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- EMDB-22129: Nodavirus RNA replication complex crown -

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Basic information

Entry
Database: EMDB / ID: EMD-22129
TitleNodavirus RNA replication complex crown
Map data
Sample
  • Complex: Nodavirus RNA replication crown complex
Biological speciesFlock House virus
Methodsubtomogram averaging / cryo EM / Resolution: 8.45 Å
AuthorsUnchwaniwala N / Zhan H / Pennington J / Horswill M / den Boon J / Ahlquist P
Funding support United States, 2 items
OrganizationGrant numberCountry
Howard Hughes Medical Institute (HHMI) United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)U24GM129547 United States
CitationJournal: Proc Natl Acad Sci U S A / Year: 2020
Title: Subdomain cryo-EM structure of nodaviral replication protein A crown complex provides mechanistic insights into RNA genome replication.
Authors: Nuruddin Unchwaniwala / Hong Zhan / Janice Pennington / Mark Horswill / Johan A den Boon / Paul Ahlquist /
Abstract: For positive-strand RNA [(+)RNA] viruses, the major target for antiviral therapies is genomic RNA replication, which occurs at poorly understood membrane-bound viral RNA replication complexes. Recent ...For positive-strand RNA [(+)RNA] viruses, the major target for antiviral therapies is genomic RNA replication, which occurs at poorly understood membrane-bound viral RNA replication complexes. Recent cryoelectron microscopy (cryo-EM) of nodavirus RNA replication complexes revealed that the viral double-stranded RNA replication template is coiled inside a 30- to 90-nm invagination of the outer mitochondrial membrane, whose necked aperture to the cytoplasm is gated by a 12-fold symmetric, 35-nm diameter "crown" complex that contains multifunctional viral RNA replication protein A. Here we report optimizing cryo-EM tomography and image processing to improve crown resolution from 33 to 8.5 Å. This resolves the crown into 12 distinct vertical segments, each with 3 major subdomains: A membrane-connected basal lobe and an apical lobe that together comprise the ∼19-nm-diameter central turret, and a leg emerging from the basal lobe that connects to the membrane at ∼35-nm diameter. Despite widely varying replication vesicle diameters, the resulting two rings of membrane interaction sites constrain the vesicle neck to a highly uniform shape. Labeling protein A with a His-tag that binds 5-nm Ni-nanogold allowed cryo-EM tomography mapping of the C terminus of protein A to the apical lobe, which correlates well with the predicted structure of the C-proximal polymerase domain of protein A. These and other results indicate that the crown contains 12 copies of protein A arranged basally to apically in an N-to-C orientation. Moreover, the apical polymerase localization has significant mechanistic implications for template RNA recruitment and (-) and (+)RNA synthesis.
History
DepositionJun 9, 2020-
Header (metadata) releaseJul 22, 2020-
Map releaseJul 22, 2020-
UpdateAug 19, 2020-
Current statusAug 19, 2020Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 4.2
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 4.2
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_22129.png

Downloads & links

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Map

FileDownload / File: emd_22129.map.gz / Format: CCP4 / Size: 88.3 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
2.16 Å/pix.
x 285 pix.
= 614.46 Å		2.16 Å/pix.
x 285 pix.
= 614.46 Å		2.16 Å/pix.
x 285 pix.
= 614.46 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 2.156 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 4.2 / Movie #1: 4.2
Minimum - Maximum-2.7461774 - 9.614704
Average (Standard dev.)0.12877443 (±0.6963113)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions285285285
Spacing285285285
CellA=B=C: 614.46 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z2.1562.1562.156
M x/y/z285285285
origin x/y/z0.0000.0000.000
length x/y/z614.460614.460614.460
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ510510510
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS285285285
D min/max/mean-2.7469.6150.129

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Supplemental data

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Sample components

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Entire : Nodavirus RNA replication crown complex

EntireName: Nodavirus RNA replication crown complex
Components
  • Complex: Nodavirus RNA replication crown complex

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Supramolecule #1: Nodavirus RNA replication crown complex

SupramoleculeName: Nodavirus RNA replication crown complex / type: complex / ID: 1 / Parent: 0
Source (natural)Organism: Flock House virus

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Experimental details

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Structure determination

Methodcryo EM
Processingsubtomogram averaging
Aggregation statecell

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Sample preparation

BufferpH: 7.4
GridModel: Quantifoil R2/2 / Material: COPPER / Mesh: 200 / Support film - Material: CARBON / Support film - topology: HOLEY / Support film - Film thickness: 12.0 nm / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Atmosphere: AIR
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 295.15 K
DetailsMitochondrial fraction

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Average exposure time: 0.67 sec. / Average electron dose: 4.86 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: SPOT SCAN / Imaging mode: BRIGHT FIELD
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Final reconstructionApplied symmetry - Point group: C12 (12 fold cyclic) / Algorithm: BACK PROJECTION / Resolution.type: BY AUTHOR / Resolution: 8.45 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: emClarity (ver. 1.4.3) / Number subtomograms used: 4640
ExtractionNumber tomograms: 59 / Number images used: 4640
Software: (Name: PEET (ver. 4.9.12), emClarity (ver. 1.4.3))
CTF correctionSoftware - Name: IMOD (ver. 4.9.12)
Final angle assignmentType: NOT APPLICABLE

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