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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: EMD-0012
TitleCryoEM structure of the MDA5-dsRNA filament in complex with AMPPNP
Map data
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 / homology
Function and homology information


MDA-5 signaling pathway / positive regulation of interferon-beta secretion / positive regulation of response to cytokine stimulus / positive regulation of tumor necrosis factor secretion / positive regulation of interferon-alpha secretion / positive regulation of interferon-alpha production / cellular response to exogenous dsRNA / positive regulation of interferon-beta production / positive regulation of interleukin-6 secretion / protein sumoylation ...MDA-5 signaling pathway / positive regulation of interferon-beta secretion / positive regulation of response to cytokine stimulus / positive regulation of tumor necrosis factor secretion / positive regulation of interferon-alpha secretion / positive regulation of interferon-alpha production / cellular response to exogenous dsRNA / positive regulation of interferon-beta production / positive regulation of interleukin-6 secretion / protein sumoylation / ribonucleoprotein complex binding / single-stranded RNA binding / response to virus / double-stranded RNA binding / RNA helicase / defense response to virus / RNA helicase activity / innate immune response / DNA binding / zinc ion binding / ATP binding / identical protein binding / nucleus / cytoplasm
Helicase/UvrB, N-terminal / Death-like domain superfamily / Helicase, C-terminal / Helicase superfamily 1/2, ATP-binding domain / RIG-I-like receptor, C-terminal regulatory domain / RIG-I receptor, C-terminal / RIG-I-like receptor, C-terminal domain superfamily / Caspase recruitment domain / P-loop containing nucleoside triphosphate hydrolase
Interferon-induced helicase C domain-containing protein 1
Biological speciesMus musculus (house mouse) / Pseudomonas savastanoi pv. phaseolicola (bacteria) / Pseudomonas phage phi6 (bacteriophage)
Methodhelical reconstruction / cryo EM / Resolution: 4.06 Å
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
History
Header (metadata) releaseApr 11, 2018-
DepositionMay 17, 2018-
Map releaseNov 21, 2018-
UpdateJan 2, 2019-
Current statusJan 2, 2019Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.0495
  • Imaged by UCSF Chimera
  • Download
  • 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
  • Download
  • 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

FileDownload / File: emd_0012.map.gz / Format: CCP4 / Size: 42.9 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.07 Å/pix.
x 224 pix.
= 239.68 Å		1.07 Å/pix.
x 224 pix.
= 239.68 Å		1.07 Å/pix.
x 224 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 LevelBy AUTHOR: 0.0495 / Movie #1: 0.0495
Minimum - Maximum-0.075805224 - 0.18155327
Average (Standard dev.)0.0010824882 (±0.009103025)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions224224224
Spacing224224224
CellA=B=C: 239.68001 Å
α=β=γ: 90.0 °

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
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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Segmentation: #1

Fileemd_0012_msk_1.map
Projections & Slices
AxesZYX

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Histogram

Histogram (log scale)

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Half map: Half map 1

Fileemd_0012_half_map_1.map
AnnotationHalf map 1
Projections & Slices
AxesZYX

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Histogram

Histogram (log scale)

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Half map: Half map 2

Fileemd_0012_half_map_2.map
AnnotationHalf map 2
Projections & Slices
AxesZYX

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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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