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- EMDB-0023: CryoEM structure of the MDA5-dsRNA filament in complex with ADP-AlF4 -

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Basic information

Entry
Database: EMDB / ID: EMD-0023
TitleCryoEM structure of the MDA5-dsRNA filament in complex with ADP-AlF4
Map dataVolume used in reconstruction
Sample
  • Complex: MDA5-dsRNA helical filament in complex with ADP-AlF4
    • Complex: MDA5 bound to ADP-AlF4
      • Protein or peptide: Interferon-induced helicase C domain-containing protein 1
    • Complex: Double-stranded RNA from bacteriophage Phi6
      • RNA: RNA (5'-R(P*GP*UP*CP*AP*AP*GP*CP*CP*GP*AP*GP*GP*AP*GP*A)-3')
      • RNA: RNA (5'-R(P*UP*CP*UP*CP*CP*UP*CP*GP*GP*CP*UP*UP*GP*AP*C)-3')
  • Ligand: ADENOSINE-5'-DIPHOSPHATE
  • Ligand: TETRAFLUOROALUMINATE ION
  • Ligand: MAGNESIUM ION
  • Ligand: ZINC ION
KeywordsProtein-RNA complex / helical filament / ATPase / innate immune receptor / IMMUNE SYSTEM
Function / homology
Function and homology information


MDA-5 signaling pathway / Ub-specific processing proteases / positive regulation of response to cytokine stimulus / pattern recognition receptor activity / negative regulation of viral genome replication / type I interferon-mediated signaling pathway / cellular response to exogenous dsRNA / 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 / pattern recognition receptor activity / negative regulation of viral genome replication / type I interferon-mediated signaling pathway / cellular response to exogenous dsRNA / 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 / cellular response to virus / positive regulation of interleukin-6 production / positive regulation of tumor necrosis factor production / double-stranded RNA binding / defense response to virus / RNA helicase activity / single-stranded RNA binding / RNA helicase / protein domain specific binding / 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 savastanoi pv. phaseolicola (bacteria) / Pseudomonas phage phi6 (bacteriophage)
Methodhelical reconstruction / cryo EM / Resolution: 4.06 Å
AuthorsYu Q / Qu K
Funding support United Kingdom, 2 items
OrganizationGrant numberCountry
Wellcome Trust101908/Z/13/Z United Kingdom
European Research CouncilERC-CoG-648432 MEMBRANEFUSION United Kingdom
CitationJournal: Mol Cell / Year: 2018
Title: Cryo-EM Structures of MDA5-dsRNA Filaments at Different Stages of ATP Hydrolysis.
Authors: Qin Yu / Kun Qu / Yorgo Modis /
Abstract: Double-stranded RNA (dsRNA) is a potent proinflammatory signature of viral infection. Long cytosolic dsRNA is recognized by MDA5. The cooperative assembly of MDA5 into helical filaments on dsRNA ...Double-stranded RNA (dsRNA) is a potent proinflammatory signature of viral infection. Long cytosolic dsRNA is recognized by MDA5. The cooperative assembly of MDA5 into helical filaments on dsRNA nucleates the assembly of a multiprotein type I interferon signaling platform. Here, we determined cryoelectron microscopy (cryo-EM) structures of MDA5-dsRNA filaments with different helical twists and bound nucleotide analogs at resolutions sufficient to build and refine atomic models. The structures identify the filament-forming interfaces, which encode the dsRNA binding cooperativity and length specificity of MDA5. The predominantly hydrophobic interface contacts confer flexibility, reflected in the variable helical twist within filaments. Mutation of filament-forming residues can result in loss or gain of signaling activity. Each MDA5 molecule spans 14 or 15 RNA base pairs, depending on the twist. Variations in twist also correlate with variations in the occupancy and type of nucleotide in the active site, providing insights on how ATP hydrolysis contributes to MDA5-dsRNA recognition.
History
Header (metadata) releaseApr 11, 2018-
DepositionMay 21, 2018-
Map releaseNov 21, 2018-
UpdateNov 20, 2024-
Current statusNov 20, 2024Processing site: PDBe / Status: Released

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Structure visualization

Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_0023.png

Downloads & links

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Map

FileDownload / File: emd_0023.map.gz / Format: CCP4 / Size: 42.9 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationVolume used in reconstruction
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.07 Å/pix.
x 224 pix.
= 239.68 Å		1.07 Å/pix.
x 224 pix.
= 239.68 Å		1.07 Å/pix.
x 224 pix.
= 239.68 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

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Images are generated by Spider.

Voxel sizeX=Y=Z: 1.07 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.0554 / Movie #1: 0
Minimum - Maximum-0.14012551 - 0.25628585
Average (Standard dev.)0.0012399934 (±0.011302313)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions224224224
Spacing224224224
CellA=B=C: 239.68001 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_0023_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map 1

Fileemd_0023_half_map_1.map
AnnotationHalf map 1
Projections & Slices
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Histogram

Histogram (log scale)

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Half map: Half map 2

Fileemd_0023_half_map_2.map
AnnotationHalf map 2
Projections & Slices
AxesZYX

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Histogram (log scale)

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Sample components

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Entire : MDA5-dsRNA helical filament in complex with ADP-AlF4

EntireName: MDA5-dsRNA helical filament in complex with ADP-AlF4
Components
  • Complex: MDA5-dsRNA helical filament in complex with ADP-AlF4
    • Complex: MDA5 bound to ADP-AlF4
      • Protein or peptide: Interferon-induced helicase C domain-containing protein 1
    • Complex: Double-stranded RNA from bacteriophage Phi6
      • RNA: RNA (5'-R(P*GP*UP*CP*AP*AP*GP*CP*CP*GP*AP*GP*GP*AP*GP*A)-3')
      • RNA: RNA (5'-R(P*UP*CP*UP*CP*CP*UP*CP*GP*GP*CP*UP*UP*GP*AP*C)-3')
  • Ligand: ADENOSINE-5'-DIPHOSPHATE
  • Ligand: TETRAFLUOROALUMINATE ION
  • Ligand: MAGNESIUM ION
  • Ligand: ZINC ION

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Supramolecule #1: MDA5-dsRNA helical filament in complex with ADP-AlF4

SupramoleculeName: MDA5-dsRNA helical filament in complex with ADP-AlF4 / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#3
Molecular weightTheoretical: 2.162 kDa/nm

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Supramolecule #2: MDA5 bound to ADP-AlF4

SupramoleculeName: MDA5 bound to ADP-AlF4 / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1
Details: DExD/H-box helicase consisting of Hel1, Hel2, Hel2i, and pincer domains, followed by a C-terminal domain
Source (natural)Organism: Mus musculus (house mouse)

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Supramolecule #3: Double-stranded RNA from bacteriophage Phi6

SupramoleculeName: Double-stranded RNA from bacteriophage Phi6 / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #2-#3
Source (natural)Organism: Pseudomonas savastanoi pv. phaseolicola (bacteria)

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Macromolecule #1: Interferon-induced helicase C domain-containing protein 1

MacromoleculeName: Interferon-induced helicase C domain-containing protein 1
type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO / EC number: RNA helicase
Source (natural)Organism: Mus musculus (house mouse)
Molecular weightTheoretical: 114.214477 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: MSIVCSAEDS FRNLILFFRP RLKMYIQVEP VLDHLIFLSA ETKEQILKKI NTCGNTSAAE LLLSTLEQGQ WPLGWTQMFV EALEHSGNP LAARYVKPTL TDLPSPSSET AHDECLHLLT LLQPTLVDKL LINDVLDTCF EKGLLTVEDR NRISAAGNSG N ESGVRELL ...String:
MSIVCSAEDS FRNLILFFRP RLKMYIQVEP VLDHLIFLSA ETKEQILKKI NTCGNTSAAE LLLSTLEQGQ WPLGWTQMFV EALEHSGNP LAARYVKPTL TDLPSPSSET AHDECLHLLT LLQPTLVDKL LINDVLDTCF EKGLLTVEDR NRISAAGNSG N ESGVRELL RRIVQKENWF STFLDVLRQT GNDALFQELT GGGCPEDNTD LANSSHRDGP AANECLLPAV DESSLETEAW NV DDILPEA SCTDSSVTTE SDTSLAEGSV SCFDESLGHN SNMGRDSGTM GSDSDESVIQ TKRVSPEPEL QLRPYQMEVA QPA LDGKNI IICLPTGSGK TRVAVYITKD HLDKKKQASE SGKVIVLVNK VMLAEQLFRK EFNPYLKKWY RIIGLSGDTQ LKIS FPEVV KSYDVIISTA QILENSLLNL ESGDDDGVQL SDFSLIIIDE CHHTNKEAVY NNIMRRYLKQ KLRNNDLKKQ NKPAI PLPQ ILGLTASPGV GAAKKQSEAE KHILNICANL DAFTIKTVKE NLGQLKHQIK EPCKKFVIAD DTRENPFKEK LLEIMA SIQ TYCQKSPMSD FGTQHYEQWA IQMEKKAAKD GNRKDRVCAE HLRKYNEALQ INDTIRMIDA YSHLETFYTD EKEKKFA VL NDSKKSLKLD ETDEFLMNLF FDNKKMLKKL AENPKYENEK LIKLRNTILE QFTRSEESSR GIIFTKTRQS TYALSQWI M ENAKFAEVGV KAHHLIGAGH SSEVKPMTQT EQKEVISKFR TGEINLLIAT TVAEEGLDIK ECNIVIRYGL VTNEIAMVQ ARGRARADES TYVLVTSSGS GVTEREIVND FREKMMYKAI NRVQNMKPEE YAHKILELQV QSILEKKMKV KRSIAKQYND NPSLITLLC KNCSMLVCSG ENIHVIEKMH HVNMTPEFKG LYIVRENKAL QKKFADYQTN GEIICKCGQA WGTMMVHKGL D LPCLKIRN FVVNFKNNSP KKQYKKWVEL PIRFPDLDYS EYCLYSDED

UniProtKB: Interferon-induced helicase C domain-containing protein 1

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Macromolecule #2: RNA (5'-R(P*GP*UP*CP*AP*AP*GP*CP*CP*GP*AP*GP*GP*AP*GP*A)-3')

MacromoleculeName: RNA (5'-R(P*GP*UP*CP*AP*AP*GP*CP*CP*GP*AP*GP*GP*AP*GP*A)-3')
type: rna / ID: 2 / Number of copies: 1
Source (natural)Organism: Pseudomonas phage phi6 (bacteriophage)
Molecular weightTheoretical: 4.894018 KDa
SequenceString:
GUCAAGCCGA GGAGA

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Macromolecule #3: RNA (5'-R(P*UP*CP*UP*CP*CP*UP*CP*GP*GP*CP*UP*UP*GP*AP*C)-3')

MacromoleculeName: RNA (5'-R(P*UP*CP*UP*CP*CP*UP*CP*GP*GP*CP*UP*UP*GP*AP*C)-3')
type: rna / ID: 3 / Number of copies: 1
Source (natural)Organism: Pseudomonas phage phi6 (bacteriophage)
Molecular weightTheoretical: 4.681785 KDa
SequenceString:
UCUCCUCGGC UUGAC

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Macromolecule #4: ADENOSINE-5'-DIPHOSPHATE

MacromoleculeName: ADENOSINE-5'-DIPHOSPHATE / type: ligand / ID: 4 / Number of copies: 1 / Formula: ADP
Molecular weightTheoretical: 427.201 Da
Chemical component information

ChemComp-ADP:
ADENOSINE-5'-DIPHOSPHATE / ADP, energy-carrying molecule*YM

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Macromolecule #5: TETRAFLUOROALUMINATE ION

MacromoleculeName: TETRAFLUOROALUMINATE ION / type: ligand / ID: 5 / Number of copies: 1 / Formula: ALF
Molecular weightTheoretical: 102.975 Da
Chemical component information

ChemComp-ALF:
TETRAFLUOROALUMINATE ION

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Macromolecule #6: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 6 / Number of copies: 1 / Formula: MG
Molecular weightTheoretical: 24.305 Da

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Macromolecule #7: ZINC ION

MacromoleculeName: ZINC ION / type: ligand / ID: 7 / Number of copies: 1 / Formula: ZN
Molecular weightTheoretical: 65.409 Da

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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

Concentration0.5 mg/mL
BufferpH: 7.7
Component:
ConcentrationNameFormula
20.0 mMHEPES
0.1 Mpotassium chlorideKCl
5.0 mMmagnesium chlorideMgCl2
2.0 mMDTT
GridModel: Quantifoil R1.2/1.3 / Material: GOLD / Mesh: 300 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 60 sec. / Pretreatment - Atmosphere: AIR / Details: 25 mA
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV
DetailsSamples were diluted twofold from 1 mg/ml to 0.5 mg/ml immediately prior to plunge freezing

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: FEI FALCON III (4k x 4k) / Detector mode: COUNTING / Digitization - Dimensions - Width: 4096 pixel / Digitization - Dimensions - Height: 4096 pixel / Average electron dose: 29.85 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 75000 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: -2.7 µm / Nominal defocus min: -1.8 µm
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: 46.5105 Å
Applied symmetry - Helical parameters - Δ&Phi: 87.8315 °
Applied symmetry - Helical parameters - Axial symmetry: C1 (asymmetric)
Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 4.06 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 2.1.0) / Number images used: 31556
Segment selectionNumber selected: 234835 / Software - Name: RELION (ver. 2.1.0)
Startup modelType of model: INSILICO MODEL
In silico model: A cylinder was used as the initial model for 3D image reconstruction
Final angle assignmentType: NOT APPLICABLE / Software - Name: RELION (ver. 2.1.0)
FSC plot (resolution estimation)

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Atomic model buiding 1

RefinementSpace: REAL / Protocol: FLEXIBLE FIT / Overall B value: 175 / Target criteria: Cross-correlation coefficient
Output model

PDB-6gkh:
CryoEM structure of the MDA5-dsRNA filament in complex with ADP-AlF4

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