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- EMDB-8439: CryoEM reconstruction of hIKK1 in the most open state, conformation 1 -

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Basic information

Entry
Database: EMDB / ID: EMD-8439
TitleCryoEM reconstruction of hIKK1 in the most open state, conformation 1
Map dataCryoEM reconstruction of human IKK1, hexameric composition, final map
Sample
  • Complex: Inhibitor of KappaB Kinase 1 hexamer
    • Protein or peptide: Inhibitor of Kappa B Kinase 1
Function / homology
Function and homology information


response to acetate / IkappaB kinase / IkappaB kinase activity / IKBKB deficiency causes SCID / IKBKG deficiency causes anhidrotic ectodermal dysplasia with immunodeficiency (EDA-ID) (via TLR) / response to cholecystokinin / IkappaB kinase complex / I-kappaB phosphorylation / transferrin receptor binding / IkBA variant leads to EDA-ID ...response to acetate / IkappaB kinase / IkappaB kinase activity / IKBKB deficiency causes SCID / IKBKG deficiency causes anhidrotic ectodermal dysplasia with immunodeficiency (EDA-ID) (via TLR) / response to cholecystokinin / IkappaB kinase complex / I-kappaB phosphorylation / transferrin receptor binding / IkBA variant leads to EDA-ID / CD40 receptor complex / NF-kB activation through FADD/RIP-1 pathway mediated by caspase-8 and -10 / RIP-mediated NFkB activation via ZBP1 / response to hydroperoxide / AKT phosphorylates targets in the cytosol / toll-like receptor 4 signaling pathway / negative regulation of NF-kappaB transcription factor activity / non-canonical NF-kappaB signal transduction / Constitutive Signaling by AKT1 E17K in Cancer / positive regulation of interferon-alpha production / TRAF6 mediated NF-kB activation / Rho protein signal transduction / skeletal muscle contraction / anatomical structure morphogenesis / response to amino acid / canonical NF-kappaB signal transduction / IRAK1 recruits IKK complex / IRAK1 recruits IKK complex upon TLR7/8 or 9 stimulation / cellular response to cadmium ion / tumor necrosis factor-mediated signaling pathway / striated muscle cell differentiation / MAP3K8 (TPL2)-dependent MAPK1/3 activation / TICAM1, RIP1-mediated IKK complex recruitment / IKK complex recruitment mediated by RIP1 / TNFR1-induced NF-kappa-B signaling pathway / Regulation of NF-kappa B signaling / NIK-->noncanonical NF-kB signaling / Dectin-1 mediated noncanonical NF-kB signaling / Regulation of TNFR1 signaling / Activation of NF-kappaB in B cells / response to virus / TAK1-dependent IKK and NF-kappa-B activation / NOD1/2 Signaling Pathway / PKR-mediated signaling / cytoplasmic side of plasma membrane / response to toxic substance / CLEC7A (Dectin-1) signaling / cellular response to virus / cellular response to reactive oxygen species / FCERI mediated NF-kB activation / Interleukin-1 signaling / Downstream TCR signaling / cellular response to tumor necrosis factor / positive regulation of NF-kappaB transcription factor activity / ER-Phagosome pathway / scaffold protein binding / positive regulation of canonical NF-kappaB signal transduction / response to lipopolysaccharide / protein kinase activity / immune response / response to xenobiotic stimulus / inflammatory response / protein heterodimerization activity / protein phosphorylation / innate immune response / protein serine/threonine kinase activity / protein-containing complex binding / SARS-CoV-2 activates/modulates innate and adaptive immune responses / positive regulation of DNA-templated transcription / protein homodimerization activity / positive regulation of transcription by RNA polymerase II / nucleoplasm / ATP binding / cytosol / cytoplasm
Similarity search - Function
I-kappa-kinase-beta NEMO binding domain / IKBKB, scaffold dimerization domain / IKBKB, scaffold dimerization domain superfamily / I-kappa-kinase-beta NEMO binding domain / IQBAL scaffold dimerization domain / I-kappa-kinase-beta NEMO binding domain / Serine/threonine-protein kinase, active site / Serine/Threonine protein kinases active-site signature. / Protein kinase domain / Serine/Threonine protein kinases, catalytic domain ...I-kappa-kinase-beta NEMO binding domain / IKBKB, scaffold dimerization domain / IKBKB, scaffold dimerization domain superfamily / I-kappa-kinase-beta NEMO binding domain / IQBAL scaffold dimerization domain / I-kappa-kinase-beta NEMO binding domain / Serine/threonine-protein kinase, active site / Serine/Threonine protein kinases active-site signature. / Protein kinase domain / Serine/Threonine protein kinases, catalytic domain / Protein kinase, ATP binding site / Protein kinases ATP-binding region signature. / Protein kinase domain profile. / Protein kinase domain / Protein kinase-like domain superfamily
Similarity search - Domain/homology
Inhibitor of nuclear factor kappa-B kinase subunit alpha
Similarity search - Component
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 5.9 Å
AuthorsLyumkis D / Ghosh G / Polley S / Biswath T / Huang D / Passos DO
Funding support United States, 4 items
OrganizationGrant numberCountry
National Institutes of Health/Office of the DirectorDP5 OD021396-01 United States
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)AI064326 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM071862 United States
National Institutes of Health/National Cancer Institute (NIH/NCI)CA141722 United States
CitationJournal: Cell Rep / Year: 2016
Title: Structural Basis for the Activation of IKK1/α.
Authors: Smarajit Polley / Dario Oliveira Passos / De-Bin Huang / Maria Carmen Mulero / Anup Mazumder / Tapan Biswas / Inder M Verma / Dmitry Lyumkis / Gourisankar Ghosh /
Abstract: Distinct signaling pathways activate the NF-κB family of transcription factors. The canonical NF-κB-signaling pathway is mediated by IκB kinase 2/β (IKK2/β), while the non-canonical pathway ...Distinct signaling pathways activate the NF-κB family of transcription factors. The canonical NF-κB-signaling pathway is mediated by IκB kinase 2/β (IKK2/β), while the non-canonical pathway depends on IKK1/α. The structural and biochemical bases for distinct signaling by these otherwise highly similar IKKs are unclear. We report single-particle cryoelectron microscopy (cryo-EM) and X-ray crystal structures of human IKK1 in dimeric (∼150 kDa) and hexameric (∼450 kDa) forms. The hexamer, which is the representative form in the crystal but comprises only ∼2% of the particles in solution by cryo-EM, is a trimer of IKK1 dimers. While IKK1 hexamers are not detectable in cells, the surface that supports hexamer formation is critical for IKK1-dependent cellular processing of p100 to p52, the hallmark of non-canonical NF-κB signaling. Comparison of this surface to that in IKK2 indicates significant divergence, and it suggests a fundamental role for this surface in signaling by these kinases through distinct pathways.
History
DepositionOct 24, 2016-
Header (metadata) releaseNov 9, 2016-
Map releaseNov 9, 2016-
UpdateJan 29, 2020-
Current statusJan 29, 2020Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.035
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 0.035
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_8439.png

Downloads & links

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Map

FileDownload / File: emd_8439.map.gz / Format: CCP4 / Size: 125 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationCryoEM reconstruction of human IKK1, hexameric composition, final map
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.31 Å/pix.
x 320 pix.
= 419.2 Å		1.31 Å/pix.
x 320 pix.
= 419.2 Å		1.31 Å/pix.
x 320 pix.
= 419.2 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.31 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.035 / Movie #1: 0.035
Minimum - Maximum-0.07212129 - 0.13111335
Average (Standard dev.)-0.00032097686 (±0.0055240183)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions320320320
Spacing320320320
CellA=B=C: 419.19998 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.311.311.31
M x/y/z320320320
origin x/y/z0.0000.0000.000
length x/y/z419.200419.200419.200
α/β/γ90.00090.00090.000
start NX/NY/NZ-38-19-20
NX/NY/NZ858082
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS320320320
D min/max/mean-0.0720.131-0.000

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Supplemental data

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Half map: CryoEM reconstruction of human IKK1, hexameric composition, half...

Fileemd_8439_half_map_1.map
AnnotationCryoEM reconstruction of human IKK1, hexameric composition, half map 2, unfiltered and unmasked
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: CryoEM reconstruction of human IKK1, hexameric composition, half...

Fileemd_8439_half_map_2.map
AnnotationCryoEM reconstruction of human IKK1, hexameric composition, half map 1, unfiltered and unmasked
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Inhibitor of KappaB Kinase 1 hexamer

EntireName: Inhibitor of KappaB Kinase 1 hexamer
Components
  • Complex: Inhibitor of KappaB Kinase 1 hexamer
    • Protein or peptide: Inhibitor of Kappa B Kinase 1

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Supramolecule #1: Inhibitor of KappaB Kinase 1 hexamer

SupramoleculeName: Inhibitor of KappaB Kinase 1 hexamer / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all / Details: hexamer
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm) / Recombinant cell: SF9 / Recombinant plasmid: pFastBacHTa
Molecular weightExperimental: 450 KDa

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Macromolecule #1: Inhibitor of Kappa B Kinase 1

MacromoleculeName: Inhibitor of Kappa B Kinase 1 / type: protein_or_peptide / ID: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: DPEFGAGGPW EMRERLGTGG FGNVCLYQHR ELDLKIAIKS CRLELSTKNR ERWCHEIQI MKKLNHANVV KACDVPEELN ILIHDVPLLA MEYCSGGDLR K LLNKPENC CGLKESQILS LLSDIGSGIR YLHENKIIHR DLKPENIVLQ DV GGKIIHK IIDLGYAKDV ...String:
DPEFGAGGPW EMRERLGTGG FGNVCLYQHR ELDLKIAIKS CRLELSTKNR ERWCHEIQI MKKLNHANVV KACDVPEELN ILIHDVPLLA MEYCSGGDLR K LLNKPENC CGLKESQILS LLSDIGSGIR YLHENKIIHR DLKPENIVLQ DV GGKIIHK IIDLGYAKDV DQGELCTEFV GTLQYLAPEL FENKPYTATV DYW SFGTMV FECIAGYRPF LHHLQPFTWH EKIKKKDPKC IFACEEMSGE VRFS SHLPQ PNSLCSLIVE PMENWLQLML NWDPQQRGGP VDLTLKQPRC FVLMD HILN LKIVHILNMT SAKIISFLLP PDESLHSLQS RIERETGINT GSQELL SET GISLDPRKPA SQCVLDGVRG CDSYMVYLFD KSKTVYEGPF ASRSLSD CV NYIVQDSKIQ LPIIQLRKVW AEAVHYVSGL KEDYSRLFQG QRAAMLSL L RYNANLTKMK NTLISASQQL KAKLEFFHKS IQLDLERYSE QMTYGISSE KMLKAWKEME EKAIHYAEVG VIGYLEDQIM SLHAEIMELQ KSPYGRRQGD LMESLEQRA IDLYKQLKHR PSDHSYSDST EMVKIIVHTV QSQDRVLKEL F GHLSKLLG CKQKIIDLLP KVEVALSNIK EADNTVMFMQ GKRQKEIWHL LK IACTQ

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration2.5 mg/mL
BufferpH: 7.5
Component:
ConcentrationFormulaName
150.0 mMNaClSodium chloridesodium chloride
20.0 mMC4H12ClNO3Tris-HClTris
5.0 mMC4H10O2S2DTT
5.0 %C3H8O3Glycerol
GridModel: C-flat-1.2/1.3 / Material: COPPER / Mesh: 400 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: PLASMA CLEANING / Pretreatment - Atmosphere: AIR
VitrificationCryogen name: ETHANE / Chamber humidity: 80 % / Chamber temperature: 277 K / Instrument: HOMEMADE PLUNGER
Details: Sample containing IKK1 dimers in SEC buffer was applied onto freshly plasma-treated (6 seconds, Gatan Solarus plasma cleaner) holey carbon C-flat grids (Protochips), adsorbed for 30 seconds, ...Details: Sample containing IKK1 dimers in SEC buffer was applied onto freshly plasma-treated (6 seconds, Gatan Solarus plasma cleaner) holey carbon C-flat grids (Protochips), adsorbed for 30 seconds, and then plunged into liquid ethane using a manual cryo-plunger in an ambient environment of 4 degrees C..

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 100.0 µm / Calibrated defocus max: 5.2 µm / Calibrated defocus min: 1.1 µm / Calibrated magnification: 38167 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal magnification: 22500
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
TemperatureMin: 70.0 K / Max: 70.0 K
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 - Sampling interval: 5.0 µm / Digitization - Frames/image: 1-50 / Number grids imaged: 1 / Number real images: 2918 / Average exposure time: 10.0 sec. / Average electron dose: 40.0 e/Å2
Details: The dose was fractionated over 50 raw frames collected over a 10 second exposure time (200 ms per frame) on the Gatan K2 Summit direct detection device, with each frame receiving a dose of ...Details: The dose was fractionated over 50 raw frames collected over a 10 second exposure time (200 ms per frame) on the Gatan K2 Summit direct detection device, with each frame receiving a dose of ~6.5 e-/pixel/sec. 2918 movies were collected and recorded at a nominal magnification of 22,500, corresponding to a pixel size of 1.31 A at the specimen level. The individual frames were gain-corrected, then aligned and summed using a GPU-enabled whole frame alignment program (Li et al., 2013), and exposure-filtered (Grant and Grigorieff, 2015).
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 230242
CTF correctionSoftware - Name: CTFFIND / Software - details: within Appion / Details: within Relion and Frealign
Startup modelType of model: INSILICO MODEL
Details: common lines model generated using OptiMod and EMAN1
Initial angle assignmentType: PROJECTION MATCHING / Software - Name: RELION (ver. 1.3)
Final 3D classificationSoftware - Name: FREALIGN (ver. 9.11)
Details: During 2D alignment and classification, we noticed that a small percentage of particles apparently formed multiple dimers stacked side-by-side. After several rounds of sub-classification, it ...Details: During 2D alignment and classification, we noticed that a small percentage of particles apparently formed multiple dimers stacked side-by-side. After several rounds of sub-classification, it became apparent that this small percentage of particles actually corresponded to an oligomer of IKK1 dimers. An ab initio initial model was generated from the subset of particles using OptiMod, which showed that the oligomer was clearly hexameric in nature. 3D classifications and refinements were performed in a similar fashion as for the dimers. Only one conformational state was identified (despite asking for the recovery of up to 5 classes), which was refined to ~5.9 A resolution
Final angle assignmentType: PROJECTION MATCHING / Software - Name: FREALIGN (ver. 9.11)
Final reconstructionApplied symmetry - Point group: D3 (2x3 fold dihedral) / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 5.9 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: FREALIGN (ver. 9.11) / Number images used: 1432
FSC plot (resolution estimation)

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Atomic model buiding 1

RefinementOverall B value: 380

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