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- PDB-9f2l: Cryo-EM structure of the I923V MDA5-dsRNA filament with ADP-AlF4 ... -

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Basic information

Entry
Database: PDB / ID: 9f2l
TitleCryo-EM structure of the I923V MDA5-dsRNA filament with ADP-AlF4 bound and 73-degree helical twist
Components
  • Interferon-induced helicase C domain-containing protein 1
  • RNA (5'-R(P*CP*AP*AP*GP*CP*CP*GP*AP*GP*GP*AP*GP*AP*G)-3')
  • RNA (5'-R(P*CP*UP*CP*UP*CP*CP*UP*CP*GP*GP*CP*UP*UP*G)-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 / 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 / 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: 3.86 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
CitationJournal: 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 23, 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

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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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*G)-3')
Y: RNA (5'-R(P*CP*UP*CP*UP*CP*CP*UP*CP*GP*GP*CP*UP*UP*G)-3')
hetero molecules


Theoretical massNumber of molelcules
Total (without water)126,2196
Polymers125,6233
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*G)-3')
Y: RNA (5'-R(P*CP*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*G)-3')
Y: RNA (5'-R(P*CP*UP*CP*UP*CP*CP*UP*CP*GP*GP*CP*UP*UP*G)-3')
hetero molecules


Theoretical massNumber of molelcules
Total (without water)252,43812
Polymers251,2476
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, delta646-663
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 / Plasmid: pET28a / Production host: Escherichia coli (E. coli) / Strain (production host): Rosetta(DE3)pLysS / References: UniProt: Q8R5F7, RNA helicase

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RNA chain , 2 types, 2 molecules XY

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


Mass: 4587.852 Da / Num. of mol.: 1 / Source method: obtained synthetically / Details: In vitro transcribed RNA CAAGCCGAGGAGAG / Source: (synth.) Mus musculus (house mouse)
#3: RNA chain RNA (5'-R(P*CP*UP*CP*UP*CP*CP*UP*CP*GP*GP*CP*UP*UP*G)-3')


Mass: 4352.580 Da / Num. of mol.: 1 / Source method: obtained synthetically / Details: In vitro transcribed RNA CUCUCCUCGGCUUG / Source: (synth.) Mus musculus (house mouse)

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Non-polymers , 3 types, 3 molecules

#4: Chemical ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Zn
#5: 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
#6: Chemical ChemComp-ALF / TETRAFLUOROALUMINATE ION


Mass: 102.975 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: AlF4 / Feature type: SUBJECT OF INVESTIGATION

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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 chlorideKCl1
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 Chimeramodel fitting
9RELION4initial Euler assignment
10RELION4final Euler assignment
11RELION4classification
12RELION43D reconstruction
13PHENIX1.21model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Helical symmertyAngular rotation/subunit: 73.94 ° / Axial rise/subunit: 42.78 Å / Axial symmetry: C1
Particle selectionNum. of particles selected: 1406812
3D reconstructionResolution: 3.86 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 53484 / Algorithm: BACK PROJECTION / Symmetry type: HELICAL
Atomic model buildingB value: 50 / 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: 7BKP

7bkp


Pdb chain-ID: A / Accession code: 7BKP / Source name: PDB / Type: experimental model
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0036283
ELECTRON MICROSCOPYf_angle_d0.5728589
ELECTRON MICROSCOPYf_dihedral_angle_d14.0011162
ELECTRON MICROSCOPYf_chiral_restr0.039992
ELECTRON MICROSCOPYf_plane_restr0.006985

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