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- EMDB-6842: Structure of RIP2 CARD domain -

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Basic information

Entry
Database: EMDB / ID: EMD-6842
TitleStructure of RIP2 CARD domain
Map data200 A long segment of RIP-CARD filament
Sample
  • Organelle or cellular component: Active RIP2 signaling complex
    • Protein or peptide: Receptor-interacting serine/threonine-protein kinase 2
KeywordsInnate Immune signaling complex / IMMUNE SYSTEM / TRANSFERASE
Function / homology
Function and homology information


response to interleukin-18 / positive regulation of T-helper 1 cell differentiation / toll-like receptor 2 signaling pathway / positive regulation of cytokine-mediated signaling pathway / immature T cell proliferation in thymus / positive regulation of T-helper 1 type immune response / LIM domain binding / positive regulation of xenophagy / CD4-positive, alpha-beta T cell proliferation / xenophagy ...response to interleukin-18 / positive regulation of T-helper 1 cell differentiation / toll-like receptor 2 signaling pathway / positive regulation of cytokine-mediated signaling pathway / immature T cell proliferation in thymus / positive regulation of T-helper 1 type immune response / LIM domain binding / positive regulation of xenophagy / CD4-positive, alpha-beta T cell proliferation / xenophagy / nucleotide-binding oligomerization domain containing 1 signaling pathway / positive regulation of protein K63-linked ubiquitination / cellular response to muramyl dipeptide / caspase binding / positive regulation of immature T cell proliferation in thymus / CARD domain binding / JUN kinase kinase kinase activity / positive regulation of CD4-positive, alpha-beta T cell proliferation / cellular response to peptidoglycan / response to interleukin-12 / activation of cysteine-type endopeptidase activity / nucleotide-binding oligomerization domain containing 2 signaling pathway / positive regulation of macrophage cytokine production / toll-like receptor 4 signaling pathway / response to exogenous dsRNA / positive regulation of interferon-alpha production / cellular response to lipoteichoic acid / positive regulation of chemokine production / JNK cascade / signaling adaptor activity / lipopolysaccharide-mediated signaling pathway / ERK1 and ERK2 cascade / p75NTR recruits signalling complexes / positive regulation of interferon-beta production / positive regulation of interleukin-12 production / response to interleukin-1 / JNK (c-Jun kinases) phosphorylation and activation mediated by activated human TAK1 / positive regulation of interleukin-2 production / positive regulation of peptidyl-threonine phosphorylation / canonical NF-kappaB signal transduction / positive regulation of interleukin-1 beta production / positive regulation of protein ubiquitination / activated TAK1 mediates p38 MAPK activation / positive regulation of JNK cascade / non-specific protein-tyrosine kinase / TAK1-dependent IKK and NF-kappa-B activation / NOD1/2 Signaling Pathway / non-membrane spanning protein tyrosine kinase activity / protein homooligomerization / Interleukin-1 signaling / positive regulation of interleukin-6 production / cytokine-mediated signaling pathway / positive regulation of type II interferon production / positive regulation of peptidyl-tyrosine phosphorylation / Ovarian tumor domain proteases / positive regulation of tumor necrosis factor production / Downstream TCR signaling / positive regulation of peptidyl-serine phosphorylation / positive regulation of NF-kappaB transcription factor activity / T cell receptor signaling pathway / positive regulation of protein binding / positive regulation of canonical NF-kappaB signal transduction / vesicle / adaptive immune response / positive regulation of ERK1 and ERK2 cascade / cytoskeleton / non-specific serine/threonine protein kinase / defense response to Gram-positive bacterium / defense response to bacterium / inflammatory response / positive regulation of apoptotic process / phosphorylation / signaling receptor binding / protein serine kinase activity / innate immune response / protein serine/threonine kinase activity / apoptotic process / SARS-CoV-2 activates/modulates innate and adaptive immune responses / endoplasmic reticulum / signal transduction / protein homodimerization activity / positive regulation of transcription by RNA polymerase II / protein-containing complex / ATP binding / identical protein binding / plasma membrane / cytosol / cytoplasm
Similarity search - Function
Receptor-interacting serine/threonine-protein kinase 2 / RIP2, CARD domain / CARD domain / CARD caspase recruitment domain profile. / Caspase recruitment domain / Death-like domain superfamily / Protein tyrosine and serine/threonine kinase / Serine-threonine/tyrosine-protein kinase, catalytic domain / Serine/threonine-protein kinase, active site / Serine/Threonine protein kinases active-site signature. ...Receptor-interacting serine/threonine-protein kinase 2 / RIP2, CARD domain / CARD domain / CARD caspase recruitment domain profile. / Caspase recruitment domain / Death-like domain superfamily / Protein tyrosine and serine/threonine kinase / Serine-threonine/tyrosine-protein kinase, catalytic domain / Serine/threonine-protein kinase, active site / Serine/Threonine protein kinases active-site signature. / Serine/Threonine protein kinases, catalytic domain / Protein kinase domain profile. / Protein kinase domain / Protein kinase-like domain superfamily
Similarity search - Domain/homology
Receptor-interacting serine/threonine-protein kinase 2
Similarity search - Component
Biological speciesHomo sapiens (human)
Methodhelical reconstruction / cryo EM / Resolution: 4.1 Å
AuthorsWu B / Gong Q
Funding support Singapore, 1 items
OrganizationGrant numberCountry
Ministry of Education (Singapore)NAP SUG Singapore
CitationJournal: Nat Commun / Year: 2018
Title: Structural basis of RIP2 activation and signaling.
Authors: Qin Gong / Ziqi Long / Franklin L Zhong / Daniel Eng Thiam Teo / Yibo Jin / Zhan Yin / Zhao Zhi Boo / Yaming Zhang / Jiawen Zhang / Renliang Yang / Shashi Bhushan / Bruno Reversade / Zongli Li / Bin Wu /
Abstract: Signals arising from bacterial infections are detected by pathogen recognition receptors (PRRs) and are transduced by specialized adapter proteins in mammalian cells. The Receptor-interacting- ...Signals arising from bacterial infections are detected by pathogen recognition receptors (PRRs) and are transduced by specialized adapter proteins in mammalian cells. The Receptor-interacting-serine/threonine-protein kinase 2 (RIPK2 or RIP2) is such an adapter protein that is critical for signal propagation of the Nucleotide-binding-oligomerization-domain-containing proteins 1/2 (NOD1 and NOD2). Dysregulation of this signaling pathway leads to defects in bacterial detection and in some cases autoimmune diseases. Here, we show that the Caspase-activation-and-recruitment-domain (CARD) of RIP2 (RIP2-CARD) forms oligomeric structures upon stimulation by either NOD1-CARD or NOD2-2CARD. We reconstitute this complex, termed the RIPosome in vitro and solve the cryo-EM filament structure of the active RIP2-CARD complex at 4.1 Å resolution. The structure suggests potential mechanisms by which CARD domains from NOD1 and NOD2 initiate the oligomerization process of RIP2-CARD. Together with structure guided mutagenesis experiments at the CARD-CARD interfaces, we demonstrate molecular mechanisms how RIP2 is activated and self-propagating such signal.
History
DepositionNov 9, 2017-
Header (metadata) releaseNov 14, 2018-
Map releaseNov 14, 2018-
UpdateMar 27, 2024-
Current statusMar 27, 2024Processing site: PDBj / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.029
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.029
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-5yrn
  • Surface level: 0.029
  • Imaged by UCSF Chimera
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  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-5yrn
  • Imaged by Jmol
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

Downloads & links

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Map

FileDownload / File: emd_6842.map.gz / Format: CCP4 / Size: 30.5 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotation200 A long segment of RIP-CARD filament
Voxel sizeX=Y=Z: 1.23 Å
Density
Contour LevelBy AUTHOR: 0.029 / Movie #1: 0.029
Minimum - Maximum-0.031169191 - 0.08184105
Average (Standard dev.)0.0011434028 (±0.005348552)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-69-69-69
Dimensions200200200
Spacing200200200
CellA=B=C: 246.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.231.231.23
M x/y/z200200200
origin x/y/z0.0000.0000.000
length x/y/z246.000246.000246.000
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS-69-69-69
NC/NR/NS200200200
D min/max/mean-0.0310.0820.001

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Supplemental data

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Additional map: Cropped out central segment of 60 A, where...

Fileemd_6842_additional.map
AnnotationCropped out central segment of 60 A, where 12 RIP2-CARD were built into.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

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Half map: unfiltered half 2 of 200 A RIP2-CARD filament

Fileemd_6842_half_map_1.map
Annotationunfiltered half 2 of 200 A RIP2-CARD filament
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

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Half map: unfiltered half 1 of 200 A RIP2-CARD filament

Fileemd_6842_half_map_2.map
Annotationunfiltered half 1 of 200 A RIP2-CARD filament
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

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Sample components

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Entire : Active RIP2 signaling complex

EntireName: Active RIP2 signaling complex
Components
  • Organelle or cellular component: Active RIP2 signaling complex
    • Protein or peptide: Receptor-interacting serine/threonine-protein kinase 2

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Supramolecule #1: Active RIP2 signaling complex

SupramoleculeName: Active RIP2 signaling complex / type: organelle_or_cellular_component / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Homo sapiens (human)

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Macromolecule #1: Receptor-interacting serine/threonine-protein kinase 2

MacromoleculeName: Receptor-interacting serine/threonine-protein kinase 2
type: protein_or_peptide / ID: 1 / Number of copies: 12 / Enantiomer: LEVO / EC number: non-specific serine/threonine protein kinase
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 12.621438 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
TNSAGIAQQW IQSKREDIVN QMTEACLNQS LDALLSRDLI MKEDYELVST KPTRTSKVRQ LLDTTDIQGE EFAKVIVQKL KDNKQMGLQ PYPEILVVSR SPSLNLLQNK SM

UniProtKB: Receptor-interacting serine/threonine-protein kinase 2

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Experimental details

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Structure determination

Methodcryo EM
Processinghelical reconstruction
Aggregation statefilament

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Sample preparation

Concentration0.2 mg/mL
BufferpH: 7.4
GridMaterial: COPPER / Support film - Material: CARBON / Support film - topology: HOLEY
VitrificationCryogen name: METHANE

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Electron microscopy

MicroscopeFEI POLARA 300
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Number grids imaged: 3 / Number real images: 3100 / Average electron dose: 8.0 e/Å2
Experimental equipment
Model: Tecnai Polara / Image courtesy: FEI Company

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Image processing

Segment selectionNumber selected: 658160
Startup modelType of model: OTHER
Details: A tubular cylinder with 80 A width was used as the starting model.
Final angle assignmentType: NOT APPLICABLE / Software - Name: RELION (ver. 2.0beta)
Final reconstructionApplied symmetry - Helical parameters - Δz: 4.936 Å
Applied symmetry - Helical parameters - Δ&Phi: -101.373 °
Applied symmetry - Helical parameters - Axial symmetry: C1 (asymmetric)
Resolution.type: BY AUTHOR / Resolution: 4.1 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 2.0beta)
Details: Model building was based on a cropped-out density segment. Pseudo-crystallographic refinement showed that the central segment of the map is good until 3.5-3.7 A.
Number images used: 142330
FSC plot (resolution estimation)

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Atomic model buiding 1

RefinementSpace: RECIPROCAL / Protocol: RIGID BODY FIT / Overall B value: 202.7 / Target criteria: Rfree value
Output model

PDB-5yrn:
Structure of RIP2 CARD domain

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