed during an initial transmission electron microscopy (TEM) analysis. These very unusual filaments are ordered at three annular positions along the contractile sheath, as well as around the capsid, and may be involved in host interaction.

Ultrasonographic 3D Evaluation in the Diagnosis of Bladder Endometriosis: A Prospective Comparative Diagnostic Accuracy Study

2021 ◽  
pp. 1-8
Author(s):  
Fabio Barra ◽  
Franco Alessandri ◽  
Carolina Scala ◽  
Simone Ferrero
Keyword(s):  
Diagnostic Accuracy ◽  
Three Dimensional ◽  
3D Ultrasound ◽  
Diagnostic Accuracy Study ◽  
Extensive Experience ◽  
Deep Endometriosis ◽  
3D Reconstructions ◽  
Analysis Group ◽  
Bladder Endometriosis ◽  
Accuracy Study

<b><i>Objective:</i></b> The use of three-dimensional (3D) transvaginal ultrasonography (TVS) has been investigated for the diagnosis of deep endometriosis (DE). This study aimed to evaluate if 3D reconstructions improve the performance of TVS) in assessing the presence and characteristics of bladder endometriosis (BE). <b><i>Design:</i></b> This was a single-center comparative diagnostic accuracy study. <b><i>Participants/Materials, Setting, Methods:</i></b> Patients referred to our institution (Piazza della Vittoria 14 Srl, Genova, Italy) with clinical suspicion of DE were included. In case of surgery, women underwent systematic preoperative ultrasonographic imaging; an experienced sonographer performed a conventional TVS; another experienced sonographer, blinded to results of the previous exam, performed TVS, with the addition of 3D modality. The presence and characteristics of BE nodules were described in accord with International DE Analysis group consensus. Ultrasound data were compared with surgical and histological results. <b><i>Results:</i></b> Overall, BE was intraoperatively found in 34 out of 194 women who underwent surgery for DE (17.5%; 95% confidence interval: 12.8–23.5%). TVS without and with 3D reconstructions were able to detect endometriotic BE in 82.2% (<i>n</i> = 28/34) and 85.3% (<i>n</i> = 29/34) of the cases (<i>p</i> = 0.125). Both the exams similarly estimated the largest diameter of BE (<i>p</i> = 0.652) and the distance between the endometriotic nodule and the closest ureteral meatus (<i>p</i> = 0.341). However, TVS with 3D reconstructions was more precise in estimating the volume of BE (<i>p</i> = 0.031). In one case (2.9%), TVS without and with 3D reconstructions detected the infiltration of the intramural ureter, which was confirmed at surgery and required laparoscopic ureterovesical reimplantation. <b><i>Limitations:</i></b> The extensive experience of the gynecologists performing the ultrasonographic scans, the lack of prestudy power analysis, and the population selected, which may have been influenced by the position of the institution as a referral center specialized in the treatment of severe endometriosis, are limitations of the current study. <b><i>Conclusion:</i></b> Our results demonstrated the high accuracy of ultrasound for diagnosing BE. The addition of 3D reconstructions does not improve the performance of TVS in diagnosing the presence and characteristics of BE. However, the volume of BE may be more precisely assessed by 3D ultrasound.

scholarly journals The double-stranded DNA-binding proteins TEBP-1 and TEBP-2 form a telomeric complex with POT-1

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Sabrina Dietz ◽  
Miguel Vasconcelos Almeida ◽  
Emily Nischwitz ◽  
Jan Schreier ◽  
Nikenza Viceconte ◽  
...  
Keyword(s):  
Quantitative Proteomics ◽  
Wild Type ◽  
C Elegans ◽  
Double Stranded Dna ◽  
Telomere Sequence ◽  
Proteomics Approach ◽  
Telomeric Protein ◽  
Regulate Telomere Length

AbstractTelomeres are bound by dedicated proteins, which protect them from DNA damage and regulate telomere length homeostasis. In the nematode Caenorhabditis elegans, a comprehensive understanding of the proteins interacting with the telomere sequence is lacking. Here, we harnessed a quantitative proteomics approach to identify TEBP-1 and TEBP-2, two paralogs expressed in the germline and embryogenesis that associate to telomeres in vitro and in vivo. tebp-1 and tebp-2 mutants display strikingly distinct phenotypes: tebp-1 mutants have longer telomeres than wild-type animals, while tebp-2 mutants display shorter telomeres and a Mortal Germline. Notably, tebp-1;tebp-2 double mutant animals have synthetic sterility, with germlines showing signs of severe mitotic and meiotic arrest. Furthermore, we show that POT-1 forms a telomeric complex with TEBP-1 and TEBP-2, which bridges TEBP-1/-2 with POT-2/MRT-1. These results provide insights into the composition and organization of a telomeric protein complex in C. elegans.

scholarly journals Deciphering the Role of Residues Involved in Disorder-To-Order Transition Regions in Archaeal tRNA Methyltransferase 5

Genes ◽  
2021 ◽  
Vol 12 (3) ◽  
pp. 399
Author(s):  
Ambuj Srivastava ◽  
Dhanusha Yesudhas ◽  
Shandar Ahmad ◽  
M. Michael Gromiha
Keyword(s):  
Three Dimensional ◽  
Md Simulations ◽  
Order Transition ◽  
Free Form ◽  
Wild Type ◽  
Intrinsically Disordered ◽  
Intrinsically Disordered Regions ◽  
Type Complex ◽  
In The Wild ◽  
Trna Methyltransferase

tRNA methyltransferase 5 (Trm5) enzyme is an S-adenosyl methionine (AdoMet)-dependent methyltransferase which methylates the G37 nucleotide at the N1 atom of the tRNA. The free form of Trm5 enzyme has three intrinsically disordered regions, which are highly flexible and lack stable three-dimensional structures. These regions gain ordered structures upon the complex formation with tRNA, also called disorder-to-order transition (DOT) regions. In this study, we performed molecular dynamics (MD) simulations of archaeal Trm5 in free and complex forms and observed that the DOT residues are highly flexible in free proteins and become stable in complex structures. The energetic contributions show that DOT residues are important for stabilising the complex. The DOT1 and DOT2 are mainly observed to be important for stabilising the complex, while DOT3 is present near the active site to coordinate the interactions between methyl-donating ligands and G37 nucleotides. In addition, mutational studies on the Trm5 complex showed that the wild type is more stable than the G37A tRNA mutant complex. The loss of productive interactions upon G37A mutation drives the AdoMet ligand away from the 37th nucleotide, and Arg145 in DOT3 plays a crucial role in stabilising the ligand, as well as the G37 nucleotide, in the wild-type complex. Further, the overall energetic contribution calculated using MMPBSA corroborates that the wild-type complex has a better affinity between Trm5 and tRNA. Overall, our study reveals that targeting DOT regions for binding could improve the inhibition of Trm5.

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