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- EMDB-50175: Cryo-EM structure of the A946T MDA5-dsRNA filament -

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Basic information

Entry
Database: EMDB / ID: EMD-50175
TitleCryo-EM structure of the A946T MDA5-dsRNA filament
Map dataPostprocessed map with masking applied
Sample
  • Complex: Mouse MDA5 A946T in complex with double stranded RNA
    • Complex: Mouse MDA5 A946T
      • Protein or peptide: Interferon-induced helicase C domain-containing protein 1
    • Complex: double stranded RNA
      • RNA: RNA (5'-R(P*GP*UP*CP*AP*AP*GP*CP*CP*GP*AP*GP*GP*AP*GP*A)-3')
      • RNA: RNA (5'-R(P*UP*CP*UP*CP*CP*UP*CP*GP*GP*CP*UP*UP*GP*AP*C)-3')
  • Ligand: ZINC ION
KeywordsPROTEIN-RNA COMPLEX / HELICAL FILAMENT / ATPASE / INNATE IMMUNE RECEPTOR / IMMUNE SYSTEM
Function / homology
Function and homology information


MDA-5 signaling pathway / Ub-specific processing proteases / positive regulation of response to cytokine stimulus / pattern recognition receptor activity / negative regulation of viral genome replication / type I interferon-mediated signaling pathway / cellular response to exogenous dsRNA / protein complex oligomerization / positive regulation of interferon-alpha production / protein sumoylation ...MDA-5 signaling pathway / Ub-specific processing proteases / positive regulation of response to cytokine stimulus / pattern recognition receptor activity / negative regulation of viral genome replication / type I interferon-mediated signaling pathway / cellular response to exogenous dsRNA / protein complex oligomerization / positive regulation of interferon-alpha production / protein sumoylation / ribonucleoprotein complex binding / antiviral innate immune response / positive regulation of interferon-beta production / response to virus / cellular response to virus / positive regulation of interleukin-6 production / positive regulation of tumor necrosis factor production / double-stranded RNA binding / defense response to virus / single-stranded RNA binding / RNA helicase activity / RNA helicase / protein domain specific binding / innate immune response / ATP hydrolysis activity / mitochondrion / DNA 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 ...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)
Methodhelical reconstruction / cryo EM / Resolution: 3.97 Å
AuthorsSingh R / Herrero del Valle A / Modis Y
Funding support United Kingdom, France, 4 items
OrganizationGrant numberCountry
Wellcome Trust101908/Z/13/Z United Kingdom
Wellcome Trust217191/Z/19/Z United Kingdom
Wellcome Trust215378/Z/19/Z United Kingdom
Human Frontier Science Program (HFSP)LT000454/2021-L France
CitationJournal: Biorxiv / Year: 2024
Title: Molecular basis of autoimmune disease protection by MDA5 variants
Authors: Singh R / Herrero del Valle A / Joiner JD / Zwaagstra M / Ferguson BJ / van Kuppeveld FJM / Modis Y
History
DepositionApr 25, 2024-
Header (metadata) releaseFeb 26, 2025-
Map releaseFeb 26, 2025-
UpdateFeb 26, 2025-
Current statusFeb 26, 2025Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_50175.png

Downloads & links

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Map

FileDownload / File: emd_50175.map.gz / Format: CCP4 / Size: 83.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationPostprocessed map with masking applied
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.82 Å/pix.
x 280 pix.
= 230.16 Å		0.82 Å/pix.
x 280 pix.
= 230.16 Å		0.82 Å/pix.
x 280 pix.
= 230.16 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.822 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.006
Minimum - Maximum-0.0142177055 - 0.032133833
Average (Standard dev.)0.00018571707 (±0.0011216654)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions280280280
Spacing280280280
CellA=B=C: 230.16 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_50175_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map 2

Fileemd_50175_half_map_1.map
AnnotationHalf map 2
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map 1

Fileemd_50175_half_map_2.map
AnnotationHalf map 1
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Mouse MDA5 A946T in complex with double stranded RNA

EntireName: Mouse MDA5 A946T in complex with double stranded RNA
Components
  • Complex: Mouse MDA5 A946T in complex with double stranded RNA
    • Complex: Mouse MDA5 A946T
      • Protein or peptide: Interferon-induced helicase C domain-containing protein 1
    • Complex: double stranded RNA
      • RNA: RNA (5'-R(P*GP*UP*CP*AP*AP*GP*CP*CP*GP*AP*GP*GP*AP*GP*A)-3')
      • RNA: RNA (5'-R(P*UP*CP*UP*CP*CP*UP*CP*GP*GP*CP*UP*UP*GP*AP*C)-3')
  • Ligand: ZINC ION

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Supramolecule #1: Mouse MDA5 A946T in complex with double stranded RNA

SupramoleculeName: Mouse MDA5 A946T in complex with double stranded RNA / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#3
Details: Helical filament. Structure determined with helical symmetry averaging.
Molecular weightTheoretical: 28.03 kDa/nm

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Supramolecule #2: Mouse MDA5 A946T

SupramoleculeName: Mouse MDA5 A946T / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1
Source (natural)Organism: Mus musculus (house mouse)

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Supramolecule #3: double stranded RNA

SupramoleculeName: double stranded RNA / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #2-#3 / Details: In vitro transcribed RNA
Source (natural)Organism: Mus musculus (house mouse)

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Macromolecule #1: Interferon-induced helicase C domain-containing protein 1

MacromoleculeName: Interferon-induced helicase C domain-containing protein 1
type: protein_or_peptide / ID: 1 / Details: N-terminal hexahistidine tag and TEV cleavage site / Number of copies: 1 / Enantiomer: LEVO / EC number: RNA helicase
Source (natural)Organism: Mus musculus (house mouse)
Molecular weightTheoretical: 116.727109 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MGSSHHHHHH SSGRENLYFQ GHMIVCSAED SFRNLILFFR PRLKMYIQVE PVLDHLIFLS AETKEQILKK INTCGNTSAA ELLLSTLEQ GQWPLGWTQM FVEALEHSGN PLAARYVKPT LTDLPSPSSE TAHDECLHLL TLLQPTLVDK LLINDVLDTC F EKGLLTVE ...String:
MGSSHHHHHH SSGRENLYFQ GHMIVCSAED SFRNLILFFR PRLKMYIQVE PVLDHLIFLS AETKEQILKK INTCGNTSAA ELLLSTLEQ GQWPLGWTQM FVEALEHSGN PLAARYVKPT LTDLPSPSSE TAHDECLHLL TLLQPTLVDK LLINDVLDTC F EKGLLTVE DRNRISAAGN SGNESGVREL LRRIVQKENW FSTFLDVLRQ TGNDALFQEL TGGGCPEDNT DLANSSHRDG PA ANECLLP AVDESSLETE AWNVDDILPE ASCTDSSVTT ESDTSLAEGS VSCFDESLGH NSNMGRDSGT MGSDSDESVI QTK RVSPEP ELQLRPYQME VAQPALDGKN IIICLPTGSG KTRVAVYITK DHLDKKKQAS ESGKVIVLVN KVMLAEQLFR KEFN PYLKK WYRIIGLSGD TQLKISFPEV VKSYDVIIST AQILENSLLN LESGDDDGVQ LSDFSLIIID ECHHTNKEAV YNNIM RRYL KQKLRNNDLK KQNKPAIPLP QILGLTASPG VGAAKKQSEA EKHILNICAN LDAFTIKTVK ENLGQLKHQI KEPCKK FVI ADDTRENPFK EKLLEIMASI QTYCQKSPMS DFGTQHYEQW AIQMEKKAAK DGNRKDRVCA EHLRKYNEAL QINDTIR MI DAYSHLETFY TDEKEKKFAV LNDSKKSLKL DETDEFLMNL FFDNKKMLKK LAENPKYENE KLIKLRNTIL EQFTRSEE S SRGIIFTKTR QSTYALSQWI MENAKFAEVG VKAHHLIGAG HSSEVKPMTQ TEQKEVISKF RTGEINLLIA TTVAEEGLD IKECNIVIRY GLVTNEIAMV QARGRARADE STYVLVTSSG SGVTEREIVN DFREKMMYKA INRVQNMKPE EYAHKILELQ VQSILEKKM KVKRSIAKQY NDNPSLITLL CKNCSMLVCS GENIHVIEKM HHVNMTPEFK GLYIVRENKT LQKKFADYQT N GEIICKCG QAWGTMMVHK GLDLPCLKIR NFVVNFKNNS PKKQYKKWVE LPIRFPDLDY SEYCLYSDED

UniProtKB: Interferon-induced helicase C domain-containing protein 1

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Macromolecule #2: RNA (5'-R(P*GP*UP*CP*AP*AP*GP*CP*CP*GP*AP*GP*GP*AP*GP*A)-3')

MacromoleculeName: RNA (5'-R(P*GP*UP*CP*AP*AP*GP*CP*CP*GP*AP*GP*GP*AP*GP*A)-3')
type: rna / ID: 2 / Details: In vitro transcribed RNA / Number of copies: 1
Source (natural)Organism: Mus musculus (house mouse)
Molecular weightTheoretical: 4.894018 KDa
SequenceString:
GUCAAGCCGA GGAGA

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Macromolecule #3: RNA (5'-R(P*UP*CP*UP*CP*CP*UP*CP*GP*GP*CP*UP*UP*GP*AP*C)-3')

MacromoleculeName: RNA (5'-R(P*UP*CP*UP*CP*CP*UP*CP*GP*GP*CP*UP*UP*GP*AP*C)-3')
type: rna / ID: 3 / Details: In vitro transcribed RNA / Number of copies: 1
Source (natural)Organism: Mus musculus (house mouse)
Molecular weightTheoretical: 4.681785 KDa
SequenceString:
UCUCCUCGGC UUGAC

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Macromolecule #4: ZINC ION

MacromoleculeName: ZINC ION / type: ligand / ID: 4 / Number of copies: 1 / Formula: ZN
Molecular weightTheoretical: 65.409 Da

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Experimental details

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Structure determination

Methodcryo EM
Processinghelical reconstruction
Aggregation statefilament

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Sample preparation

Concentration0.5 mg/mL
BufferpH: 7.7
Component:
ConcentrationFormulaName
20.0 mMC8H18N2O4SHEPES
0.1 MKCpotassium chloride
5.0 mMMgCl2magnesium chloride
2.0 mMC4H10O2S2DTT
10.0 mMC10H17N6O12P3AMP-PNP
GridModel: Quantifoil R1.2/1.3 / Material: GOLD / Mesh: 300 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 60 sec. / Details: Edwards 12E6/531 glow discharger at 30 mA
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV / Details: 2-4 s blotting, 15 s wait time.
Details1 mg/ml MDA5 protein was incubated with 0.05 mg/ml 1-kb dsRNA on ice for 2 min. Samples were diluted twofold with buffer and applied to grids.

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Specialist opticsEnergy filter - Name: GIF Bioquantum / Energy filter - Slit width: 20 eV
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Average electron dose: 44.0 e/Å2 / Details: Six shots per hole
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: DARK FIELD / Cs: 2.7 mm / Nominal defocus max: 2.5 µm / Nominal defocus min: 0.5 µm / Nominal magnification: 96000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Final reconstructionApplied symmetry - Helical parameters - Δz: 44.58 Å
Applied symmetry - Helical parameters - Δ&Phi: 89.44 °
Applied symmetry - Helical parameters - Axial symmetry: C1 (asymmetric)
Algorithm: BACK PROJECTION / Resolution.type: BY AUTHOR / Resolution: 3.97 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 4.0) / Number images used: 234719
Segment selectionNumber selected: 1378603 / Software - Name: crYOLO
Startup modelType of model: EMDB MAP
EMDB ID:

0024

Final angle assignmentType: NOT APPLICABLE / Software - Name: RELION (ver. 4.0)
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

9f0j


Chain - Chain ID: A / Chain - Source name: PDB / Chain - Initial model type: experimental model
DetailsInitial local fitting with Fit-in-Map in Chimera followed by real space refinement in Phenix.
RefinementSpace: REAL / Protocol: RIGID BODY FIT / Overall B value: 90 / Target criteria: Cross-correlation coefficient
Output model

PDB-9f3p:
Cryo-EM structure of the A946T MDA5-dsRNA filament

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