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

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Basic information

Entry
Database: PDB / ID: 6gkh
TitleCryoEM structure of the MDA5-dsRNA filament in complex with ADP-AlF4
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')
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 / cellular response to exogenous dsRNA / pattern recognition receptor activity / negative regulation of viral genome replication / type I interferon-mediated signaling pathway / positive regulation of interferon-alpha production / protein complex oligomerization / protein sumoylation ...MDA-5 signaling pathway / Ub-specific processing proteases / positive regulation of response to cytokine stimulus / cellular response to exogenous dsRNA / pattern recognition receptor activity / negative regulation of viral genome replication / type I interferon-mediated signaling pathway / positive regulation of interferon-alpha production / protein complex oligomerization / 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 / Caspase recruitment domain ...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 / 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)
Pseudomonas phage phi6 (bacteriophage)
MethodELECTRON MICROSCOPY / helical reconstruction / cryo EM / Resolution: 4.06 Å
AuthorsYu, Q. / Qu, K. / Modis, Y.
Funding support United Kingdom, 2items
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
DepositionMay 21, 2018Deposition site: PDBE / Processing site: PDBE
Revision 1.0Nov 21, 2018Provider: repository / Type: Initial release
Revision 1.1Nov 28, 2018Group: Data collection / Database references / Category: citation
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_ASTM / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year
Revision 1.2Jan 2, 2019Group: Data collection / Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first / _citation.page_last
Revision 1.3Nov 18, 2020Group: Data collection / Category: em_imaging_optics
Item: _em_imaging_optics.chr_aberration_corrector / _em_imaging_optics.phase_plate / _em_imaging_optics.sph_aberration_corrector

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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)124,4107
Polymers123,7903
Non-polymers6204
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: cross-linking, chemical cross-linking, microscopy, negative-stain electron microscopy
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area6740 Å2
ΔGint-58 kcal/mol
Surface area37030 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: 114214.477 Da / Num. of mol.: 1 / Mutation: Residues 646-663 deleted
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mus musculus (house mouse) / Gene: Ifih1 / Plasmid: pET28a / 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 / Source: (synth.) Pseudomonas phage phi6 (bacteriophage)
#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 / Source: (synth.) Pseudomonas phage phi6 (bacteriophage)

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Non-polymers , 4 types, 4 molecules

#4: Chemical ChemComp-ADP / ADENOSINE-5'-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-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Mg
#7: Chemical ChemComp-ZN / ZINC ION


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

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Experimental details

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Experiment

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

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Sample preparation

Component
IDNameTypeEntity IDParent-IDSourceDetails
1MDA5-dsRNA helical filament in complex with ADP-AlF4COMPLEX#1-#30MULTIPLE SOURCES
2MDA5 bound to ADP-AlF4COMPLEX#11RECOMBINANTDExD/H-box helicase consisting of Hel1, Hel2, Hel2i, and pincer domains, followed by a C-terminal domain
3Double-stranded RNA from bacteriophage Phi6COMPLEX#2-#31NATURAL
Molecular weight
IDEntity assembly-IDValue (°)Experimental value
1126.41 kDa/nmNO
210.114 MDaNO
312.162 kDa/nmNO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
12Mus musculus (house mouse)10090
23Pseudomonas savastanoi pv. phaseolicola (bacteria)10879
Source (recombinant)Organism: Escherichia coli (E. coli) / Strain: BL21(DE3)
Buffer solutionpH: 7.7
Buffer component
IDConc.NameFormulaBuffer-ID
120 mMHEPES1
20.1 Mpotassium chlorideKCl1
35 mMmagnesium chlorideMgCl21
42 mMDTT1
SpecimenConc.: 0.5 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Details: Samples were diluted twofold from 1 mg/ml to 0.5 mg/ml immediately prior to plunge freezing
Specimen supportDetails: 25 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

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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 FIELD / Calibrated magnification: 75000 X / Nominal defocus max: -2700 nm / Nominal defocus min: -1800 nm / Alignment procedure: ZEMLIN TABLEAU
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 29.85 e/Å2 / Detector mode: COUNTING / Film or detector model: FEI FALCON III (4k x 4k)
Image scansWidth: 4096 / Height: 4096

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Processing

EM software
IDNameVersionCategory
1RELION2.1.0particle selection
2EPU1.9.1image acquisition
4Gctf1.06CTF correction
7Coot0.8.9model fitting
9PHENIX1.13model refinement
10RELION2.1.0initial Euler assignment
11RELION2.1.0final Euler assignment
12RELION2.1.0classification
13RELION2.1.03D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Helical symmertyAngular rotation/subunit: 87.8315 ° / Axial rise/subunit: 46.5105 Å / Axial symmetry: C1
Particle selectionNum. of particles selected: 234835
3D reconstructionResolution: 4.06 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 31556 / Algorithm: FOURIER SPACE / Num. of class averages: 1 / Symmetry type: HELICAL
Atomic model buildingB value: 175 / Protocol: FLEXIBLE FIT / Space: REAL / Target criteria: Cross-correlation coefficient

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