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

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Basic information

Entry
Database: PDB / ID: 7nic
TitleCryoEM structure of disease related M854K MDA5-dsRNA filament in complex with ADP-AlF4(minor class)
Components
  • Interferon-induced helicase C domain-containing protein 1
  • RNA (5'-R(P*GP*UP*CP*AP*AP*GP*CP*CP*GP*AP*GP*GP*AP*GP*A)-3')
  • RNA (5'-R(P*UP*CP*UP*CP*CP*UP*CP*GP*GP*CP*UP*UP*GP*AP*C)-3')
KeywordsANTIVIRAL PROTEIN / PROTEIN-RNA COMPLEX / HELICAL FILAMENT / ATPASE / INNATE IMMUNE RECEPTOR / IMMUNE SYSTEM
Function / homology
Function and homology information


adenyl ribonucleotide binding / MDA-5 signaling pathway / purine ribonucleoside triphosphate binding / positive regulation of response to cytokine stimulus / Ub-specific processing proteases / pattern recognition receptor activity / negative regulation of viral genome replication / cellular response to exogenous dsRNA / type I interferon-mediated signaling pathway / positive regulation of interferon-alpha production ...adenyl ribonucleotide binding / MDA-5 signaling pathway / purine ribonucleoside triphosphate binding / positive regulation of response to cytokine stimulus / Ub-specific processing proteases / pattern recognition receptor activity / negative regulation of viral genome replication / cellular response to exogenous dsRNA / type I interferon-mediated signaling pathway / positive regulation of interferon-alpha production / antiviral innate immune response / protein sumoylation / ribonucleoprotein complex binding / positive regulation of interferon-beta production / response to virus / cellular response to virus / positive regulation of interleukin-6 production / double-stranded RNA binding / positive regulation of tumor necrosis factor production / protein complex oligomerization / 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 / RNA 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 / Type III restriction enzyme, res subunit ...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)
synthetic construct (others)
MethodELECTRON MICROSCOPY / helical reconstruction / cryo EM / Resolution: 4.3 Å
AuthorsYu, 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
DepositionFeb 11, 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

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Structure visualization

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Assembly

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


Theoretical massNumber of molelcules
Total (without water)126,2946
Polymers125,6983
Non-polymers5963
Water0
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: microscopy
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area6530 Å2
ΔGint-54 kcal/mol
Surface area35990 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
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 XY

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


Mass: 4894.018 Da / Num. of mol.: 1 / Source method: obtained synthetically / Details: Pseudomonas virus phi6 Tax ID 10879 / Source: (synth.) synthetic construct (others)
#3: RNA chain RNA (5'-R(P*UP*CP*UP*CP*CP*UP*CP*GP*GP*CP*UP*UP*GP*AP*C)-3')


Mass: 4681.785 Da / Num. of mol.: 1 / Source method: obtained synthetically / Details: Pseudomonas virus phi6 Tax ID 10879 / Source: (synth.) synthetic construct (others)

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Non-polymers , 3 types, 3 molecules

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


Mass: 427.201 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C10H15N5O10P2 / 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
#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 interestN

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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
1Disease mutant M854K MDA5-dsRNA filament in complex with ADP-AlF4COMPLEX#1-#30RECOMBINANT
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
12Mus musculus (house mouse)10090
23synthetic construct (others)32630
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-ID
12Escherichia coli BL21(DE3) (bacteria)469008
23synthetic construct (others)32630
Buffer solutionpH: 7.7
Buffer component
IDConc.NameFormulaBuffer-ID
120 mMHEPES1
20.1 MKCl1
35 mMMgCl21
42 mMDithiothreitol1
51 mMADP-AlF41
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: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / 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: BRIGHT FIELDBright-field microscopy
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 60 sec. / Electron dose: 29.6 e/Å2 / Detector mode: COUNTING / Film or detector model: FEI FALCON III (4k x 4k)
EM imaging opticsPhase plate: OTHER / Spherical aberration corrector: none

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Processing

Software
NameVersionClassificationNB
phenix.real_space_refine1.18.2_3874refinement
PHENIX1.18.2_3874refinement
EM software
IDNameCategory
1RELIONparticle selection
2EPUimage acquisition
4CTFFINDCTF correction
7UCSF Chimeramodel fitting
9RELIONinitial Euler assignment
10RELIONfinal Euler assignment
11RELIONclassification
12RELION3D reconstruction
13PHENIXmodel refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Helical symmertyAngular rotation/subunit: 88.0297 ° / Axial rise/subunit: 45.0795 Å / Axial symmetry: C1
Particle selectionNum. of particles selected: 731263
3D reconstructionResolution: 4.3 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 62028 / Num. of class averages: 1 / Symmetry type: HELICAL
Atomic model buildingB value: 147 / Protocol: FLEXIBLE FIT / Space: REAL
Atomic model buildingPDB-ID: 6GKH

6gkh

RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 125.79 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00826327
ELECTRON MICROSCOPYf_angle_d0.73638660
ELECTRON MICROSCOPYf_chiral_restr0.072998
ELECTRON MICROSCOPYf_plane_restr0.0033987
ELECTRON MICROSCOPYf_dihedral_angle_d12.92032531

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