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- EMDB-0012: CryoEM structure of the MDA5-dsRNA filament in complex with AMPPNP -

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Basic information

Entry
Database: EMDB / ID: 0012
TitleCryoEM structure of the MDA5-dsRNA filament in complex with AMPPNP
Map dataMDA5-dsRNA helical reconstruction in the presence of 2.5 mM AMPPNP
SampleMDA5-dsRNA helical filament in complex with AMPPNP
  • MDA5 bound to AMPPNP
  • Double-stranded RNA from bacteriophage Phi6RNA
  • Interferon-induced helicase C domain-containing protein 1
  • (nucleic-acidNucleic acid) x 2
  • (ligand) x 2
Function / homologyP-loop containing nucleoside triphosphate hydrolase / Caspase recruitment domain / RIG-I-like receptor, C-terminal regulatory domain / Helicase superfamily 1/2, ATP-binding domain / Death-like domain superfamily / Helicase/UvrB, N-terminal / Helicase, C-terminal / RIG-I-like receptor, C-terminal domain superfamily / Helicase conserved C-terminal domain / Type III restriction enzyme, res subunit ...P-loop containing nucleoside triphosphate hydrolase / Caspase recruitment domain / RIG-I-like receptor, C-terminal regulatory domain / Helicase superfamily 1/2, ATP-binding domain / Death-like domain superfamily / Helicase/UvrB, N-terminal / Helicase, C-terminal / 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 / Superfamilies 1 and 2 helicase ATP-binding type-1 domain profile. / Ub-specific processing proteases / RIG-I-like receptor (RLR) C-terminal regulatory (CTR) domain profile. / Caspase recruitment domain / Superfamilies 1 and 2 helicase C-terminal domain profile. / 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 production / positive regulation of interferon-alpha secretion / cellular response to exogenous dsRNA / positive regulation of interleukin-6 secretion / positive regulation of interferon-beta production / ribonucleoprotein complex binding / single-stranded RNA binding / helicase activity / response to virus / RNA helicase / double-stranded RNA binding / 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
SourceMus musculus (house mouse) / Pseudomonas savastanoi pv. phaseolicola (bacteria) / Pseudomonas phage phi6 (bacteriophage)
Methodhelical reconstruction / cryo EM / 4.06 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 ReportPDB-ID: 6gjz

SummaryFull reportAbout validation report
DateDeposition: May 17, 2018 / Header (metadata) release: Apr 11, 2018 / Map release: Nov 21, 2018 / Last update: Jan 2, 2019

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.0495
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.0495
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: : PDB-6gjz
  • Surface level: 0.0495
  • Imaged by UCSF Chimera
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  • Simplified surface model + fitted atomic model
  • Atomic models: PDB-6gjz
  • Imaged by Jmol
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_0012.png

Downloads & links

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Map

Fileemd_0012.map.gz (map file in CCP4 format, 44958 KB)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
224 pix
1.07 Å/pix.
= 239.68 Å		224 pix
1.07 Å/pix.
= 239.68 Å		224 pix
1.07 Å/pix.
= 239.68 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.07 Å
Density

Histogram

Histogram (log scale)
Contour Level:0.0495 (by author), 0.0495 (movie #1):
Minimum - Maximum-0.075805224 - 0.18155327
Average (Standard dev.)0.0010824882 (0.009103025)
Details

EMDB XML:

Space Group Number1
Map Geometry
Axis orderXYZ
Dimensions224224224
Origin0.00.00.0
Limit223.0223.0223.0
Spacing224224224
CellA=B=C: 239.68001 Å
α=β=γ: 90.0 deg.

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.071.071.07
M x/y/z224224224
origin x/y/z0.0000.0000.000
length x/y/z239.680239.680239.680
α/β/γ90.00090.00090.000
start NX/NY/NZ
NX/NY/NZ
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS224224224
D min/max/mean-0.0760.1820.001

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Supplemental data

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Mask #1

Fileemd_0012_msk_1.map
Projections & Slices
AxesZYX
Projections
Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire MDA5-dsRNA helical filament in complex with AMPPNP

EntireName: MDA5-dsRNA helical filament in complex with AMPPNP / Number of components: 8

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Component #1: protein, MDA5-dsRNA helical filament in complex with AMPPNP

ProteinName: MDA5-dsRNA helical filament in complex with AMPPNP / Recombinant expression: No
MassTheoretical: 2.079 MDa

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Component #2: protein, MDA5 bound to AMPPNP

ProteinName: MDA5 bound to AMPPNP
Details: DExD/H-box helicase consisting of Hel1, Hel2, Hel2i, and pincer domains, followed by a C-terminal domain
Recombinant expression: No
SourceSpecies: Mus musculus (house mouse)
Source (engineered)Expression System: Escherichia coli (E. coli) / Strain: BL21(DE3)

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Component #3: protein, Double-stranded RNA from bacteriophage Phi6

ProteinName: Double-stranded RNA from bacteriophage Phi6RNA / Recombinant expression: No
SourceSpecies: Pseudomonas savastanoi pv. phaseolicola (bacteria)

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Component #4: protein, Interferon-induced helicase C domain-containing protein 1

ProteinName: Interferon-induced helicase C domain-containing protein 1
Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 114.214477 kDa
SourceSpecies: Mus musculus (house mouse)
Source (engineered)Expression System: Escherichia coli BL21(DE3) (bacteria)

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Component #5: nucleic-acid, RNA (5'-R(P*CP*AP*AP*GP*CP*CP*GP*AP*GP*GP*AP*GP*AP*...

Nucleic-acidName: RNA (5'-R(P*CP*AP*AP*GP*CP*CP*GP*AP*GP*GP*AP*GP*AP*G)-3')
Class: RNA / Structure: OTHER / Synthetic: No
Sequence:
CAAGCCGAGG AGAG
MassTheoretical: 4.587852 kDa
SourceSpecies: Pseudomonas phage phi6 (bacteriophage)

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Component #6: nucleic-acid, RNA (5'-R(P*CP*UP*CP*UP*CP*CP*UP*CP*GP*GP*CP*UP*UP*...

Nucleic-acidName: RNA (5'-R(P*CP*UP*CP*UP*CP*CP*UP*CP*GP*GP*CP*UP*UP*G)-3')
Class: RNA / Structure: OTHER / Synthetic: No
Sequence:
CUCUCCUCGG CUUG
MassTheoretical: 4.35258 kDa
SourceSpecies: Pseudomonas phage phi6 (bacteriophage)

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Component #7: ligand, ZINC ION

LigandName: ZINC ION / Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 6.540905 MDa

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Component #8: ligand, PHOSPHOAMINOPHOSPHONIC ACID-ADENYLATE ESTER

LigandName: PHOSPHOAMINOPHOSPHONIC ACID-ADENYLATE ESTER / Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 0.506196 kDa

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Experimental details

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Sample preparation

SpecimenSpecimen state: helical array / Method: cryo EM
Helical parametersAxial symmetry: C1 (asymmetric) / Delta z: 43.1057 Å / Delta phi: 76.7759 deg.
Sample solutionSpecimen conc.: 0.5 mg/ml / pH: 7.7
Support film25 mA
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Temperature: 277 K / Humidity: 100 %

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
ImagingMicroscope: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Electron dose: 29.33 e/Å2 / Illumination mode: FLOOD BEAM
LensMagnification: 75000.0 X (calibrated) / Imaging mode: BRIGHT FIELD / Defocus: -1800.0 - -2700.0 nm
Specimen HolderModel: FEI TITAN KRIOS AUTOGRID HOLDER
CameraDetector: FEI FALCON III (4k x 4k)

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Image processing

ProcessingMethod: helical reconstruction
3D reconstructionAlgorithm: FOURIER SPACE / Software: RELION / Resolution: 4.06 Å / Resolution method: FSC 0.143 CUT-OFF
FSC plot
(resolution estimation)

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Atomic model buiding

Modeling #1Refinement protocol: flexible / Target criteria: Cross-correlation coefficient / Refinement space: REAL / Overall bvalue: 125
Output model

6gjz

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