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- PDB-7bkp: CryoEM structure of disease related M854K MDA5-dsRNA filament in ... -

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Basic information

Entry
Database: PDB / ID: 7bkp
TitleCryoEM structure of disease related M854K MDA5-dsRNA filament in complex with ATP
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 / positive regulation of response to cytokine stimulus / Ub-specific processing proteases / negative regulation of viral genome replication / type I interferon-mediated signaling pathway / pattern recognition receptor activity / cellular response to exogenous dsRNA / protein complex oligomerization / positive regulation of interferon-alpha production / protein sumoylation ...MDA-5 signaling pathway / positive regulation of response to cytokine stimulus / Ub-specific processing proteases / negative regulation of viral genome replication / type I interferon-mediated signaling pathway / 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 / cellular response to virus / positive regulation of interleukin-6 production / response to virus / 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
ADENOSINE-5'-TRIPHOSPHATE / RNA / RNA (> 10) / Interferon-induced helicase C domain-containing protein 1
Similarity search - Component
Biological speciesMus musculus (house mouse)
Pseudomonas virus phi6
MethodELECTRON MICROSCOPY / helical reconstruction / cryo EM / Resolution: 2.8 Å
AuthorsSingh, R. / Herrero del Valle, A. / Yu, Q. / Modis, Y.
Funding support United Kingdom, 2items
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
DepositionJan 16, 2021Deposition site: PDBE / Processing site: PDBE
Revision 1.0Nov 17, 2021Provider: repository / Type: Initial release
Revision 1.1Dec 1, 2021Group: Database references / Category: citation / citation_author
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_PubMed / _citation.title / _citation_author.identifier_ORCID / _citation_author.name
Revision 1.2Oct 16, 2024Group: Data collection / Refinement description / Structure summary
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / em_3d_fitting_list / em_admin / pdbx_entry_details / pdbx_initial_refinement_model / pdbx_modification_feature
Item: _em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id ..._em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id / _em_3d_fitting_list.source_name / _em_3d_fitting_list.type / _em_admin.last_update / _pdbx_entry_details.has_protein_modification

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Structure visualization

Movie
  • Deposited structure unit
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  • EMDB-12213
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Structure viewerMolecule:
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Assembly

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


Theoretical massNumber of molelcules
Total (without water)125,6606
Polymers125,0633
Non-polymers5973
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: microscopy, CryoEM
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area6300 Å2
ΔGint-71 kcal/mol
Surface area38480 Å2
MethodPISA

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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: 116122.359 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Details: ATP / Source: (gene. exp.) Mus musculus (house mouse) / Gene: Ifih1 / Production host: Escherichia coli BL21(DE3) (bacteria) / Strain (production host): BL21(DE3) / References: UniProt: Q8R5F7, RNA helicase

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RNA chain , 2 types, 2 molecules ZX

#2: 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 / Source: (synth.) Pseudomonas virus phi6
#3: 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 / Source: (synth.) Pseudomonas virus phi6

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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-ATP / ADENOSINE-5'-TRIPHOSPHATE


Mass: 507.181 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C10H16N5O13P3 / Comment: ATP, energy-carrying molecule*YM
#6: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Mg

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Details

Has ligand of interestN
Has protein modificationY

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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
IDNameTypeEntity IDParent-IDSource
1M854K MDA5-dsRNA filament in complex with ATPCOMPLEX#1-#30MULTIPLE SOURCES
2INTERFERON-INDUCED HELICASE C DOMAIN-CONTAINING PROTEINCOMPLEX#11RECOMBINANT
3RNACOMPLEX#2-#31RECOMBINANT
Molecular weight
IDEntity assembly-IDValue (°)Experimental value
1128.2 kDa/nmYES
2126.36 kDa/nmYES
311.91 kDa/nmYES
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
22Mus musculus (house mouse)10090
33Pseudomonas virus phi610879
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-ID
22Escherichia coli BL21(DE3) (bacteria)469008
33synthetic construct (others)32630
Buffer solutionpH: 7.8
Buffer component
IDConc.NameFormulaBuffer-ID
130 mMHEPES1
20.15 MKCl1
31 mMDithiothreitol1
410 mMATP1
SpecimenConc.: 1 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: machine step 6 / Grid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: UltrAuFoil
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE-PROPANE / Humidity: 100 % / Chamber temperature: 277.2 K
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
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: DARK FIELD / Nominal defocus max: -2500 nm / Nominal defocus min: -1200 nm / Calibrated defocus min: -250 nm / Calibrated defocus max: -4000 nm / Cs: 2.7 mm / C2 aperture diameter: 70 µm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 40 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) / Num. of real images: 7147
EM imaging opticsEnergyfilter slit width: 20 eV / Phase plate: OTHER / Spherical aberration corrector: none
Image scansWidth: 5760 / Height: 4092

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Processing

Software
NameVersionClassification
phenix.real_space_refine1.18.2_3874refinement
PHENIX1.18.2_3874refinement
EM software
IDNameVersionCategory
1RELION3.1particle selection
2EPUimage acquisition
4CTFFINDCTF correction
7UCSF Chimera1.13.1model fitting
9PHENIX1.18.2model refinement
10RELION3.1initial Euler assignment
11RELION3.1final Euler assignment
12RELION3.1classification
13RELION3.13D reconstruction
Image processingDetails: The images were motion-corrected
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Helical symmertyAngular rotation/subunit: 74.63 ° / Axial rise/subunit: 43.92 Å / Axial symmetry: C1
Particle selectionNum. of particles selected: 1068108
3D reconstructionResolution: 2.8 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 383128 / Num. of class averages: 1 / Symmetry type: HELICAL
Atomic model buildingB value: 50 / Protocol: OTHER / Space: RECIPROCAL / Target criteria: Correlation coefficient
Atomic model buildingPDB-ID: 6G19

6g19


Accession code: 6G19 / Source name: PDB / Type: experimental model
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 40 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00926317
ELECTRON MICROSCOPYf_angle_d0.75438635
ELECTRON MICROSCOPYf_chiral_restr0.0483995
ELECTRON MICROSCOPYf_plane_restr0.0038991
ELECTRON MICROSCOPYf_dihedral_angle_d15.0212524

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