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- PDB-6gkm: CryoEM structure of the MDA5-dsRNA filament in complex with ATP (... -

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Basic information

Entry
Database: PDB / ID: 6gkm
TitleCryoEM structure of the MDA5-dsRNA filament in complex with ATP (10 mM)
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 / homologyRIG-I receptor C-terminal domain / Helicase, C-terminal / Helicase/UvrB, N-terminal / Death-like domain superfamily / Helicase superfamily 1/2, ATP-binding domain / RIG-I-like receptor, C-terminal regulatory domain / P-loop containing nucleoside triphosphate hydrolase / Caspase recruitment domain / RIG-I-like receptor, C-terminal domain superfamily / Helicase conserved C-terminal domain ...RIG-I receptor C-terminal domain / Helicase, C-terminal / Helicase/UvrB, N-terminal / Death-like domain superfamily / Helicase superfamily 1/2, ATP-binding domain / RIG-I-like receptor, C-terminal regulatory domain / P-loop containing nucleoside triphosphate hydrolase / Caspase recruitment domain / RIG-I-like receptor, C-terminal domain superfamily / Helicase conserved C-terminal domain / Type III restriction enzyme, res subunit / C-terminal domain of RIG-I / Caspase recruitment domain / Superfamilies 1 and 2 helicase ATP-binding type-1 domain profile. / Superfamilies 1 and 2 helicase C-terminal domain profile. / RIG-I-like receptor (RLR) C-terminal regulatory (CTR) domain profile. / Ub-specific processing proteases / Caspase recruitment domain / MDA-5 signaling pathway / positive regulation of interferon-beta secretion / positive regulation of response to cytokine stimulus / positive regulation of tumor necrosis factor secretion / protein sumoylation / positive regulation of interferon-alpha secretion / cellular response to exogenous dsRNA / positive regulation of interferon-alpha production / positive regulation of interleukin-6 secretion / positive regulation of interferon-beta production / ribonucleoprotein complex binding / single-stranded RNA binding / helicase activity / response to virus / double-stranded RNA binding / RNA helicase / defense response to virus / innate immune response / DNA binding / zinc ion binding / ATP binding / identical protein binding / nucleus / cytoplasm / Interferon-induced helicase C domain-containing protein 1
Function and homology information
Specimen sourceMus musculus (house mouse)
Pseudomonas phage phi6 (bacteriophage)
MethodELECTRON MICROSCOPY / helical reconstruction / cryo EM / 3.87 Å resolution
AuthorsYu, Q. / Qu, K. / Modis, Y.
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.
Validation Report
SummaryFull reportAbout validation report
DateDeposition: May 21, 2018 / Release: Nov 21, 2018
RevisionDateData content typeGroupCategoryItemProviderType
1.0Nov 21, 2018Structure modelrepositoryInitial release
1.1Nov 28, 2018Structure modelData collection / Database referencescitation_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

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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)123,7275
Polyers123,1553
Non-polymers5732
Water0
1


  • idetical with deposited unit
  • defined by author&software
  • Evidence: cross-linking, Chemical crosslinking, microscopy, Negative stain electron microscopy
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TypeNameSymmetry operationNumber
identity operation1_5551
Buried area (Å2)6620
ΔGint (kcal/M)-56
Surface area (Å2)36970
MethodPISA

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Components

#1: Protein/peptide Interferon-induced helicase C domain-containing 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: (gene. exp.) Mus musculus (house mouse) / Gene: Ifih1 / Plasmid name: pET28a / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: Q8R5F7, RNA helicase
#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: (synth.) Pseudomonas phage phi6 (bacteriophage)
#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: (synth.) Pseudomonas phage phi6 (bacteriophage)
#4: Chemical ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 1 / Formula: Zn / Zinc
#5: Chemical ChemComp-ATP / ADENOSINE-5'-TRIPHOSPHATE


Mass: 507.181 Da / Num. of mol.: 1 / Formula: C10H16N5O13P3 / Adenosine triphosphate / Comment: ATP (energy-carrying molecule) *YM

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Experimental details

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Experiment

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

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Sample preparation

Component

Type: COMPLEX

IDNameDetailsEntity IDParent IDSource
1MDA5-dsRNA helical filament in complex with ATPFilaments formed in the presence of 10 mM ATP and frozen 7.5 min after addition of ATP1, 2, 30MULTIPLE SOURCES
2MDA5 bound to ATPDExD/H-box helicase consisting of Hel1, Hel2, Hel2i, and pincer domains, followed by a C-terminal domain11RECOMBINANT
3Double-stranded RNA from bacteriophage Phi62, 31NATURAL
Molecular weight
IDValueEntity assembly IDExperimental value
128.53 kDa/nm1NO
20.114 MDa1NO
32.171 kDa/nm1NO
Source (natural)
IDEntity assembly IDNcbi tax IDOrganism
1210090Mus musculus (house mouse)
2310879Pseudomonas savastanoi pv. phaseolicola (bacteria)
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
Details: Samples were diluted twofold from 1 mg/ml to 0.5 mg/ml immediately prior to plunge freezing
Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: 25 mA / Grid material: GOLD / Grid mesh size: 300 / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 kelvins

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyMicroscope model: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Calibrated magnification: 75000 / Nominal defocus max: -3100 nm / Nominal defocus min: -1700 nm / Alignment procedure: ZEMLIN TABLEAU
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 30.24 e/Å2 / Detector mode: COUNTING / Film or detector model: FEI FALCON III (4k x 4k)
EM imaging opticsChr aberration corrector: None / Phase plate: None / Sph aberration corrector: None
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
9RELION2.1.0initial Euler assignment
10RELION2.1.0final Euler assignment
11RELION2.1.0classification
12RELION2.1.03D reconstruction
13PHENIX1.13model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Helical symmertyAngular rotation/subunit: 72.8171 deg. / Axial rise/subunit: 43.0617 Å / Axial symmetry: C1
Particle selectionNumber of particles selected: 526596
3D reconstructionResolution: 3.87 Å / Resolution method: FSC 0.143 CUT-OFF / Number of particles: 100482 / Algorithm: FOURIER SPACE / Number of class averages: 1 / Symmetry type: HELICAL
Atomic model buildingOverall b value: 175 / Ref protocol: FLEXIBLE FIT / Ref space: REAL / Target criteria: Cross-correlation coefficient

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