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- EMDB-11937: CryoEM structure of the MDA5-dsRNA filament in complex with ADP w... -

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Basic information

Entry
Database: EMDB / ID: EMD-11937
TitleCryoEM structure of the MDA5-dsRNA filament in complex with ADP with 92-degree helical twist
Map data
Sample
  • Complex: MDA5-dsRNA filament in complex with ADP
    • Complex: INTERFERON-INDUCED HELICASE C DOMAIN-CONTAINING PROTEIN
      • Protein or peptide: INTERFERON-INDUCED HELICASE C DOMAIN-CONTAINING PROTEIN
    • Complex: RNA
      • RNA: RNA
      • RNA: RNA
Function / homology
Function and homology information


adenyl ribonucleotide binding / MDA-5 signaling pathway / purine ribonucleoside triphosphate binding / positive regulation of response to cytokine stimulus / Ub-specific processing proteases / pattern recognition receptor activity / negative regulation of viral genome replication / cellular response to exogenous dsRNA / type I interferon-mediated signaling pathway / positive regulation of interferon-alpha production ...adenyl ribonucleotide binding / MDA-5 signaling pathway / purine ribonucleoside triphosphate binding / positive regulation of response to cytokine stimulus / Ub-specific processing proteases / pattern recognition receptor activity / negative regulation of viral genome replication / cellular response to exogenous dsRNA / type I interferon-mediated signaling pathway / positive regulation of interferon-alpha production / antiviral innate immune response / protein sumoylation / ribonucleoprotein complex binding / positive regulation of interferon-beta production / response to virus / cellular response to virus / positive regulation of interleukin-6 production / double-stranded RNA binding / positive regulation of tumor necrosis factor production / protein complex oligomerization / 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 / RNA 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 / Helicase/UvrB, N-terminal / Type III restriction enzyme, res subunit ...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 / 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
Interferon-induced helicase C domain-containing protein 1
Similarity search - Component
Biological speciesMus musculus (house mouse) / Pseudomonas virus phi6 (bacteriophage)
Methodhelical reconstruction / cryo EM / Resolution: 3.4 Å
AuthorsYu Q / Modis Y
Funding support United Kingdom, 2 items
OrganizationGrant numberCountry
Wellcome Trust217191/Z/19/Z United Kingdom
Wellcome Trust101908/Z/13/Z United Kingdom
CitationJournal: Nat Commun / Year: 2021
Title: MDA5 disease variant M854K prevents ATP-dependent structural discrimination of viral and cellular RNA.
Authors: Qin Yu / Alba Herrero Del Valle / Rahul Singh / Yorgo Modis /
Abstract: Our innate immune responses to viral RNA are vital defenses. Long cytosolic double-stranded RNA (dsRNA) is recognized by MDA5. The ATPase activity of MDA5 contributes to its dsRNA binding selectivity. ...Our innate immune responses to viral RNA are vital defenses. Long cytosolic double-stranded RNA (dsRNA) is recognized by MDA5. The ATPase activity of MDA5 contributes to its dsRNA binding selectivity. Mutations that reduce RNA selectivity can cause autoinflammatory disease. Here, we show how the disease-associated MDA5 variant M854K perturbs MDA5-dsRNA recognition. M854K MDA5 constitutively activates interferon signaling in the absence of exogenous RNA. M854K MDA5 lacks ATPase activity and binds more stably to synthetic Alu:Alu dsRNA. CryoEM structures of MDA5-dsRNA filaments at different stages of ATP hydrolysis show that the K854 sidechain forms polar bonds that constrain the conformation of MDA5 subdomains, disrupting key steps in the ATPase cycle- RNA footprint expansion and helical twist modulation. The M854K mutation inhibits ATP-dependent RNA proofreading via an allosteric mechanism, allowing MDA5 to form signaling complexes on endogenous RNAs. This work provides insights on how MDA5 recognizes dsRNA in health and disease.
History
DepositionNov 15, 2020-
Header (metadata) releaseNov 24, 2021-
Map releaseNov 24, 2021-
UpdateDec 1, 2021-
Current statusDec 1, 2021Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.02
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.02
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-7bkq
  • Surface level: 0.02
  • Imaged by UCSF Chimera
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  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-7bkq
  • Imaged by Jmol
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_11937.png

Downloads & links

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Map

FileDownload / File: emd_11937.map.gz / Format: CCP4 / Size: 47.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 1.04 Å
Density
Contour LevelBy AUTHOR: 0.02 / Movie #1: 0.02
Minimum - Maximum-0.04012276 - 0.094014786
Average (Standard dev.)0.00039355905 (±0.0032461553)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions232232232
Spacing232232232
CellA=B=C: 241.28 Å
α=β=γ: 90.0 °

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
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS232232232
D min/max/mean-0.0400.0940.000

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Supplemental data

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Mask #1

Fileemd_11937_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #2

Fileemd_11937_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_11937_half_map_2.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 filament in complex with ADP

EntireName: MDA5-dsRNA filament in complex with ADP
Components
  • Complex: MDA5-dsRNA filament in complex with ADP
    • Complex: INTERFERON-INDUCED HELICASE C DOMAIN-CONTAINING PROTEIN
      • Protein or peptide: INTERFERON-INDUCED HELICASE C DOMAIN-CONTAINING PROTEIN
    • Complex: RNA
      • RNA: RNA
      • RNA: RNA

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Supramolecule #1: MDA5-dsRNA filament in complex with ADP

SupramoleculeName: MDA5-dsRNA filament in complex with ADP / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Molecular weightExperimental: 30 kDa/nm

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Supramolecule #2: INTERFERON-INDUCED HELICASE C DOMAIN-CONTAINING PROTEIN

SupramoleculeName: INTERFERON-INDUCED HELICASE C DOMAIN-CONTAINING PROTEIN
type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1
Source (natural)Organism: Mus musculus (house mouse)
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)

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Supramolecule #3: RNA

SupramoleculeName: RNA / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #2-#3
Source (natural)Organism: Pseudomonas virus phi6 (bacteriophage)

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Macromolecule #1: INTERFERON-INDUCED HELICASE C DOMAIN-CONTAINING PROTEIN

MacromoleculeName: INTERFERON-INDUCED HELICASE C DOMAIN-CONTAINING PROTEIN
type: protein_or_peptide / ID: 1 / Enantiomer: LEVO / EC number: RNA helicase
Source (natural)Organism: Mus musculus (house mouse)
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: LQLRPYQMEV AQPALDGKNI IICLPTGSGK TRVAVYITKD HLDKKKQASE SGKVIVLVNK VMLAEQLFRK EFNPYLKKWY RIIGLSGDTQ LKISFPEVVK SYDVIISTAQ ILENSLLNLE SGDDDGVQLS DFSLIIIDEC HHTNKEAVYN NIMRRYLKQK LRNNDLKKQN ...String:
LQLRPYQMEV AQPALDGKNI IICLPTGSGK TRVAVYITKD HLDKKKQASE SGKVIVLVNK VMLAEQLFRK EFNPYLKKWY RIIGLSGDTQ LKISFPEVVK SYDVIISTAQ ILENSLLNLE SGDDDGVQLS DFSLIIIDEC HHTNKEAVYN NIMRRYLKQK LRNNDLKKQN KPAIPLPQIL GLTASPGVGA AKKQSEAEKH ILNICANLDA FTIKTVKENL GQLKHQIKEP CKKFVIADDT RENPFKEKLL EIMASIQTYC QKSPMSDFGT QHYEQWAIQM EKKAAKDGNR KDRVCAEHLR KYNEALQIND TIRMIDAYSH LETFYTDEKE KKFAVLNDSK KSLKLDETDE FLMNLFFDNK KMLKKLAENP KYENEKLIKL RNTILEQFTR SEESSRGIIF TKTRQSTYAL SQWIMENAKF AEVGVKAHHL IGAGHSSEVK PMTQTEQKEV ISKFRTGEIN LLIATTVAEE GLDIKECNIV IRYGLVTNEI AMVQARGRAR ADESTYVLVT SSGSGVTERE IVNDFREKMM YKAINRVQNM KPEEYAHKIL ELQVQSILEK KMKVKRSIAK QYNDNPSLIT LLCKNCSMLV CSGENIHVIE KMHHVNMTPE FKGLYIVREN KALQKKFADY QTNGEIICKC GQAWGTMMVH KGLDLPCLKI RNFVVNFKNN SPKKQYKKWV ELPIRFPDLD YSEYCL

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Macromolecule #2: RNA

MacromoleculeName: RNA / type: rna / ID: 2
Source (natural)Organism: Pseudomonas virus phi6 (bacteriophage)
SequenceString:
UCCAUGCGCA UGACG

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Macromolecule #3: RNA

MacromoleculeName: RNA / type: rna / ID: 3
Source (natural)Organism: Pseudomonas virus phi6 (bacteriophage)
SequenceString:
CGUCAUGCGC AUGGA

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Experimental details

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Structure determination

Methodcryo EM
Processinghelical reconstruction
Aggregation statefilament

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Sample preparation

Concentration1 mg/mL
BufferpH: 7.7
Component:
ConcentrationNameFormula
20.0 mMHEPES
0.1 MKCl
5.0 mMMgCl2
2.0 mMDithiothreitol
2.0 mMADPAdenosine diphosphate
GridModel: Quantifoil R1.2/1.3 / Material: GOLD / Mesh: 300 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Details: machine step 6
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277.2 K / Instrument: FEI VITROBOT MARK IV
Details: Grids were blotted for 3 s; blot force 10, 5, 0, -5, -10.

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 2.7 µm / Nominal defocus min: 1.8 µm
Specialist opticsPhase plate: OTHER / Spherical aberration corrector: None / Energy filter - Slit width: 20 eV
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Digitization - Dimensions - Width: 3838 pixel / Digitization - Dimensions - Height: 3710 pixel / Digitization - Frames/image: 1-40 / Number grids imaged: 1 / Number real images: 4680 / Average electron dose: 1.1125 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Segment selectionNumber selected: 1063529 / Software - Name: RELION (ver. 3.1)
CTF correctionSoftware - Name: Gctf (ver. 0.5)
Startup modelType of model: PDB ENTRY
PDB model - PDB ID:

6h66


Details: Model was low-passed filtered to 30 A resolution
Final angle assignmentType: NOT APPLICABLE / Software - Name: RELION (ver. 3.1)
Final reconstructionNumber classes used: 2
Applied symmetry - Helical parameters - Δz: 44.6 Å
Applied symmetry - Helical parameters - Δ&Phi: 91.7 °
Applied symmetry - Helical parameters - Axial symmetry: C1 (asymmetric)
Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 3.4 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.1) / Number images used: 64992
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

6h66


Chain - Chain ID: A
RefinementSpace: REAL / Protocol: OTHER / Overall B value: 118 / Target criteria: Correlation coefficient
Output model

PDB-7bkq:
CryoEM structure of MDA5-dsRNA filament in complex with ADP with 92-degree helical twist

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