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- EMDB-5948: Three-dimensional structure of a protozoal dsRNA virus that infec... -

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Database: EMDB / ID: 5948
TitleThree-dimensional structure of a protozoal dsRNA virus that infects enteric pathogen Giardia lamblia
Map dataIcosahedral reconstruction of giardiavirus
SampleGLV virion:
virus
KeywordsGiardia lamblia virus / protozoal virus / dsRNA virus / icosahedral image reconstruction / cryoTEM / infects Giardia lamblia
SourceGiardia lamblia virus (GLV)
Methodsingle particle reconstruction / cryo EM / 6 Å resolution
AuthorsJanssen ME / Takagi Y / Parent KN / Cardone G / Fichorova RN / Nibert ML / Baker TS
CitationJournal: J. Virol. / Year: 2015
Title: Three-dimensional structure of a protozoal double-stranded RNA virus that infects the enteric pathogen Giardia lamblia.
Authors: Mandy E W Janssen / Yuko Takagi / Kristin N Parent / Giovanni Cardone / Max L Nibert / Timothy S Baker
Abstract: Giardia lamblia virus (GLV) is a small, nonenveloped, nonsegmented double-stranded RNA (dsRNA) virus infecting Giardia lamblia, the most common protozoan pathogen of the human intestine and a major ...Giardia lamblia virus (GLV) is a small, nonenveloped, nonsegmented double-stranded RNA (dsRNA) virus infecting Giardia lamblia, the most common protozoan pathogen of the human intestine and a major agent of waterborne diarrheal disease worldwide. GLV (genus Giardiavirus) is a member of family Totiviridae, along with several other groups of protozoal or fungal viruses, including Leishmania RNA viruses and Trichomonas vaginalis viruses. Interestingly, GLV is more closely related than other Totiviridae members to a group of recently discovered metazoan viruses that includes penaeid shrimp infectious myonecrosis virus (IMNV). Moreover, GLV is the only known protozoal dsRNA virus that can transmit efficiently by extracellular means, also like IMNV. In this study, we used transmission electron cryomicroscopy and icosahedral image reconstruction to examine the GLV virion at an estimated resolution of 6.0 Å. Its outermost diameter is 485 Å, making it the largest totivirus capsid analyzed to date. Structural comparisons of GLV and other totiviruses highlighted a related "T=2" capsid organization and a conserved helix-rich fold in the capsid subunits. In agreement with its unique capacity as a protozoal dsRNA virus to survive and transmit through extracellular environments, GLV was found to be more thermoresistant than Trichomonas vaginalis virus 1, but no specific protein machinery to mediate cell entry, such as the fiber complexes in IMNV, could be localized. These and other structural and biochemical findings provide a basis for future work to dissect the cell entry mechanism of GLV into a "primitive" (early-branching) eukaryotic host and an important enteric pathogen of humans.
Importance: Numerous pathogenic bacteria, including Corynebacterium diphtheriae, Salmonella enterica, and Vibrio cholerae, are infected with lysogenic bacteriophages that contribute significantly to bacterial virulence. In line with this phenomenon, several pathogenic protozoa, including Giardia lamblia, Leishmania species, and Trichomonas vaginalis are persistently infected with dsRNA viruses, and growing evidence indicates that at least some of these protozoal viruses can likewise enhance the pathogenicity of their hosts. Understanding of these protozoal viruses, however, lags far behind that of many bacteriophages. Here, we investigated the dsRNA virus that infects the widespread enteric parasite Giardia lamblia. Using electron cryomicroscopy and icosahedral image reconstruction, we determined the virion structure of Giardia lamblia virus, obtaining new information relating to its assembly, stability, functions in cell entry and transcription, and similarities and differences with other dsRNA viruses. The results of our study set the stage for further mechanistic work on the roles of these viruses in protozoal virulence.
DateDeposition: Apr 16, 2014 / Header (metadata) release: Apr 30, 2014 / Map release: Nov 12, 2014 / Last update: May 27, 2015

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 1.1
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 1.1
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 1.1
  • Imaged by UCSF Chimera
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Structure viewerEM map:
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Supplemental images

Downloads & links

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Map

Fileemd_5948.map.gz (map file in CCP4 format, 847977 KB)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
601 pix
1.09 Å/pix.
= 655.09 Å
601 pix
1.09 Å/pix.
= 655.09 Å
601 pix
1.09 Å/pix.
= 655.09 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.09 Å
Density
Contour Level:1.1 (by author), 1.1 (movie #1):
Minimum - Maximum-3.38433194 - 6.48850393
Average (Standard dev.)0.05024409 (0.66671234)
Details

EMDB XML:

Space Group Number1
Map Geometry
Axis orderXYZ
Dimensions601601601
Origin000
Limit600600600
Spacing601601601
CellA=B=C: 655.09 Å
α=β=γ: 90.0 deg.

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.091.091.09
M x/y/z601601601
origin x/y/z0.0000.0000.000
length x/y/z655.090655.090655.090
α/β/γ90.00090.00090.000
start NX/NY/NZ
NX/NY/NZ
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS601601601
D min/max/mean-3.3846.4890.050

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Supplemental data

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Sample components

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Entire GLV virion

EntireName: GLV virion / Number of components: 1 / Oligomeric State: icosahedral

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Component #1: virus, Giardia lamblia virus

VirusName: Giardia lamblia virus / a.k.a: GLV / Class: VIRION / Empty: No / Enveloped: No / Isolate: STRAIN
SpeciesSpecies: Giardia lamblia virus (GLV) / Strain: WBI
Source (natural)Host Species: Giardia intestinalis (eukaryote) / Host category: PROTOZOA / Host species strain: WBI

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Experimental details

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Sample preparation

SpecimenSpecimen state: particle / Method: cryo EM
Sample solutionSpecimen conc.: 0.02 mg/ml / Buffer solution: 50 mM HEPES, 500 mM NaCl, 20 mM MgCl2 / pH: 7.2
Support filmQuantifoil R2/2
VitrificationInstrument: HOMEMADE PLUNGER / Cryogen name: ETHANE / Temperature: 90 K / Humidity: 99 % / Method: Blot 5 sec before plunging

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Electron microscopy imaging

Experimental equipment
Model: Tecnai Polara / Image courtesy: FEI Company
ImagingMicroscope: FEI POLARA 300 / Date: Apr 18, 2012
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Electron dose: 25 e/Å2 / Illumination mode: FLOOD BEAM
LensMagnification: 59000 X (nominal), 58050 X (calibrated)
Astigmatism: Objective lens astigmatism was corrected at 135,000 times magnification.
Cs: 2.26 mm / Imaging mode: BRIGHT FIELD / Defocus: 780 - 3410 nm
Specimen HolderModel: OTHER / Temperature: 93 K ( 90 - 96 K)
CameraDetector: KODAK SO-163 FILM

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Image acquisition

Image acquisitionNumber of digital images: 101 / Scanner: NIKON SUPER COOLSCAN 8000 / Sampling size: 6.35 microns / Bit depth: 8

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Image processing

ProcessingMethod: single particle reconstruction / Applied symmetry: I (icosahedral) / Number of projections: 14125
Details: Particles were selected and preprocessed using RobEM. Image reconstruction was performed using Auto3DEM.
3D reconstructionAlgorithm: reference projection / Software: auto3dem / CTF correction: robem
Details: Particles were selected and preprocessed using RobEM.
Resolution: 6 Å / Resolution method: FSC 0.5, gold-standard

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