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- PDB-5tqx: CryoEM reconstruction of human IKK1, intermediate conformation 2 -

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Entry
Database: PDB / ID: 5tqx
TitleCryoEM reconstruction of human IKK1, intermediate conformation 2
ComponentsInhibitor of nuclear factor kappa-B kinase subunit alpha
KeywordsTRANSFERASE / kinase / conserved helix-loop-helix / transcription / oncogene
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 / Constitutive Signaling by AKT1 E17K in Cancer / non-canonical NF-kappaB signal transduction / 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)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 5.4 Å
AuthorsLyumkis, D. / Ghosh, G. / Polley, S. / Biswath, T. / Huang, D. / Passos, D.O.
Funding support United States, 4items
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 Cancer Institute (NIH/NCI)CA141722 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM071862 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, 2016Deposition site: RCSB / Processing site: RCSB
Revision 1.0Nov 9, 2016Provider: repository / Type: Initial release
Revision 1.1Nov 30, 2016Group: Database references
Revision 1.2Sep 13, 2017Group: Author supporting evidence / Data collection / Category: em_software / pdbx_audit_support
Item: _em_software.name / _pdbx_audit_support.funding_organization
Revision 1.3Jul 18, 2018Group: Data collection / Experimental preparation / Category: em_sample_support / Item: _em_sample_support.grid_type
Revision 1.4Dec 4, 2019Group: Author supporting evidence / Category: pdbx_audit_support / Item: _pdbx_audit_support.funding_organization
Revision 1.5Mar 13, 2024Group: Data collection / Database references / Category: chem_comp_atom / chem_comp_bond / database_2
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession

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Structure visualization

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Assembly

Deposited unit
A: Inhibitor of nuclear factor kappa-B kinase subunit alpha
B: Inhibitor of nuclear factor kappa-B kinase subunit alpha


Theoretical massNumber of molelcules
Total (without water)150,2942
Polymers150,2942
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by author
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area2400 Å2
ΔGint-24 kcal/mol
Surface area65430 Å2

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Components

#1: Protein Inhibitor of nuclear factor kappa-B kinase subunit alpha / IkappaB kinase / Conserved helix-loop-helix ubiquitous kinase / I-kappa-B kinase 1 / IKK1 / Nuclear ...IkappaB kinase / Conserved helix-loop-helix ubiquitous kinase / I-kappa-B kinase 1 / IKK1 / Nuclear factor NF-kappa-B inhibitor kinase alpha / NFKBIKA / Transcription factor 16 / TCF-16


Mass: 75146.945 Da / Num. of mol.: 2 / Mutation: S176E, S180E
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: CHUK, IKKA, TCF16 / Plasmid: pFastBacHTa / Production host: Spodoptera frugiperda (fall armyworm) / Strain (production host): SF9 / References: UniProt: O15111, IkappaB kinase

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: Inhibitor of KappaB Kinase 1 dimer / Type: COMPLEX / Details: dimer / Entity ID: all / Source: RECOMBINANT
Molecular weightValue: 0.15 MDa / Experimental value: YES
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Spodoptera frugiperda (fall armyworm) / Cell: SF9 / Plasmid: pFastBacHTa
Buffer solutionpH: 7.5
Buffer component
IDConc.NameFormulaBuffer-ID
1150 mMsodium chlorideNaClSodium chloride1
220 mMTris-HClTrisC4H12ClNO31
35 mMDTTC4H10O2S21
45 %GlycerolC3H8O31
SpecimenConc.: 2.5 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 400 divisions/in. / Grid type: C-flat-1.2/1.3
VitrificationInstrument: HOMEMADE PLUNGER / Cryogen name: ETHANE / Humidity: 80 % / Chamber temperature: 277 K
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 imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 22500 X / Calibrated magnification: 38167 X / Calibrated defocus min: 1100 nm / Calibrated defocus max: 5200 nm / Cs: 2.7 mm / C2 aperture diameter: 100 µm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Temperature (max): 70 K / Temperature (min): 70 K
Image recordingAverage exposure time: 10 sec. / Electron dose: 40 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 2918
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).
Image scansSampling size: 5 µm / Width: 3838 / Height: 3710 / Movie frames/image: 50 / Used frames/image: 1-50

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Processing

SoftwareName: PHENIX / Version: dev_2499: / Classification: refinement
EM software
IDNameVersionCategoryDetails
1FindEMparticle selectionimplemented within Appion
2Leginon3.1image acquisition
4CTFFINDCTF correctionwithin Appion
9RELION1.3initial Euler assignment
10FREALIGN9.11final Euler assignment
11FREALIGN9.11classification
12FREALIGN9.113D reconstruction
13Rosetta2015.12.57698model refinementsequence mapped to IKK2, then refined to density
14PHENIXdev-2499model refinementreal-space refined
CTF correctionDetails: within Relion and Frealign / Type: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 230242
SymmetryPoint symmetry: C2 (2 fold cyclic)
3D reconstructionResolution: 5.4 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 30540 / Algorithm: FOURIER SPACE / Symmetry type: POINT
Atomic model buildingB value: 340 / Protocol: FLEXIBLE FIT / Space: REAL / Target criteria: FSC 0.5
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0059326
ELECTRON MICROSCOPYf_angle_d0.82112538
ELECTRON MICROSCOPYf_dihedral_angle_d4.1745752
ELECTRON MICROSCOPYf_chiral_restr0.0491396
ELECTRON MICROSCOPYf_plane_restr0.0071574

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