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- EMDB-0145: CryoEM structure of the MDA5-dsRNA filament with 93 degree twist ... -

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Basic information

Entry
Database: EMDB / ID: 0145
TitleCryoEM structure of the MDA5-dsRNA filament with 93 degree twist and without nucleotide
Map datacryoEM reconstruction map
SampleMDA5-dsRNA helical filament without nucleotide
  • MDA5 (IFIH1)
  • Double-stranded RNA from bacteriophage Phi6, generated by n vitro translationRNA
  • Interferon-induced helicase C domain-containing protein 1
  • (nucleic-acidNucleic acid) x 2
  • ligand
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 / cellular response to exogenous dsRNA / positive regulation of interferon-alpha secretion / 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 / 4.16 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: 6h66

SummaryFull reportAbout validation report
DateDeposition: Jul 26, 2018 / Header (metadata) release: Apr 11, 2018 / Map release: Nov 21, 2018 / Last update: Nov 28, 2018

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.052
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 0.052
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: : PDB-6h66
  • Surface level: 0.052
  • Imaged by UCSF Chimera
  • Download
  • Simplified surface model + fitted atomic model
  • Atomic models: PDB-6h66
  • Imaged by Jmol
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

Downloads & links

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Map

Fileemd_0145.map.gz (map file in CCP4 format, 49949 KB)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
232 pix
1.04 Å/pix.
= 241.28 Å
232 pix
1.04 Å/pix.
= 241.28 Å
232 pix
1.04 Å/pix.
= 241.28 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.04 Å
Density
Contour Level:0.052 (by author), 0.052 (movie #1):
Minimum - Maximum-0.023099303 - 0.06743383
Average (Standard dev.)0.00055849756 (0.0048806244)
Details

EMDB XML:

Space Group Number1
Map Geometry
Axis orderXYZ
Dimensions232232232
Origin0.00.00.0
Limit231.0231.0231.0
Spacing232232232
CellA=B=C: 241.28 Å
α=β=γ: 90.0 deg.

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.041.041.04
M x/y/z232232232
origin x/y/z0.0000.0000.000
length x/y/z241.280241.280241.280
α/β/γ90.00090.00090.000
start NX/NY/NZ
NX/NY/NZ
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS232232232
D min/max/mean-0.1410.2860.001

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Supplemental data

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Mask #1

Fileemd_0145_msk_1.map
Projections & Slices
AxesZYX
Projections
Slices (1/2)
Density Histograms

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Sample components

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Entire MDA5-dsRNA helical filament without nucleotide

EntireName: MDA5-dsRNA helical filament without nucleotide / Details: Filaments formed without nucleotide / Number of components: 7

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Component #1: protein, MDA5-dsRNA helical filament without nucleotide

ProteinName: MDA5-dsRNA helical filament without nucleotide / Details: Filaments formed without nucleotide / Recombinant expression: No
MassTheoretical: 2.23 MDa

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Component #2: protein, MDA5 (IFIH1)

ProteinName: MDA5 (IFIH1)
Details: RIG-I-like Superfamily 2 (SF2) RNA 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 BL21(DE3) (bacteria)

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

ProteinName: Double-stranded RNA from bacteriophage Phi6, generated by n vitro translationRNA
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*UP*CP*CP*AP*UP*GP*CP*GP*CP*AP*UP*GP*AP*...

Nucleic-acidName: RNA (5'-R(P*UP*CP*CP*AP*UP*GP*CP*GP*CP*AP*UP*GP*AP*CP*G)-3')
Class: RNA / Structure: OTHER / Synthetic: No
Sequence:
UCCAUGCGCA UGACG
MassTheoretical: 4.767889 kDa
SourceSpecies: Pseudomonas phage phi6 (bacteriophage)

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

Nucleic-acidName: RNA (5'-R(P*CP*GP*UP*CP*AP*UP*GP*CP*GP*CP*AP*UP*GP*GP*A)-3')
Class: RNA / Structure: OTHER / Synthetic: No
Sequence:
CGUCAUGCGC AUGGA
MassTheoretical: 4.807914 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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Experimental details

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Sample preparation

SpecimenSpecimen state: helical array / Method: cryo EM
Helical parametersAxial symmetry: C1 (asymmetric) / Delta z: 44.366 Å / Delta phi: 93.0596 deg.
Sample solutionSpecimen conc.: 0.5 mg/ml / pH: 7.7
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: 27 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 reconstructionSoftware: RELION / Resolution: 4.16 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
Input PDB model: 6G1X
Overall bvalue: 175
Output model

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