3D structure of three jumbo phage heads

Jumbo phages are bacteriophages that carry more than 200 kbp of DNA. In this study we characterized two jumbo phages (ΦRSL2 and ΦXacN1) and one semi-jumbo phage (ΦRP13) at the structural level by cryo-electron microscopy. Focusing on their capsids, three-dimensional structures of the heads at resolutions ranging from 16 to 9 Å were calculated. Based on these structures we determined the geometrical basis on which the icosahedral capsids of these phages are constructed, which includes the accessory and decorative proteins that complement them. A triangulation number novel to Myoviridae (ΦRP13; T=21) was discovered as well as two others, which are more common for jumbo phages (T=27 and T=28). Based on one of the structures we also provide evidence that accessory or decorative proteins are not a prerequisite for maintaining the structural integrity of very large capsids.

Download Full-text

Two conformations of DNA polymerase D-PCNA-DNA, an archaeal replisome complex, revealed by cryo-electron microscopy

BMC Biology ◽

10.1186/s12915-020-00889-y ◽

2020 ◽

Vol 18 (1) ◽

Author(s):

Kouta Mayanagi ◽

Keisuke Oki ◽

Naoyuki Miyazaki ◽

Sonoko Ishino ◽

Takeshi Yamagami ◽

...

Keyword(s):

Electron Microscopy ◽

Dna Polymerase ◽

Structural Model ◽

Contact Point ◽

Dna Polymerases ◽

3D Structure ◽

Nuclear Antigen ◽

Structural Level ◽

Cryo Electron Microscopy ◽

Dna Complex

Abstract Background DNA polymerase D (PolD) is the representative member of the D family of DNA polymerases. It is an archaea-specific DNA polymerase required for replication and unrelated to other known DNA polymerases. PolD consists of a heterodimer of two subunits, DP1 and DP2, which contain catalytic sites for 3′-5′ editing exonuclease and DNA polymerase activities, respectively, with both proteins being mutually required for the full activities of each enzyme. However, the processivity of the replicase holoenzyme has additionally been shown to be enhanced by the clamp molecule proliferating cell nuclear antigen (PCNA), making it crucial to elucidate the interaction between PolD and PCNA on a structural level for a full understanding of its functional relevance. We present here the 3D structure of a PolD-PCNA-DNA complex from Thermococcus kodakarensis using single-particle cryo-electron microscopy (EM). Results Two distinct forms of the PolD-PCNA-DNA complex were identified by 3D classification analysis. Fitting the reported crystal structures of truncated forms of DP1 and DP2 from Pyrococcus abyssi onto our EM map showed the 3D atomic structural model of PolD-PCNA-DNA. In addition to the canonical interaction between PCNA and PolD via PIP (PCNA-interacting protein)-box motif, we found a new contact point consisting of a glutamate residue at position 171 in a β-hairpin of PCNA, which mediates interactions with DP1 and DP2. The DNA synthesis activity of a mutant PolD with disruption of the E171-mediated PCNA interaction was not stimulated by PCNA in vitro. Conclusions Based on our analyses, we propose that glutamate residues at position 171 in each subunit of the PCNA homotrimer ring can function as hooks to lock PolD conformation on PCNA for conversion of its activity. This hook function of the clamp molecule may be conserved in the three domains of life.

Download Full-text

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.

Download Full-text

Synthesis of Three-Dimensional Fe3O4/Graphene Aerogels for the Removal of Arsenic Ions from Water

Journal of Nanomaterials ◽

10.1155/2015/864864 ◽

2015 ◽

Vol 2015 ◽

pp. 1-6 ◽

Cited By ~ 10

Author(s):

Yan Ye ◽

Da Yin ◽

Bin Wang ◽

Qingwen Zhang

Keyword(s):

Electron Microscopy ◽

Photoelectron Spectroscopy ◽

3D Structure ◽

Three Dimensional ◽

X Ray Diffraction ◽

X Ray ◽

Graphene Aerogels ◽

Transmission Electron ◽

Potential Applications ◽

Removal Of Arsenic

We report the synthesis of three-dimensional Fe3O4/graphene aerogels (GAs) and their application for the removal of arsenic (As) ions from water. The morphology and properties of Fe3O4/GAs have been characterized by scanning electron microscopy, transmission electron microscopy, X-ray diffraction, X-ray photoelectron spectroscopy, and superconducting quantum inference device. The 3D nanostructure shows that iron oxide nanoparticles are decorated on graphene with an interconnected network structure. It is found that Fe3O4/GAs own a capacity of As(V) ions adsorption up to 40.048 mg/g due to their remarkable 3D structure and existence of magnetic Fe3O4nanoparticles for separation. The adsorption isotherm matches well with the Langmuir model and kinetic analysis suggests that the adsorption process is pseudo-second-ordered. In addition to the excellent adsorption capability, Fe3O4/GAs can be easily and effectively separated from water, indicating potential applications in water treatment.

Download Full-text

The Core and Holoenzyme Forms of RNA Polymerase from Mycobacterium smegmatis

Journal of Bacteriology ◽

10.1128/jb.00583-18 ◽

2018 ◽

Vol 201 (4) ◽

Cited By ~ 1

Author(s):

Tomáš Kouba ◽

Jiří Pospíšil ◽

Jarmila Hnilicová ◽

Hana Šanderová ◽

Ivan Barvík ◽

...

Keyword(s):

Electron Microscopy ◽

Rna Polymerase ◽

Conformational Changes ◽

Mycobacterium Smegmatis ◽

Drug Target ◽

Conformational Dynamics ◽

Three Dimensional ◽

Cryo Electron Microscopy ◽

Bacterial Rna ◽

High Degree

ABSTRACT Bacterial RNA polymerase (RNAP) is essential for gene expression and as such is a valid drug target. Hence, it is imperative to know its structure and dynamics. Here, we present two as-yet-unreported forms of Mycobacterium smegmatis RNAP: core and holoenzyme containing σA but no other factors. Each form was detected by cryo-electron microscopy in two major conformations. Comparisons of these structures with known structures of other RNAPs reveal a high degree of conformational flexibility of the mycobacterial enzyme and confirm that region 1.1 of σA is directed into the primary channel of RNAP. Taken together, we describe the conformational changes of unrestrained mycobacterial RNAP. IMPORTANCE We describe here three-dimensional structures of core and holoenzyme forms of mycobacterial RNA polymerase (RNAP) solved by cryo-electron microscopy. These structures fill the thus-far-empty spots in the gallery of the pivotal forms of mycobacterial RNAP and illuminate the extent of conformational dynamics of this enzyme. The presented findings may facilitate future designs of antimycobacterial drugs targeting RNAP.

Download Full-text

Representation Theoretic Patterns in Three-Dimensional Cryo-Electron Microscopy II—The Class Averaging Problem

Foundations of Computational Mathematics ◽

10.1007/s10208-011-9095-3 ◽

2011 ◽

Vol 11 (5) ◽

pp. 589-616 ◽

Cited By ~ 12

Author(s):

Ronny Hadani ◽

Amit Singer

Keyword(s):

Electron Microscopy ◽

Three Dimensional ◽

Cryo Electron Microscopy ◽

Averaging Problem

Download Full-text

Cryomesh™: A New Substrate for Cryo-Electron Microscopy

Microscopy and Microanalysis ◽

10.1017/s1431927609991310 ◽

2010 ◽

Vol 16 (1) ◽

pp. 43-53 ◽

Cited By ~ 31

Author(s):

Craig Yoshioka ◽

Bridget Carragher ◽

Clinton S. Potter

Keyword(s):

Electron Microscopy ◽

Semiconductor Industry ◽

Three Dimensional ◽

Quality Of Data ◽

Temperature Conductivity ◽

Cryo Electron Microscopy ◽

Transmission Electron ◽

Using Data ◽

Collection Methods

AbstractHere we evaluate a new grid substrate developed by ProtoChips Inc. (Raleigh, NC) for cryo-transmission electron microscopy. The new grids are fabricated from doped silicon carbide using processes adapted from the semiconductor industry. A major motivating purpose in the development of these grids was to increase the low-temperature conductivity of the substrate, a characteristic that is thought to affect the appearance of beam-induced movement (BIM) in transmission electron microscope (TEM) images of biological specimens. BIM degrades the quality of data and is especially severe when frozen biological specimens are tilted in the microscope. Our results show that this new substrate does indeed have a significant impact on reducing the appearance and severity of beam-induced movement in TEM images of tilted cryo-preserved samples. Furthermore, while we have not been able to ascertain the exact causes underlying the BIM phenomenon, we have evidence that the rigidity and flatness of these grids may play a major role in its reduction. This improvement in the reliability of imaging at tilt has a significant impact on using data collection methods such as random conical tilt or orthogonal tilt reconstruction with cryo-preserved samples. Reduction in BIM also has the potential for improving the resolution of three-dimensional cryo-reconstructions in general.

Download Full-text

Cryo-electron Microscopy Structures of Novel Viruses from Mud Crab Scylla paramamosain with Multiple Infections

Journal of Virology ◽

10.1128/jvi.02255-18 ◽

2019 ◽

Vol 93 (7) ◽

Cited By ~ 3

Author(s):

Yuanzhu Gao ◽

Shanshan Liu ◽

Jiamiao Huang ◽

Qianqian Wang ◽

Kunpeng Li ◽

...

Keyword(s):

Electron Microscopy ◽

3D Structure ◽

Structural Features ◽

Economic Losses ◽

Scylla Paramamosain ◽

Multiple Infections ◽

Mud Crab ◽

Cryo Electron Microscopy ◽

Novel Virus ◽

Icosahedral Capsid

ABSTRACT Viruses associated with sleeping disease (SD) in crabs cause great economic losses to aquaculture, and no effective measures are available for their prevention. In this study, to help develop novel antiviral strategies, single-particle cryo-electron microscopy was applied to investigate viruses associated with SD. The results not only revealed the structure of mud crab dicistrovirus (MCDV) but also identified a novel mud crab tombus-like virus (MCTV) not previously detected using molecular biology methods. The structure of MCDV at a 3.5-Å resolution reveals three major capsid proteins (VP1 to VP3) organized into a pseudo-T=3 icosahedral capsid, and affirms the existence of VP4. Unusually, MCDV VP3 contains a long C-terminal region and forms a novel protrusion that has not been observed in other dicistrovirus. Our results also reveal that MCDV can release its genome via conformation changes of the protrusions when viral mixtures are heated. The structure of MCTV at a 3.3-Å resolution reveals a T= 3 icosahedral capsid with common features of both tombusviruses and nodaviruses. Furthermore, MCTV has a novel hydrophobic tunnel beneath the 5-fold vertex and 30 dimeric protrusions composed of the P-domains of the capsid protein at the 2-fold axes that are exposed on the virion surface. The structural features of MCTV are consistent with a novel type of virus. IMPORTANCE Pathogen identification is vital for unknown infectious outbreaks, especially for dual or multiple infections. Sleeping disease (SD) in crabs causes great economic losses to aquaculture worldwide. Here we report the discovery and identification of a novel virus in mud crabs with multiple infections that was not previously detected by molecular, immune, or traditional electron microscopy (EM) methods. High-resolution structures of pathogenic viruses are essential for a molecular understanding and developing new disease prevention methods. The three-dimensional (3D) structure of the mud crab tombus-like virus (MCTV) and mud crab dicistrovirus (MCDV) determined in this study could assist the development of antiviral inhibitors. The identification of a novel virus in multiple infections previously missed using other methods demonstrates the usefulness of this strategy for investigating multiple infectious outbreaks, even in humans and other animals.

Download Full-text