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- EMDB-11937: CryoEM structure of the MDA5-dsRNA filament in complex with ADP w... -

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Basic information

Entry
Database: EMDB / ID: EMD-11937
TitleCryoEM structure of the MDA5-dsRNA filament in complex with ADP with 92-degree helical twist
Map data
SampleMDA5-dsRNA filament in complex with ADP
  • (INTERFERON-INDUCED HELICASE C DOMAIN-CONTAINING ...) x 2
  • RNA
  • (nucleic-acidNucleic acid) x 2
Function / homology
Function and homology information


MDA-5 signaling pathway / positive regulation of response to cytokine stimulus / Ub-specific processing proteases / cellular response to exogenous dsRNA / protein sumoylation / negative regulation of viral genome replication / ribonucleoprotein complex binding / positive regulation of interferon-alpha production / positive regulation of interferon-beta production / response to virus ...MDA-5 signaling pathway / positive regulation of response to cytokine stimulus / Ub-specific processing proteases / cellular response to exogenous dsRNA / protein sumoylation / negative regulation of viral genome replication / ribonucleoprotein complex binding / positive regulation of interferon-alpha production / positive regulation of interferon-beta production / response to virus / positive regulation of interleukin-6 production / positive regulation of tumor necrosis factor production / : / double-stranded RNA binding / single-stranded RNA binding / defense response to virus / RNA helicase / RNA helicase activity / innate immune response / mitochondrion / RNA binding / DNA binding / zinc ion binding / ATP binding / identical protein binding / nucleus / cytoplasm
Similarity search - Function
RIG-I receptor, C-terminal / RIG-I receptor C-terminal domain / RIG-I-like receptor (RLR) C-terminal regulatory (CTR) domain profile. / RIG-I-like receptor, C-terminal regulatory domain / C-terminal domain of RIG-I / RIG-I-like receptor, C-terminal domain superfamily / Caspase recruitment domain / Caspase recruitment domain / Helicase/UvrB, N-terminal / Type III restriction enzyme, res subunit ...RIG-I receptor, C-terminal / RIG-I receptor C-terminal domain / RIG-I-like receptor (RLR) C-terminal regulatory (CTR) domain profile. / RIG-I-like receptor, C-terminal regulatory domain / C-terminal domain of RIG-I / RIG-I-like receptor, C-terminal domain superfamily / Caspase recruitment domain / Caspase recruitment domain / Helicase/UvrB, N-terminal / Type III restriction enzyme, res subunit / Death-like domain superfamily / Helicase conserved C-terminal domain / helicase superfamily c-terminal domain / Superfamilies 1 and 2 helicase C-terminal domain profile. / Superfamilies 1 and 2 helicase ATP-binding type-1 domain profile. / Helicase, C-terminal / DEAD-like helicases superfamily / Helicase superfamily 1/2, ATP-binding domain / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
Interferon-induced helicase C domain-containing protein 1
Similarity search - Component
Biological speciesMus musculus (house mouse) / Pseudomonas virus phi6 (bacteriophage)
Methodhelical reconstruction / cryo EM / Resolution: 3.4 Å
AuthorsYu Q / Modis Y
Funding support United Kingdom, 2 items
OrganizationGrant numberCountry
Wellcome Trust217191/Z/19/Z United Kingdom
Wellcome Trust101908/Z/13/Z United Kingdom
CitationJournal: Nat Commun / Year: 2021
Title: MDA5 disease variant M854K prevents ATP-dependent structural discrimination of viral and cellular RNA.
Authors: Qin Yu / Alba Herrero Del Valle / Rahul Singh / Yorgo Modis /
Abstract: Our innate immune responses to viral RNA are vital defenses. Long cytosolic double-stranded RNA (dsRNA) is recognized by MDA5. The ATPase activity of MDA5 contributes to its dsRNA binding selectivity. ...Our innate immune responses to viral RNA are vital defenses. Long cytosolic double-stranded RNA (dsRNA) is recognized by MDA5. The ATPase activity of MDA5 contributes to its dsRNA binding selectivity. Mutations that reduce RNA selectivity can cause autoinflammatory disease. Here, we show how the disease-associated MDA5 variant M854K perturbs MDA5-dsRNA recognition. M854K MDA5 constitutively activates interferon signaling in the absence of exogenous RNA. M854K MDA5 lacks ATPase activity and binds more stably to synthetic Alu:Alu dsRNA. CryoEM structures of MDA5-dsRNA filaments at different stages of ATP hydrolysis show that the K854 sidechain forms polar bonds that constrain the conformation of MDA5 subdomains, disrupting key steps in the ATPase cycle- RNA footprint expansion and helical twist modulation. The M854K mutation inhibits ATP-dependent RNA proofreading via an allosteric mechanism, allowing MDA5 to form signaling complexes on endogenous RNAs. This work provides insights on how MDA5 recognizes dsRNA in health and disease.
History
DepositionNov 15, 2020-
Header (metadata) releaseNov 24, 2021-
Map releaseNov 24, 2021-
UpdateDec 1, 2021-
Current statusDec 1, 2021Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.02
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.02
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-7bkq
  • Surface level: 0.02
  • Imaged by UCSF Chimera
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  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-7bkq
  • Imaged by Jmol
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

Downloads & links

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Map

FileDownload / File: emd_11937.map.gz / Format: CCP4 / Size: 47.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.04 Å/pix.
x 232 pix.
= 241.28 Å
1.04 Å/pix.
x 232 pix.
= 241.28 Å
1.04 Å/pix.
x 232 pix.
= 241.28 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.04 Å
Density
Contour LevelBy AUTHOR: 0.02 / Movie #1: 0.02
Minimum - Maximum-0.04012276 - 0.094014786
Average (Standard dev.)0.00039355905 (±0.0032461553)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions232232232
Spacing232232232
CellA=B=C: 241.28 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.041.041.04
M x/y/z232232232
origin x/y/z0.0000.0000.000
length x/y/z241.280241.280241.280
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS232232232
D min/max/mean-0.0400.0940.000

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Supplemental data

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Segmentation: #1

Fileemd_11937_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

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Half map: #2

Fileemd_11937_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

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Half map: #1

Fileemd_11937_half_map_2.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

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Sample components

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Entire MDA5-dsRNA filament in complex with ADP

EntireName: MDA5-dsRNA filament in complex with ADP / Number of Components: 6

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Component #1: protein, MDA5-dsRNA filament in complex with ADP

ProteinName: MDA5-dsRNA filament in complex with ADP / Recombinant expression: No
MassExperimental: 30 MDa

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Component #2: protein, INTERFERON-INDUCED HELICASE C DOMAIN-CONTAINING PROTEIN

ProteinName: INTERFERON-INDUCED HELICASE C DOMAIN-CONTAINING PROTEIN
Recombinant expression: No
SourceSpecies: Mus musculus (house mouse)
Source (engineered)Expression System: Escherichia coli BL21(DE3) (bacteria)

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Component #3: protein, RNA

ProteinName: RNA / Recombinant expression: No
SourceSpecies: Pseudomonas virus phi6 (bacteriophage)

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Component #4: protein, INTERFERON-INDUCED HELICASE C DOMAIN-CONTAINING PROTEIN

ProteinName: INTERFERON-INDUCED HELICASE C DOMAIN-CONTAINING PROTEIN
Recombinant expression: No
SourceSpecies: Mus musculus (house mouse)
Source (engineered)Expression System: Escherichia coli BL21(DE3) (bacteria)

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Component #5: nucleic-acid, RNA

nucleic acidName: RNA / Class: RNA / Structure: OTHER / Synthetic: No
Sequence:
UCCAUGCGCA UGACG
SourceSpecies: Pseudomonas virus phi6 (bacteriophage)

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Component #6: nucleic-acid, RNA

nucleic acidName: RNA / Class: RNA / Structure: OTHER / Synthetic: No
Sequence:
CGUCAUGCGC AUGGA
SourceSpecies: Pseudomonas virus phi6 (bacteriophage)

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Experimental details

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Sample preparation

SpecimenSpecimen State: Filament / Method: cryo EM
Helical ParametersAxial Symmetry: C1 (asymmetric) / Delta Z: 44.6 Å / Delta Phi: 91.7 %deg;
Sample solutionSpecimen conc.: 1 mg/mL / pH: 7.7
Support filmmachine step 6
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen Name: ETHANE / Temperature: 277.2 K / Humidity: 100 %
Details: Grids were blotted for 3 s; blot force 10, 5, 0, -5, -10.

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
ImagingMicroscope: FEI TITAN KRIOS
Electron gunElectron Source: FIELD EMISSION GUN / Accelerating Voltage: 300 kV / Electron Dose: 1.1125 e/Å2 / Illumination Mode: FLOOD BEAM
LensImaging Mode: BRIGHT FIELD / Defocus: 1800.0 - 2700.0 nm
Specimen HolderModel: FEI TITAN KRIOS AUTOGRID HOLDER
CameraDetector: GATAN K2 SUMMIT (4k x 4k)

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Image acquisition

Image acquisitionNumber of Digital Images: 4680

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Image processing

ProcessingMethod: helical reconstruction
3D reconstructionAlgorithm: FOURIER SPACE / Software: RELION / Resolution: 3.4 Å / Resolution Method: FSC 0.143 CUT-OFF
FSC plot (resolution estimation)

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Atomic model buiding

Modeling #1Target Criteria: Correlation coefficient / Refinement space: REAL
Input PDB model: 6H66
Chain ID: A

Overall BValue: 118
Output model

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