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- EMDB-17730: masked refinement giving rise to better defined protruding densit... -

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Basic information

Entry
Database: EMDB / ID: EMD-17730
Titlemasked refinement giving rise to better defined protruding densities of the potential macrodomain outside the AUD helical assemblies.
Map data
Sample
  • Complex: Helical scaffold assembly of CHIKV nsP3 mediated by its Unique alphavirus domain.
    • Protein or peptide: Chikungunya virus non structural protein 3
KeywordsHelical scaffold / Replication complex / Alpha granules / Viral factories / VIRAL PROTEIN
Biological speciesChikungunya virus strain S27-African prototype
Methodhelical reconstruction / cryo EM / Resolution: 2.98 Å
AuthorsReguera J / Hons M / Zimberger C / Ptchelkine D / Jones R / Desfosses A
Funding support France, 2 items
OrganizationGrant numberCountry
Agence Nationale de la Recherche (ANR)ANR-22-CE11-0023-01 France
ATIP-Avenir2015 France
CitationJournal: Nat Commun / Year: 2024
Title: Alphavirus nsP3 organizes into tubular scaffolds essential for infection and the cytoplasmic granule architecture.
Authors: Vasiliya Kril / Michael Hons / Celine Amadori / Claire Zimberger / Laurine Couture / Yara Bouery / Julien Burlaud-Gaillard / Andrei Karpov / Denis Ptchelkine / Alexandra L Thienel / Beate M ...Authors: Vasiliya Kril / Michael Hons / Celine Amadori / Claire Zimberger / Laurine Couture / Yara Bouery / Julien Burlaud-Gaillard / Andrei Karpov / Denis Ptchelkine / Alexandra L Thienel / Beate M Kümmerer / Ambroise Desfosses / Rhian Jones / Philippe Roingeard / Laurent Meertens / Ali Amara / Juan Reguera /
Abstract: Alphaviruses, such as chikungunya virus (CHIKV), are mosquito-borne viruses that represent a significant threat to human health due to the current context of global warming. Efficient alphavirus ...Alphaviruses, such as chikungunya virus (CHIKV), are mosquito-borne viruses that represent a significant threat to human health due to the current context of global warming. Efficient alphavirus infection relies on the activity of the non-structural protein 3 (nsP3), a puzzling multifunctional molecule whose role in infection remains largely unknown. NsP3 is a component of the plasma membrane-bound viral RNA replication complex (vRC) essential for RNA amplification and is also found in large cytoplasmic aggregates of unknown function Here, we report the cryo-electron microscopy (cryo-EM) structure of the CHIKV nsP3 at 2.35 Å resolution. We show that nsP3 assembles into tubular structures made by a helical arrangement of its alphavirus unique domain (AUD). The nsP3 helical scaffolds are consistent with crown structures found on tomographic reconstructions of the mature viral RCs. In addition, nsP3 helices assemble into cytoplasmic granules organized in a network of tubular structures that contain viral genomic RNA and capsid as well as host factors required for productive infection. Structure-guided mutagenesis identified residues that prevent or disturb nsP3 assemblies, resulting in impaired viral replication or transcription. Altogether, our results reveal an unexpected nsP3-dependent molecular organization essential for different phases of alphavirus infection.
History
DepositionJun 25, 2023-
Header (metadata) releaseJul 10, 2024-
Map releaseJul 10, 2024-
UpdateJan 22, 2025-
Current statusJan 22, 2025Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_17730.png

Downloads & links

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Map

FileDownload / File: emd_17730.map.gz / Format: CCP4 / Size: 259.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.06 Å/pix.
x 408 pix.
= 432.48 Å		1.06 Å/pix.
x 408 pix.
= 432.48 Å		1.06 Å/pix.
x 408 pix.
= 432.48 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.06 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.0014
Minimum - Maximum-0.006941898 - 0.022917496
Average (Standard dev.)-0.000020683301 (±0.00083647086)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions408408408
Spacing408408408
CellA=B=C: 432.47998 Å
α=β=γ: 90.0 °

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Supplemental data

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Additional map: Class of a mask including 16 protomers Map alligned to EMD-17729

Fileemd_17730_additional_1.map
AnnotationClass of a mask including 16 protomers Map alligned to EMD-17729
Projections & Slices
AxesZYX

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Additional map: Map alligned to EMD-17729

Fileemd_17730_additional_2.map
AnnotationMap alligned to EMD-17729
Projections & Slices
AxesZYX

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Histogram

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Half map: #1

Fileemd_17730_half_map_1.map
Projections & Slices
AxesZYX

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Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #2

Fileemd_17730_half_map_2.map
Projections & Slices
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Sample components

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Entire : Helical scaffold assembly of CHIKV nsP3 mediated by its Unique al...

EntireName: Helical scaffold assembly of CHIKV nsP3 mediated by its Unique alphavirus domain.
Components
  • Complex: Helical scaffold assembly of CHIKV nsP3 mediated by its Unique alphavirus domain.
    • Protein or peptide: Chikungunya virus non structural protein 3

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Supramolecule #1: Helical scaffold assembly of CHIKV nsP3 mediated by its Unique al...

SupramoleculeName: Helical scaffold assembly of CHIKV nsP3 mediated by its Unique alphavirus domain.
type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Chikungunya virus strain S27-African prototype
Molecular weightTheoretical: 220.25 kDa/nm

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Macromolecule #1: Chikungunya virus non structural protein 3

MacromoleculeName: Chikungunya virus non structural protein 3 / type: protein_or_peptide / ID: 1 / Details: Zn / Enantiomer: LEVO
Source (natural)Organism: Chikungunya virus strain S27-African prototype
Recombinant expressionOrganism: Trichoplusia ni (cabbage looper)
SequenceString: APSYRVKRMD IAKNDEECVV NAANPRGLPG DGVCKAVYKK WPESFKNSAT PVGTAKTVMC GTYPVIHAVG PNFSNYSESE GDRELAAAYR EVAKEVTRLG VNSVAIPLLS TGVYSGGKDR LTQSLNHLFT AMDSTDADVV IYCRDKEWEK KISEAIQMRT QVELLDEHIS ...String:
APSYRVKRMD IAKNDEECVV NAANPRGLPG DGVCKAVYKK WPESFKNSAT PVGTAKTVMC GTYPVIHAVG PNFSNYSESE GDRELAAAYR EVAKEVTRLG VNSVAIPLLS TGVYSGGKDR LTQSLNHLFT AMDSTDADVV IYCRDKEWEK KISEAIQMRT QVELLDEHIS IDCDVVRVHP DSSLAGRKGY STTEGALYSY LEGTRFHQTA VDMAEIYTMW PKQTEANEQV CLYALGESIE SIRQKCPVDD ADASSPPKTV PCLCRYAMTP ERVTRLRMNH VTSIIVCSSF PLPKYKIEGV QKVKCSKVML FDHNVPSRVS PREYRPSQES VQEASTTTSL THSQFDLSVD GKILPVPSDL DADAPALEPA LDDGAIHTLP SATGNLAAVS DWVMSTVPVA PPRRRRGRNL TVTCDEREGN ITPMASVRFF RAELCPVVQE TAETRDTAMS LQAPPSTATE LSHPPISFGA PSETFPITFG DFNEGEIESL SSELLTFGDF LPGEVDDLTD SDWSTCSDTD DEL

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Experimental details

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Structure determination

Methodcryo EM
Processinghelical reconstruction
Aggregation statehelical array

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Sample preparation

BufferpH: 7.5
VitrificationCryogen name: ETHANE-PROPANE
Detailshis sample was heterogeneous in lenght

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Average electron dose: 18.1 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.5 µm / Nominal defocus min: 0.8 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Final reconstructionApplied symmetry - Helical parameters - Δz: 2.782 Å
Applied symmetry - Helical parameters - Δ&Phi: -164.175 °
Applied symmetry - Helical parameters - Axial symmetry: C1 (asymmetric)
Resolution.type: BY AUTHOR / Resolution: 2.98 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION / Number images used: 807973
Startup modelType of model: INSILICO MODEL
In silico model: Alphafold was used to have a model of the CHIKV AUD domain
Final angle assignmentType: NOT APPLICABLE / Software - Name: RELION
FSC plot (resolution estimation)

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