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- EMDB-12294: CryoEM structure of disease related M854K MDA5-dsRNA filament in ... -

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Basic information

Entry
Database: EMDB / ID: EMD-12294
TitleCryoEM structure of disease related M854K MDA5-dsRNA filament in complex with ADP-AlF4(minor class)
Map data
Sample
  • Complex: M854K MDA5-dsRNA filament in complex with ADP-AlF4(major class)
    • Complex: INTERFERON-INDUCED HELICASE C DOMAIN-CONTAINING PROTEIN
      • Protein or peptide: INTERFERON-INDUCED HELICASE C DOMAIN-CONTAINING PROTEIN
    • Complex: RNA
      • RNA: RNA
    • Complex: RNA
      • RNA: RNA
Function / homology
Function and homology information


MDA-5 signaling pathway / Ub-specific processing proteases / positive regulation of response to cytokine stimulus / negative regulation of viral genome replication / type I interferon-mediated signaling pathway / cellular response to exogenous dsRNA / pattern recognition receptor activity / protein complex oligomerization / positive regulation of interferon-alpha production / protein sumoylation ...MDA-5 signaling pathway / Ub-specific processing proteases / positive regulation of response to cytokine stimulus / negative regulation of viral genome replication / type I interferon-mediated signaling pathway / cellular response to exogenous dsRNA / pattern recognition receptor activity / protein complex oligomerization / positive regulation of interferon-alpha production / protein sumoylation / ribonucleoprotein complex binding / antiviral innate immune response / positive regulation of interferon-beta production / response to virus / positive regulation of interleukin-6 production / cellular response to virus / positive regulation of tumor necrosis factor production / double-stranded RNA binding / defense response to virus / single-stranded RNA binding / RNA helicase activity / RNA helicase / protein domain specific binding / chromatin extrusion motor activity / ATP-dependent H2AZ histone chaperone activity / ATP-dependent H3-H4 histone complex chaperone activity / cohesin loader activity / DNA clamp loader activity / innate immune response / ATP hydrolysis activity / mitochondrion / DNA binding / zinc ion binding / ATP binding / identical protein binding / nucleus / cytoplasm
Similarity search - Function
RIG-I-like receptor, C-terminal / RIG-I receptor C-terminal domain / RIG-I-like receptor, C-terminal regulatory domain / RIG-I-like receptor, C-terminal domain superfamily / : / C-terminal domain of RIG-I / RIG-I-like receptor (RLR) C-terminal regulatory (CTR) domain profile. / Caspase recruitment domain / Caspase recruitment domain / Helicase/UvrB, N-terminal ...RIG-I-like receptor, C-terminal / RIG-I receptor C-terminal domain / RIG-I-like receptor, C-terminal regulatory domain / RIG-I-like receptor, C-terminal domain superfamily / : / C-terminal domain of RIG-I / RIG-I-like receptor (RLR) C-terminal regulatory (CTR) domain profile. / 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. / DEAD-like helicases superfamily / Helicase, C-terminal / 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: 4.3 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
DepositionFeb 4, 2021-
Header (metadata) releaseDec 1, 2021-
Map releaseDec 1, 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: 0.036
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.036
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-7nic
  • Surface level: 0.036
  • Imaged by UCSF Chimera
  • Download
  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-7nic
  • Imaged by Jmol
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_12294.png

Downloads & links

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Map

FileDownload / File: emd_12294.map.gz / Format: CCP4 / Size: 40.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.08 Å/pix.
x 220 pix.
= 238.48 Å		1.08 Å/pix.
x 220 pix.
= 238.48 Å		1.08 Å/pix.
x 220 pix.
= 238.48 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.084 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.036 / Movie #1: 0.036
Minimum - Maximum-0.067317516 - 0.14439605
Average (Standard dev.)0.00077729084 (±0.006120554)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions220220220
Spacing220220220
CellA=B=C: 238.48 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.0841.0841.084
M x/y/z220220220
origin x/y/z0.0000.0000.000
length x/y/z238.480238.480238.480
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ384384384
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS220220220
D min/max/mean-0.0670.1440.001

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Supplemental data

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Mask #1

Fileemd_12294_msk_1.map
Projections & Slices
AxesZYX

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Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_12294_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_12294_half_map_2.map
Projections & Slices
AxesZYX

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Histogram

Histogram (log scale)

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Sample components

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Entire : M854K MDA5-dsRNA filament in complex with ADP-AlF4(major class)

EntireName: M854K MDA5-dsRNA filament in complex with ADP-AlF4(major class)
Components
  • Complex: M854K MDA5-dsRNA filament in complex with ADP-AlF4(major class)
    • Complex: INTERFERON-INDUCED HELICASE C DOMAIN-CONTAINING PROTEIN
      • Protein or peptide: INTERFERON-INDUCED HELICASE C DOMAIN-CONTAINING PROTEIN
    • Complex: RNA
      • RNA: RNA
    • Complex: RNA
      • RNA: RNA

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Supramolecule #1: M854K MDA5-dsRNA filament in complex with ADP-AlF4(major class)

SupramoleculeName: M854K MDA5-dsRNA filament in complex with ADP-AlF4(major class)
type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Molecular weightExperimental: 2 kDa/nm

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Supramolecule #2: INTERFERON-INDUCED HELICASE C DOMAIN-CONTAINING PROTEIN

SupramoleculeName: INTERFERON-INDUCED HELICASE C DOMAIN-CONTAINING PROTEIN
type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1
Source (natural)Organism: Mus musculus (house mouse)
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)

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Supramolecule #3: RNA

SupramoleculeName: RNA / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #2
Source (natural)Organism: Mus musculus (house mouse)

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Supramolecule #4: RNA

SupramoleculeName: RNA / type: complex / ID: 4 / Parent: 1 / Macromolecule list: #3
Source (natural)Organism: Mus musculus (house mouse)

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Macromolecule #1: INTERFERON-INDUCED HELICASE C DOMAIN-CONTAINING PROTEIN

MacromoleculeName: INTERFERON-INDUCED HELICASE C DOMAIN-CONTAINING PROTEIN
type: protein_or_peptide / ID: 1 / Enantiomer: LEVO / EC number: RNA helicase
Source (natural)Organism: Mus musculus (house mouse)
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: LQLRPYQMEV AQPALDGKNI IICLPTGSGK TRVAVYITKD HLDKKKQASE SGKVIVLVNK VMLAEQLFRK EFNPYLKKWY RIIGLSGDTQ LKISFPEVVK SYDVIISTAQ ILENSLLNLE SGDDDGVQLS DFSLIIIDEC HHTNKEAVYN NIMRRYLKQK LRNNDLKKQN ...String:
LQLRPYQMEV AQPALDGKNI IICLPTGSGK TRVAVYITKD HLDKKKQASE SGKVIVLVNK VMLAEQLFRK EFNPYLKKWY RIIGLSGDTQ LKISFPEVVK SYDVIISTAQ ILENSLLNLE SGDDDGVQLS DFSLIIIDEC HHTNKEAVYN NIMRRYLKQK LRNNDLKKQN KPAIPLPQIL GLTASPGVGA AKKQSEAEKH ILNICANLDA FTIKTVKENL GQLKHQIKEP CKKFVIADDT RENPFKEKLL EIMASIQTYC QKSPMSDFGT QHYEQWAIQM EKKAAKDGNR KDRVCAEHLR KYNEALQIND TIRMIDAYSH LETFYTDEKE KKFAVLNDSK KSLKLDETDE FLMNLFFDNK KMLKKLAENP KYENEKLIKL RNTILEQFTR SEESSRGIIF TKTRQSTYAL SQWIMENAKF AEVGVKAHHL IGAGHSSEVK PMTQTEQKEV ISKFRTGEIN LLIATTVAEE GLDIKECNIV IRYGLVTNEI AMVQARGRAR ADESTYVLVT SSGSGVTERE IVNDFREKMK YKAINRVQNM KPEEYAHKIL ELQVQSILEK KMKVKRSIAK QYNDNPSLIT LLCKNCSMLV CSGENIHVIE KMHHVNMTPE FKGLYIVREN KALQKKFADY QTNGEIICKC GQAWGTMMVH KGLDLPCLKI RNFVVNFKNN SPKKQYKKWV ELPIRFPDLD YSEYCL

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Macromolecule #2: RNA

MacromoleculeName: RNA / type: rna / ID: 2
Source (natural)Organism: Pseudomonas virus phi6 (bacteriophage)
SequenceString:
GUCAAGCCGA GGAGA

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Macromolecule #3: RNA

MacromoleculeName: RNA / type: rna / ID: 3
Source (natural)Organism: Pseudomonas virus phi6 (bacteriophage)
SequenceString:
UCUCCUCGGC UUGAC

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Experimental details

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Structure determination

Methodcryo EM
Processinghelical reconstruction
Aggregation statefilament

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Sample preparation

Concentration1 mg/mL
BufferpH: 7.7
Component:
ConcentrationFormulaName
100.0 mMKCl
20.0 mMHEPES
5.0 mMMgCl2
2.0 mMDithiothreitol
1.0 mMADP AlF4
GridModel: Quantifoil / Material: GOLD / Mesh: 300 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Atmosphere: AIR / Details: machine step 6
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277.2 K / Instrument: FEI VITROBOT MARK IV
Details: Grids were blotted for 3 s; blot force 10, 5, 0, -5, -10.

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Specialist opticsPhase plate: OTHER
Image recordingFilm or detector model: FEI FALCON III (4k x 4k) / Detector mode: COUNTING / Digitization - Dimensions - Width: 4096 pixel / Digitization - Dimensions - Height: 4096 pixel / Number real images: 2177 / Average exposure time: 60.0 sec. / Average electron dose: 29.6 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: -3.0 µm / Nominal defocus min: -1.8 µm / Nominal magnification: 75000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Final reconstructionNumber classes used: 1
Applied symmetry - Helical parameters - Δz: 45.0795 Å
Applied symmetry - Helical parameters - Δ&Phi: 88.0297 °
Applied symmetry - Helical parameters - Axial symmetry: C1 (asymmetric)
Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 4.3 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.1) / Number images used: 62028
CTF correctionSoftware - Name: CTFFIND
Segment selectionNumber selected: 731263 / Software - Name: RELION (ver. 3.1)
Startup modelType of model: EMDB MAP
EMDB ID:

0023

Final angle assignmentType: NOT APPLICABLE / Software - Name: RELION (ver. 3.1)
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

6gkh


Chain - Chain ID: A
RefinementSpace: REAL / Protocol: FLEXIBLE FIT / Overall B value: 147
Output model

PDB-7nic:
CryoEM structure of disease related M854K MDA5-dsRNA filament in complex with ADP-AlF4(minor class)

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