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- EMDB-8976: CARD9 CARD helical filament -

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Basic information

Entry
Database: EMDB / ID: EMD-8976
TitleCARD9 CARD helical filament
Map dataNegative-stain electron microscopy reconstruction of helical filaments of the CARD9 CARD
Sample
  • Complex: Helical filament of the CARD9 CARD
    • Protein or peptide: CARD9 CARD
Biological speciesHomo sapiens (human)
Methodhelical reconstruction / negative staining / Resolution: 20.0 Å
AuthorsHolliday MJ / Estevez A / Rohou A / Dueber EC / Fairbrother WJ
CitationJournal: J Biol Chem / Year: 2018
Title: Picomolar zinc binding modulates formation of Bcl10-nucleating assemblies of the caspase recruitment domain (CARD) of CARD9.
Authors: Michael J Holliday / Ryan Ferrao / Gladys de Leon Boenig / Alberto Estevez / Elizabeth Helgason / Alexis Rohou / Erin C Dueber / Wayne J Fairbrother /
Abstract: The caspase recruitment domain-containing protein 9 (CARD9)-B-cell lymphoma/leukemia 10 (Bcl10) signaling axis is activated in myeloid cells during the innate immune response to a variety of diverse ...The caspase recruitment domain-containing protein 9 (CARD9)-B-cell lymphoma/leukemia 10 (Bcl10) signaling axis is activated in myeloid cells during the innate immune response to a variety of diverse pathogens. This signaling pathway requires a critical caspase recruitment domain (CARD)-CARD interaction between CARD9 and Bcl10 that promotes downstream activation of factors, including NF-κB and the mitogen-activated protein kinase (MAPK) p38. Despite these insights, CARD9 remains structurally uncharacterized, and little mechanistic understanding of its regulation exists. We unexpectedly found here that the CARD in CARD9 binds to Zn with picomolar affinity-a concentration comparable with the levels of readily accessible Zn in the cytosol. NMR solution structures of the CARD9-CARD in the apo and Zn-bound states revealed that Zn has little effect on the ground-state structure of the CARD; yet the stability of the domain increased considerably upon Zn binding, with a concomitant reduction in conformational flexibility. Moreover, Zn binding inhibited polymerization of the CARD9-CARD into helical assemblies. Here, we also present a 20-Å resolution negative-stain EM (NS-EM) structure of these filamentous assemblies and show that they adopt a similar helical symmetry as reported previously for filaments of the Bcl10 CARD. Using both bulk assays and direct NS-EM visualization, we further show that the CARD9-CARD assemblies can directly template and thereby nucleate Bcl10 polymerization, a capacity considered critical to propagation of the CARD9-Bcl10 signaling cascade. Our findings indicate that CARD9 is a potential target of Zn-mediated signaling that affects Bcl10 polymerization in innate immune responses.
History
DepositionJul 14, 2018-
Header (metadata) releaseAug 22, 2018-
Map releaseSep 26, 2018-
UpdateNov 7, 2018-
Current statusNov 7, 2018Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.04
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 0.04
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_8976.png

Downloads & links

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Map

FileDownload / File: emd_8976.map.gz / Format: CCP4 / Size: 2.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationNegative-stain electron microscopy reconstruction of helical filaments of the CARD9 CARD
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
2.01 Å/pix.
x 200 pix.
= 401.2 Å		2.01 Å/pix.
x 60 pix.
= 120.36 Å		2.01 Å/pix.
x 60 pix.
= 120.36 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

generated in cubic-lattice coordinate

Voxel sizeX=Y=Z: 2.006 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.04 / Movie #1: 0.04
Minimum - Maximum-0.13292108 - 1.4829998
Average (Standard dev.)0.106759265 (±0.2958757)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-30-30-100
Dimensions6060200
Spacing6060200
CellA: 120.36 Å / B: 120.36 Å / C: 401.2 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z2.0062.0062.006
M x/y/z6060200
origin x/y/z0.0000.0000.000
length x/y/z120.360120.360401.200
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ240240240
MAP C/R/S123
start NC/NR/NS-30-30-100
NC/NR/NS6060200
D min/max/mean-0.1331.4830.107

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Supplemental data

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Sample components

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Entire : Helical filament of the CARD9 CARD

EntireName: Helical filament of the CARD9 CARD
Components
  • Complex: Helical filament of the CARD9 CARD
    • Protein or peptide: CARD9 CARD

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Supramolecule #1: Helical filament of the CARD9 CARD

SupramoleculeName: Helical filament of the CARD9 CARD / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Details: Generated by stripping Zn from 0.2 mM 1:1 CARD:Zn with 0.25 mM EDTA, followed by shaking for 90 minutes at 25C
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria) / Recombinant strain: BL21(DE3)
Molecular weightExperimental: 22.4 kDa/nm

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Macromolecule #1: CARD9 CARD

MacromoleculeName: CARD9 CARD / type: protein_or_peptide / ID: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString:
GSDYENDDEC WSVLEGFRVT LTSVIDPSRI TPYLRQCKVL NPDDEEQVLS DPNLVIRKRK VGVLLDILQ RTGHKGYVAF LESLELYYPQ LYKKVTGK

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Experimental details

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Structure determination

Methodnegative staining
Processinghelical reconstruction
Aggregation statefilament

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Sample preparation

Concentration2.2 mg/mL
BufferpH: 7 / Details: 50 mM Tris, 150 mM NaCl, 0.5 mM TCEP, pH 7.0
StainingType: NEGATIVE / Material: Uranyl Acetate
GridMaterial: COPPER / Mesh: 400 / Support film - Material: CARBON / Support film - topology: CONTINUOUS / Pretreatment - Type: GLOW DISCHARGE

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Electron microscopy

MicroscopeFEI TALOS ARCTICA
Image recordingFilm or detector model: FEI CETA (4k x 4k) / Number grids imaged: 1 / Average exposure time: 2.0 sec. / Average electron dose: 2.0 e/Å2
Electron beamAcceleration voltage: 200 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: 1.2 µm / Nominal magnification: 73000
Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company

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Image processing

Final reconstructionNumber classes used: 1
Applied symmetry - Helical parameters - Δz: 5.0 Å
Applied symmetry - Helical parameters - Δ&Phi: -102 °
Applied symmetry - Helical parameters - Axial symmetry: C1 (asymmetric)
Resolution.type: BY AUTHOR / Resolution: 20.0 Å / Resolution method: OTHER / Software - Name: SPRING / Software - details: Using SPRING routine Segmentrefine3D.
Details: Approximate resolution estimated by comparison to other NS helical reconstructions
Number images used: 11226
CTF correctionSoftware - Name: SPRING / Software - details: Using SPRING routine Micctfdetermine.
Segment selectionNumber selected: 1086 / Software - Name: EMAN2 (ver. 2.2)
Software - details: Segments were manually picked in e2helixboxer
Startup modelType of model: OTHER / Details: 100 A cylinder generated by SPRING.
Final angle assignmentType: NOT APPLICABLE / Software - Name: SPRING / Software - details: Using SPRING routine Segmentrefine3D.

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Atomic model buiding 1

DetailsNo fitting was performed.

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