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- EMDB-9332: Helical assembly of the CARD9 CARD -

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Basic information

Entry
Database: EMDB / ID: EMD-9332
TitleHelical assembly of the CARD9 CARD
Map dataHelical assembly of the CARD9 CARD
Sample
  • Complex: Helical assembly of the CARD9 CARD.
    • Protein or peptide: Caspase recruitment domain-containing protein 9
KeywordsCARD / filament / helical assembly / death domain / innate immunity / SIGNALING PROTEIN
Function / homology
Function and homology information


regulation of interleukin-2 production / host-mediated regulation of intestinal microbiota composition / CBM complex / response to peptidoglycan / antifungal innate immune response / CARD domain binding / positive regulation of stress-activated MAPK cascade / neutrophil mediated immunity / positive regulation of innate immune response / positive regulation of T-helper 17 type immune response ...regulation of interleukin-2 production / host-mediated regulation of intestinal microbiota composition / CBM complex / response to peptidoglycan / antifungal innate immune response / CARD domain binding / positive regulation of stress-activated MAPK cascade / neutrophil mediated immunity / positive regulation of innate immune response / positive regulation of T-helper 17 type immune response / positive regulation of cytokine production involved in inflammatory response / positive regulation of granulocyte macrophage colony-stimulating factor production / positive regulation of macrophage cytokine production / response to aldosterone / : / response to exogenous dsRNA / positive regulation of interleukin-17 production / response to muramyl dipeptide / immunoglobulin mediated immune response / signaling adaptor activity / stress-activated MAPK cascade / positive regulation of chemokine production / JNK cascade / positive regulation of cytokine production / positive regulation of JNK cascade / NOD1/2 Signaling Pathway / protein homooligomerization / CLEC7A (Dectin-1) signaling / positive regulation of interleukin-6 production / positive regulation of tumor necrosis factor production / positive regulation of NF-kappaB transcription factor activity / defense response to virus / regulation of apoptotic process / positive regulation of canonical NF-kappaB signal transduction / positive regulation of ERK1 and ERK2 cascade / defense response to Gram-positive bacterium / response to xenobiotic stimulus / protein homodimerization activity / protein-containing complex / identical protein binding / metal ion binding / plasma membrane / cytosol / cytoplasm
Similarity search - Function
CARD9, CARD domain / CARD domain / CARD caspase recruitment domain profile. / Caspase recruitment domain / Death-like domain superfamily
Similarity search - Domain/homology
Caspase recruitment domain-containing protein 9
Similarity search - Component
Biological speciesHomo sapiens (human)
Methodhelical reconstruction / cryo EM / Resolution: 4.0 Å
AuthorsHolliday MJ / Rohou A
CitationJournal: Nat Commun / Year: 2019
Title: Structures of autoinhibited and polymerized forms of CARD9 reveal mechanisms of CARD9 and CARD11 activation.
Authors: Michael J Holliday / Axel Witt / Alejandro Rodríguez Gama / Benjamin T Walters / Christopher P Arthur / Randal Halfmann / Alexis Rohou / Erin C Dueber / Wayne J Fairbrother /
Abstract: CARD9 and CARD11 drive immune cell activation by nucleating Bcl10 polymerization, but are held in an autoinhibited state prior to stimulation. Here, we elucidate the structural basis for this ...CARD9 and CARD11 drive immune cell activation by nucleating Bcl10 polymerization, but are held in an autoinhibited state prior to stimulation. Here, we elucidate the structural basis for this autoinhibition by determining the structure of a region of CARD9 that includes an extensive interface between its caspase recruitment domain (CARD) and coiled-coil domain. We demonstrate, for both CARD9 and CARD11, that disruption of this interface leads to hyperactivation in cells and to the formation of Bcl10-templating filaments in vitro, illuminating the mechanism of action of numerous oncogenic mutations of CARD11. These structural insights enable us to characterize two similar, yet distinct, mechanisms by which autoinhibition is relieved in the course of canonical CARD9 or CARD11 activation. We also dissect the molecular determinants of helical template assembly by solving the structure of the CARD9 filament. Taken together, these findings delineate the structural mechanisms of inhibition and activation within this protein family.
History
DepositionNov 13, 2018-
Header (metadata) releaseDec 12, 2018-
Map releaseJul 3, 2019-
UpdateMar 20, 2024-
Current statusMar 20, 2024Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.033
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.033
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6n2p
  • Surface level: 0.033
  • Imaged by UCSF Chimera
  • Download
  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-6n2p
  • Imaged by Jmol
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_9332.png

Downloads & links

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Map

FileDownload / File: emd_9332.map.gz / Format: CCP4 / Size: 103 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationHelical assembly of the CARD9 CARD
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.09 Å/pix.
x 300 pix.
= 325.5 Å		1.09 Å/pix.
x 300 pix.
= 325.5 Å		1.09 Å/pix.
x 300 pix.
= 325.5 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.085 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.033 / Movie #1: 0.033
Minimum - Maximum-0.013754008 - 0.0614793
Average (Standard dev.)-0.00050711195 (±0.004071336)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions300300300
Spacing300300300
CellA=B=C: 325.5 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.0851.0851.085
M x/y/z300300300
origin x/y/z0.0000.0000.000
length x/y/z325.500325.500325.500
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ480480480
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS300300300
D min/max/mean-0.0140.061-0.001

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Supplemental data

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Additional map: CARD9 CARD helical filament, sharpened.

Fileemd_9332_additional.map
AnnotationCARD9 CARD helical filament, sharpened.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Helical assembly of the CARD9 CARD.

EntireName: Helical assembly of the CARD9 CARD.
Components
  • Complex: Helical assembly of the CARD9 CARD.
    • Protein or peptide: Caspase recruitment domain-containing protein 9

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Supramolecule #1: Helical assembly of the CARD9 CARD.

SupramoleculeName: Helical assembly of the CARD9 CARD. / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Details: Formed from CARD9 2-152 dimer with an I107E mutation, purified with 1:1 Zn. 1 mM EDTA was added to 0.5 mM protein, followed by 10 minute incubation at 25C.
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 34.0 kDa/nm

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Macromolecule #1: Caspase recruitment domain-containing protein 9

MacromoleculeName: Caspase recruitment domain-containing protein 9 / type: protein_or_peptide / ID: 1 / Number of copies: 10 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 17.351873 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString:
GSDYENDDEC WSVLEGFRVT LTSVIDPSRI TPYLRQCKVL NPDDEEQVLS DPNLVIRKRK VGVLLDILQR TGHKGYVAFL ESLELYYPQ LYKKVTGKEP ARVFSMIEDA SGESGLTQLL MTEVMKLQKK VQDLTALLSS KDDFIKELRV KDS

UniProtKB: Caspase recruitment domain-containing protein 9

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Experimental details

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Structure determination

Methodcryo EM
Processinghelical reconstruction
Aggregation statefilament

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Sample preparation

Concentration8.67 mg/mL
BufferpH: 7
Component:
ConcentrationFormulaName
50.0 mMC8H18N2O4SHEPES
150.0 mMNaClSodium Chloride
0.5 mMC9H15O6PTCEP
0.5 mMZnZinc
1.0 mMC10H16N2O8EDTA
GridModel: C-flat-2/1 / Material: COPPER / Mesh: 200 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 30 sec.
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV
Details: Sample was incubated on the grid for 1 minute, subsequently washed/blotted 6 times in buffer, followed by addition of 3.5 ul buffer, which was blotted by vitrobot for 5 seconds prior to plunging..
DetailsFormed from CARD9 2-152 dimer with an I107E mutation, purified with 1:1 Zn. 1 mM EDTA was added to 0.5 mM protein, followed by 10 minute incubation at 25C. Sample was subsequently diluted 1:5 before adding to grid.

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Specialist opticsEnergy filter - Slit width: 20 eV
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: 4403 / Average exposure time: 10.0 sec. / Average electron dose: 50.9 e/Å2 / Details: Collected in movie-mode at 4 frames per second
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 100.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 2.5 µm / Nominal defocus min: 1.25 µm / Nominal magnification: 130000
Sample stageCooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Final reconstructionApplied symmetry - Helical parameters - Δz: 5.11 Å
Applied symmetry - Helical parameters - Δ&Phi: -101.6 °
Applied symmetry - Helical parameters - Axial symmetry: C1 (asymmetric)
Resolution.type: BY AUTHOR / Resolution: 4.0 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: FREALIGN (ver. 9.11)
Details: A 5.0 A high-resolution limit was used for particle alignment in frealign.
Number images used: 31908
Segment selectionNumber selected: 141592
Software:
Namedetails
EMAN2Filaments were manually selected using e2helixboxer.py.
RELION (ver. 2.1)Segments were extracted from filaments using RELION.

Details: Filaments were manually selected using e2helixboxer.py. Segments were extracted from filaments using RELION, using a 30 A shift between particles.
Startup modelType of model: OTHER
Details: Ab initio 3D reconstruction was generated in C1 in cisTEM and used as a startup model.
Final angle assignmentType: NOT APPLICABLE / Software - Name: FREALIGN (ver. 9.11)
Details: Final assignment was performed in helical frealign using helical parameters of 5.11-A rise and 101.6 degree rotation.
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

6e26


Chain - Chain ID: A / Chain - Residue range: 8-95 / Chain - Source name: PDB / Chain - Initial model type: experimental model
Details"Fit in map" in UCSF Chimera was used to fit the previously determined, lowest energy NMR solution structure (PDB ID 6E26) into the sharpened density and to generate 9 symmetry-mates according to the determined helical parameters. The map was then iteratively refined in Phenix and Coot, maintaining strict non-crystallographic symmetry among the CARDs.
RefinementSpace: REAL / Protocol: RIGID BODY FIT
Output model

PDB-6n2p:
Helical assembly of the CARD9 CARD

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