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- PDB-9f1u: Cryo-EM structure of the I923V MDA5-dsRNA filament with ADP-AlF4 ... -

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Basic information

Entry
Database: PDB / ID: 9f1u
TitleCryo-EM structure of the I923V MDA5-dsRNA filament with ADP-AlF4 bound and 81-degree helical twist
Components
  • Interferon-induced helicase C domain-containing protein 1
  • RNA (5'-R(P*AP*UP*CP*UP*CP*CP*UP*CP*GP*GP*CP*UP*UP*G)-3')
  • RNA (5'-R(P*CP*AP*AP*GP*CP*CP*GP*AP*GP*GP*AP*GP*AP*U)-3')
KeywordsIMMUNE SYSTEM / PROTEIN-RNA COMPLEX / HELICAL FILAMENT / ATPASE / INNATE IMMUNE RECEPTOR
Function / homology
Function and homology information


MDA-5 signaling pathway / Ub-specific processing proteases / positive regulation of response to cytokine stimulus / type I interferon-mediated signaling pathway / negative regulation of viral genome replication / pattern recognition receptor activity / 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 / type I interferon-mediated signaling pathway / negative regulation of viral genome replication / pattern recognition receptor activity / 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 / single-stranded RNA binding / RNA helicase activity / 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
ADENOSINE-5'-DIPHOSPHATE / TETRAFLUOROALUMINATE ION / RNA / RNA (> 10) / Interferon-induced helicase C domain-containing protein 1
Similarity search - Component
Biological speciesMus musculus (house mouse)
MethodELECTRON MICROSCOPY / helical reconstruction / cryo EM / Resolution: 3.67 Å
AuthorsSingh, R. / Herrero del Valle, A. / Modis, Y.
Funding support United Kingdom, France, 4items
OrganizationGrant numberCountry
Wellcome Trust101908/Z/13/Z United Kingdom
Wellcome Trust217191/Z/19/Z United Kingdom
Wellcome Trust215378/Z/19/Z United Kingdom
Human Frontier Science Program (HFSP)LT000454/2021-L France
Citation
Journal: Cell Rep / Year: 2025
Title: Molecular basis of autoimmune disease protection by MDA5 variants.
Authors: Rahul Singh / Joe D Joiner / Alba Herrero Del Valle / Marleen Zwaagstra / Ida Jobe / Brian J Ferguson / Frank J M van Kuppeveld / Yorgo Modis /
Abstract: MDA5 recognizes double-stranded RNA (dsRNA) from viruses and retroelements. Cooperative filament formation and ATP-dependent proofreading confer MDA5 with the necessary sensitivity and specificity ...MDA5 recognizes double-stranded RNA (dsRNA) from viruses and retroelements. Cooperative filament formation and ATP-dependent proofreading confer MDA5 with the necessary sensitivity and specificity for dsRNA. Many MDA5 genetic variants are associated with protection from autoimmune disease while increasing the risk of infection and chronic inflammation. How these variants affect RNA sensing remains unclear. Here, we determine the consequences of autoimmune-protective variants on the molecular structure and activities of MDA5. Rare variants E627 and I923V reduce the interferon response to picornavirus infection. E627 does not bind RNA. I923V is ATPase hyperactive, causing premature dissociation from dsRNA. Cryoelectron microscopy (cryo-EM) structures of MDA5 I923V bound to dsRNA at different stages of ATP hydrolysis reveal smaller RNA binding interfaces, leading to excessive proofreading activity. Variants R843H and T946A, which are genetically linked and cause mild phenotypes, did not affect cytokine induction, suggesting an indirect disease mechanism. In conclusion, autoimmune-protective MDA5 variants dampen MDA5-dependent signaling via multiple mechanisms.
#1: Journal: Biorxiv / Year: 2024
Title: Molecular basis of autoimmune disease protection by MDA5 variants
Authors: Singh, R. / Herrero del Valle, A. / Joiner, J.D. / Zwaagstra, M. / Ferguson, B.J. / van Kuppeveld, F.J.M. / Modis, Y.
History
DepositionApr 20, 2024Deposition site: PDBE / Processing site: PDBE
Revision 1.0Feb 26, 2025Provider: repository / Type: Initial release
Revision 1.0Feb 26, 2025Data content type: EM metadata / Data content type: EM metadata / Provider: repository / Type: Initial release
Revision 1.0Feb 26, 2025Data content type: FSC / Data content type: FSC / Provider: repository / Type: Initial release
Revision 1.0Feb 26, 2025Data content type: Half map / Part number: 1 / Data content type: Half map / Provider: repository / Type: Initial release
Revision 1.0Feb 26, 2025Data content type: Half map / Part number: 2 / Data content type: Half map / Provider: repository / Type: Initial release
Revision 1.0Feb 26, 2025Data content type: Image / Data content type: Image / Provider: repository / Type: Initial release
Revision 1.0Feb 26, 2025Data content type: Mask / Part number: 1 / Data content type: Mask / Provider: repository / Type: Initial release
Revision 1.0Feb 26, 2025Data content type: Primary map / Data content type: Primary map / Provider: repository / Type: Initial release
Revision 1.1Jun 18, 2025Group: Data collection / Database references / Category: citation / citation_author / em_admin / Item: _em_admin.last_update

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Interferon-induced helicase C domain-containing protein 1
X: RNA (5'-R(P*CP*AP*AP*GP*CP*CP*GP*AP*GP*GP*AP*GP*AP*U)-3')
Z: RNA (5'-R(P*AP*UP*CP*UP*CP*CP*UP*CP*GP*GP*CP*UP*UP*G)-3')
hetero molecules


Theoretical massNumber of molelcules
Total (without water)126,2046
Polymers125,6083
Non-polymers5963
Water00
1
A: Interferon-induced helicase C domain-containing protein 1
X: RNA (5'-R(P*CP*AP*AP*GP*CP*CP*GP*AP*GP*GP*AP*GP*AP*U)-3')
Z: RNA (5'-R(P*AP*UP*CP*UP*CP*CP*UP*CP*GP*GP*CP*UP*UP*G)-3')
hetero molecules

A: Interferon-induced helicase C domain-containing protein 1
X: RNA (5'-R(P*CP*AP*AP*GP*CP*CP*GP*AP*GP*GP*AP*GP*AP*U)-3')
Z: RNA (5'-R(P*AP*UP*CP*UP*CP*CP*UP*CP*GP*GP*CP*UP*UP*G)-3')
hetero molecules


Theoretical massNumber of molelcules
Total (without water)252,40812
Polymers251,2176
Non-polymers1,1916
Water0
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
point symmetry operation1

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Components

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Protein , 1 types, 1 molecules A

#1: Protein Interferon-induced helicase C domain-containing protein 1 / Helicase with 2 CARD domains / Helicard / Interferon induced with helicase C domain protein 1 / ...Helicase with 2 CARD domains / Helicard / Interferon induced with helicase C domain protein 1 / Melanoma differentiation-associated protein 5 / MDA-5 / RIG-I-like receptor 2 / RLR-2


Mass: 116683.062 Da / Num. of mol.: 1 / Mutation: I923V
Source method: isolated from a genetically manipulated source
Details: N-terminal hexahistidine tag and TEV cleavage site / Source: (gene. exp.) Mus musculus (house mouse) / Gene: Ifih1 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: Q8R5F7, RNA helicase

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RNA chain , 2 types, 2 molecules XZ

#2: RNA chain RNA (5'-R(P*CP*AP*AP*GP*CP*CP*GP*AP*GP*GP*AP*GP*AP*U)-3')


Mass: 4548.812 Da / Num. of mol.: 1 / Source method: obtained synthetically / Details: In vitro transcribed RNA / Source: (synth.) Mus musculus (house mouse)
#3: RNA chain RNA (5'-R(P*AP*UP*CP*UP*CP*CP*UP*CP*GP*GP*CP*UP*UP*G)-3')


Mass: 4376.604 Da / Num. of mol.: 1 / Source method: obtained synthetically / Details: In vitro transcribed RNA / Source: (synth.) Mus musculus (house mouse)

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Non-polymers , 3 types, 3 molecules

#4: Chemical ChemComp-ADP / ADENOSINE-5'-DIPHOSPHATE


Mass: 427.201 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C10H15N5O10P2 / Feature type: SUBJECT OF INVESTIGATION / Comment: ADP, energy-carrying molecule*YM
#5: Chemical ChemComp-ALF / TETRAFLUOROALUMINATE ION


Mass: 102.975 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: AlF4 / Feature type: SUBJECT OF INVESTIGATION
#6: Chemical ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Zn

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Details

Has ligand of interestY
Has protein modificationN

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: FILAMENT / 3D reconstruction method: helical reconstruction

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Sample preparation

Component
IDNameTypeDetailsEntity IDParent-IDSource
1Mouse MDA5 I923V in complex with double stranded RNACOMPLEXHelical filament. Structure determined with helical symmetry averaging.#1-#30MULTIPLE SOURCES
2Mouse MDA5 I923VCOMPLEX#11RECOMBINANT
3double stranded RNACOMPLEX#2-#31RECOMBINANT
Molecular weightValue: 28.03 kDa/nm / Experimental value: YES
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
22Mus musculus (house mouse)10090
33Mus musculus (house mouse)10090
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-IDStrainPlasmid
22Escherichia coli (E. coli)562Rosetta(DE3)pLysSpET28
33synthetic construct (others)32630
Buffer solutionpH: 7.7
Buffer component
IDConc.NameFormulaBuffer-ID
120 mMHEPESC8H18N2O4S1
20.1 Mpotassium chlorideKC1
35 mMmagnesium chlorideMgCl21
42 mMDTTC4H10O2S21
510 mMADP-AlF4C10H15N5O10P2AlF41
SpecimenConc.: 0.5 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Details: 1 mg/ml MDA5 protein was incubated with 0.05 mg/ml 1-kb dsRNA on ice for 2 min. Samples were diluted twofold with buffer and applied to grids.
Specimen supportDetails: Edwards 12E6/531 glow discharger at 30 mA / Grid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 K / Details: 2-4 s blotting, 15 s wait time

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: DARK FIELD / Nominal magnification: 105000 X / Nominal defocus max: 2500 nm / Nominal defocus min: 500 nm / Cs: 2.7 mm / Alignment procedure: ZEMLIN TABLEAU
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 44 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k)
EM imaging opticsEnergyfilter name: GIF Bioquantum / Energyfilter slit width: 20 eV

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Processing

EM software
IDNameVersionCategory
1crYOLOparticle selection
2EPU3image acquisition
4CTFFIND4.1CTF correction
7UCSF Chimera8.6model fitting
9RELION4initial Euler assignment
10RELION4final Euler assignment
11RELION4classification
12RELION43D reconstruction
13PHENIX1.21model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Helical symmertyAngular rotation/subunit: 80.71 ° / Axial rise/subunit: 43.86 Å / Axial symmetry: C1
Particle selectionNum. of particles selected: 1406812
3D reconstructionResolution: 3.67 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 347343 / Symmetry type: HELICAL
Atomic model buildingB value: 90 / Protocol: RIGID BODY FIT / Space: REAL / Target criteria: Cross-correlation coefficient
Details: Initial local fitting with Fit-in-Map in Chimera followed by real space refinement in Phenix.
Atomic model buildingPDB-ID: 7NIQ

7niq


Pdb chain-ID: A / Accession code: 7NIQ / Source name: PDB / Type: experimental model
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0046133
ELECTRON MICROSCOPYf_angle_d0.6048397
ELECTRON MICROSCOPYf_dihedral_angle_d14.0211139
ELECTRON MICROSCOPYf_chiral_restr0.039975
ELECTRON MICROSCOPYf_plane_restr0.004960

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