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- EMDB-16822: Cryo-EM structure of the murine IL-12 complete extracellular sign... -

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Basic information

Entry
Database: EMDB / ID: EMD-16822
TitleCryo-EM structure of the murine IL-12 complete extracellular signaling complex (Class 1).
Map dataSharpened map of the murine IL12:IL12Rbeta1:IL12Rbeta2 complex (Class 1).
Sample
  • Complex: Murine IL-12 in complex with Strep-II tagged mIL-12Rbeta1 and mIL-12Rbeta2.
    • Protein or peptide: Murine Interleukin-12 subunit alpha (IL-12A, p35), with a C-terminal caspase-3 cleavable His tag.
  • Protein or peptide: Murine Interleukin-12 subunit beta (IL-12B, p40).
  • Protein or peptide: Murine Interleukin-12 Receptor beta 1 (domains 1 to 5) with C-terminal Strep-II tag.
  • Protein or peptide: Murine Interleukin-12 Receptor beta 2 (domains 1 to 6) with C-terminal Strep-II tag.
KeywordsComplex / Cytokine / Receptor / SIGNALING PROTEIN
Function / homology
Function and homology information


interleukin-12 beta subunit binding / Interleukin-12 signaling / Interleukin-23 signaling / interleukin-23 receptor binding / Interleukin-35 Signalling / interleukin-12 alpha subunit binding / interleukin-12 complex / interleukin-23 complex / T-helper 1 cell activation / negative regulation of vascular endothelial growth factor signaling pathway ...interleukin-12 beta subunit binding / Interleukin-12 signaling / Interleukin-23 signaling / interleukin-23 receptor binding / Interleukin-35 Signalling / interleukin-12 alpha subunit binding / interleukin-12 complex / interleukin-23 complex / T-helper 1 cell activation / negative regulation of vascular endothelial growth factor signaling pathway / positive regulation of natural killer cell mediated cytotoxicity directed against tumor cell target / negative regulation of blood vessel endothelial cell proliferation involved in sprouting angiogenesis / positive regulation of smooth muscle cell apoptotic process / positive regulation of T-helper 1 type immune response / interleukin-12 receptor binding / interleukin-23 receptor complex / T-helper cell differentiation / interleukin-12-mediated signaling pathway / positive regulation of NK T cell proliferation / negative regulation of interleukin-17 production / positive regulation of osteoclast differentiation / cytokine receptor activity / natural killer cell activation / positive regulation of granulocyte macrophage colony-stimulating factor production / response to UV-B / positive regulation of T cell differentiation / negative regulation of interleukin-10 production / defense response to protozoan / positive regulation of interleukin-17 production / positive regulation of natural killer cell proliferation / positive regulation of activated T cell proliferation / positive regulation of interleukin-10 production / negative regulation of protein secretion / cell surface receptor signaling pathway via JAK-STAT / T cell proliferation / coreceptor activity / positive regulation of defense response to virus by host / response to cytokine / positive regulation of T cell proliferation / positive regulation of interleukin-12 production / endosome lumen / cytokine activity / growth factor activity / negative regulation of inflammatory response to antigenic stimulus / negative regulation of smooth muscle cell proliferation / cellular response to type II interferon / positive regulation of T cell mediated cytotoxicity / Golgi lumen / positive regulation of type II interferon production / positive regulation of tumor necrosis factor production / cell migration / cellular response to lipopolysaccharide / response to lipopolysaccharide / defense response to Gram-negative bacterium / defense response to virus / cell surface receptor signaling pathway / cell population proliferation / immune response / endoplasmic reticulum lumen / protein heterodimerization activity / external side of plasma membrane / protein kinase binding / cell surface / extracellular space / extracellular region / identical protein binding / membrane / plasma membrane / cytoplasm
Similarity search - Function
Interleukin-12 alpha / Interleukin-12 alpha subunit / : / Interleukin-12 beta / Interleukin-12 beta, central domain / : / Cytokine interleukin-12p40 C-terminus / Long hematopoietin receptor, soluble alpha chain, conserved site / Long hematopoietin receptor, soluble alpha chains family signature. / Immunoglobulin C2-set-like, ligand-binding ...Interleukin-12 alpha / Interleukin-12 alpha subunit / : / Interleukin-12 beta / Interleukin-12 beta, central domain / : / Cytokine interleukin-12p40 C-terminus / Long hematopoietin receptor, soluble alpha chain, conserved site / Long hematopoietin receptor, soluble alpha chains family signature. / Immunoglobulin C2-set-like, ligand-binding / Ig-like C2-type domain / Long hematopoietin receptor, Gp130 family 2, conserved site / Long hematopoietin receptor, gp130 family signature. / Four-helical cytokine-like, core / Fibronectin type III domain / Fibronectin type 3 domain / Immunoglobulin subtype 2 / Immunoglobulin C-2 Type / Fibronectin type-III domain profile. / Fibronectin type III / Fibronectin type III superfamily / Ig-like domain profile. / Immunoglobulin-like domain / Immunoglobulin-like domain superfamily / Immunoglobulin-like fold
Similarity search - Domain/homology
Interleukin-12 subunit alpha / Interleukin-12 subunit beta / Interleukin-12 receptor subunit beta-2 / Interleukin-12 receptor subunit beta-1
Similarity search - Component
Biological speciesMus musculus (house mouse)
Methodsingle particle reconstruction / cryo EM / Resolution: 5.6 Å
AuthorsFelix J / Bloch Y / Savvides SN
Funding support Belgium, 2 items
OrganizationGrant numberCountry
Research Foundation - Flanders (FWO)12S0519N Belgium
Research Foundation - Flanders (FWO)G0B4918N Belgium
CitationJournal: Nat Struct Mol Biol / Year: 2024
Title: Structures of complete extracellular receptor assemblies mediated by IL-12 and IL-23.
Authors: Yehudi Bloch / Jan Felix / Romain Merceron / Mathias Provost / Royan Alipour Symakani / Robin De Backer / Elisabeth Lambert / Ahmad R Mehdipour / Savvas N Savvides /
Abstract: Cell-surface receptor complexes mediated by pro-inflammatory interleukin (IL)-12 and IL-23, both validated therapeutic targets, are incompletely understood due to the lack of structural insights into ...Cell-surface receptor complexes mediated by pro-inflammatory interleukin (IL)-12 and IL-23, both validated therapeutic targets, are incompletely understood due to the lack of structural insights into their complete extracellular assemblies. Furthermore, there is a paucity of structural details describing the IL-12-receptor interaction interfaces, in contrast to IL-23-receptor complexes. Here we report structures of fully assembled mouse IL-12/human IL-23-receptor complexes comprising the complete extracellular segments of the cognate receptors determined by electron cryo-microscopy. The structures reveal key commonalities but also surprisingly diverse features. Most notably, whereas IL-12 and IL-23 both utilize a conspicuously presented aromatic residue on their α-subunit as a hotspot to interact with the N-terminal Ig domain of their high-affinity receptors, only IL-12 juxtaposes receptor domains proximal to the cell membrane. Collectively, our findings will help to complete our understanding of cytokine-mediated assemblies of tall cytokine receptors and will enable a cytokine-specific interrogation of IL-12/IL-23 signaling in physiology and disease.
History
DepositionMar 10, 2023-
Header (metadata) releaseFeb 7, 2024-
Map releaseFeb 7, 2024-
UpdateMay 1, 2024-
Current statusMay 1, 2024Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_16822.png

Downloads & links

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Map

FileDownload / File: emd_16822.map.gz / Format: CCP4 / Size: 40.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationSharpened map of the murine IL12:IL12Rbeta1:IL12Rbeta2 complex (Class 1).
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.51 Å/pix.
x 220 pix.
= 332.2 Å		1.51 Å/pix.
x 220 pix.
= 332.2 Å		1.51 Å/pix.
x 220 pix.
= 332.2 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.51 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.13
Minimum - Maximum-0.26047748 - 0.64348257
Average (Standard dev.)0.00084523944 (±0.016497888)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions220220220
Spacing220220220
CellA=B=C: 332.2 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_16822_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map 2 of the murine IL12:IL12Rbeta1:IL12Rbeta2 complex...

Fileemd_16822_half_map_1.map
AnnotationHalf map 2 of the murine IL12:IL12Rbeta1:IL12Rbeta2 complex (Class 1).
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map 1 of the murine IL12:IL12Rbeta1:IL12Rbeta2 complex...

Fileemd_16822_half_map_2.map
AnnotationHalf map 1 of the murine IL12:IL12Rbeta1:IL12Rbeta2 complex (Class 1).
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Murine IL-12 in complex with Strep-II tagged mIL-12Rbeta1 and mIL...

EntireName: Murine IL-12 in complex with Strep-II tagged mIL-12Rbeta1 and mIL-12Rbeta2.
Components
  • Complex: Murine IL-12 in complex with Strep-II tagged mIL-12Rbeta1 and mIL-12Rbeta2.
    • Protein or peptide: Murine Interleukin-12 subunit alpha (IL-12A, p35), with a C-terminal caspase-3 cleavable His tag.
  • Protein or peptide: Murine Interleukin-12 subunit beta (IL-12B, p40).
  • Protein or peptide: Murine Interleukin-12 Receptor beta 1 (domains 1 to 5) with C-terminal Strep-II tag.
  • Protein or peptide: Murine Interleukin-12 Receptor beta 2 (domains 1 to 6) with C-terminal Strep-II tag.

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Supramolecule #1: Murine IL-12 in complex with Strep-II tagged mIL-12Rbeta1 and mIL...

SupramoleculeName: Murine IL-12 in complex with Strep-II tagged mIL-12Rbeta1 and mIL-12Rbeta2.
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: Mus musculus (house mouse)
Molecular weightTheoretical: 198 KDa

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Macromolecule #1: Murine Interleukin-12 subunit alpha (IL-12A, p35), with a C-termi...

MacromoleculeName: Murine Interleukin-12 subunit alpha (IL-12A, p35), with a C-terminal caspase-3 cleavable His tag.
type: protein_or_peptide / ID: 1 / Enantiomer: LEVO
Source (natural)Organism: Mus musculus (house mouse)
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: MCQSRYLLFL ATLALLNHLS LARVIPVSGP ARCLSQSRNL LKTTDDMVKT AREKLKHYSC TAEDIDHEDI TRDQTSTLKT CLPLELHKNE SCLATRETSS TTRGSCLPPQ KTSLMMTLCL GSIYEDLKMY QTEFQAINAA LQNHNHQQII LDKGMLVAID ELMQSLNHNG ...String:
MCQSRYLLFL ATLALLNHLS LARVIPVSGP ARCLSQSRNL LKTTDDMVKT AREKLKHYSC TAEDIDHEDI TRDQTSTLKT CLPLELHKNE SCLATRETSS TTRGSCLPPQ KTSLMMTLCL GSIYEDLKMY QTEFQAINAA LQNHNHQQII LDKGMLVAID ELMQSLNHNG ETLRQKPPVG EADPYRVKMK LCILLHAFST RVVTINRVMG YLSSAGTSDE VDGGSGGSGL NDIFEAQKIE WHEGRTKHHH HHH

UniProtKB: Interleukin-12 subunit alpha

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Macromolecule #2: Murine Interleukin-12 subunit beta (IL-12B, p40).

MacromoleculeName: Murine Interleukin-12 subunit beta (IL-12B, p40). / type: protein_or_peptide / ID: 2 / Enantiomer: LEVO
Source (natural)Organism: Mus musculus (house mouse)
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: MCPQKLTISW FAIVLLVSPL MAMWELEKDV YVVEVDWTPD APGETVNLTC DTPEEDDITW TSDQRHGVIG SGKTLTITVK EFLDAGQYTC HKGGETLSHS HLLLHKKENG IWSTEILKNF KNKTFLKCEA PNYSGRFTCS WLVQRNMDLK FNIKSSSSSP DSRAVTCGMA ...String:
MCPQKLTISW FAIVLLVSPL MAMWELEKDV YVVEVDWTPD APGETVNLTC DTPEEDDITW TSDQRHGVIG SGKTLTITVK EFLDAGQYTC HKGGETLSHS HLLLHKKENG IWSTEILKNF KNKTFLKCEA PNYSGRFTCS WLVQRNMDLK FNIKSSSSSP DSRAVTCGMA SLSAEKVTLD QRDYEKYSVS CQEDVTCPTA EETLPIELAL EARQQNKYEN YSTSFFIRDI IKPDPPKNLQ MKPLKNSQVE VSWEYPDSWS TPHSYFSLKF FVRIQRKKEK MKETEEGCNQ KGAFLVEKTS TEVQCKGGNV CVQAQDRYYN SSCSKWACVP CRVRS

UniProtKB: Interleukin-12 subunit beta

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Macromolecule #3: Murine Interleukin-12 Receptor beta 1 (domains 1 to 5) with C-ter...

MacromoleculeName: Murine Interleukin-12 Receptor beta 1 (domains 1 to 5) with C-terminal Strep-II tag.
type: protein_or_peptide / ID: 3 / Enantiomer: LEVO
Source (natural)Organism: Mus musculus (house mouse)
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: QLGASGPGDG CCVEKTSFPE GASGSPLGPR NLSCYRVSKT DYECSWQYDG PEDNVSHVLW CCFVPPNHTH TGQERCRYFS SGPDRTVQFW EQDGIPVLSK VNFWVESRLG NRTMKSQKIS QYLYNWTKTT PPLGHIKVSQ SHRQLRMDWN VSEEAGAEVQ FRRRMPTTNW ...String:
QLGASGPGDG CCVEKTSFPE GASGSPLGPR NLSCYRVSKT DYECSWQYDG PEDNVSHVLW CCFVPPNHTH TGQERCRYFS SGPDRTVQFW EQDGIPVLSK VNFWVESRLG NRTMKSQKIS QYLYNWTKTT PPLGHIKVSQ SHRQLRMDWN VSEEAGAEVQ FRRRMPTTNW TLGDCGPQVN SGSGVLGDIR GSMSESCLCP SENMAQEIQI RRRRRLSSGA PGGPWSDWSM PVCVPPEVLP QAKIKFLVEP LNQGGRRRLT MQGQSPQLAV PEGCRGRPGA QVKKHLVLVR MLSCRCQAQT SKTVPLGKKL NLSGATYDLN VLAKTRFGRS TIQKWHLPAQ ELTETRALNV SVGGNMTSMQ WAAQAPGTTY CLEWQPWFQH RNHTHCTLIV PEEEDPAKMV THSWSSKPTL EQEECYRITV FASKNPKNPM LWATVLSSYY FGGNASRAGT PRHVSVRNQT GDSVSVEWTA SQLSTCPGVL TQYVVRCEAE DGAWESEWLV PPTKTQVTLD GLRSRVMYKV QVRADTARLP GAWSHPQRFS FEGTWSHPQF EK

UniProtKB: Interleukin-12 receptor subunit beta-1

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Macromolecule #4: Murine Interleukin-12 Receptor beta 2 (domains 1 to 6) with C-ter...

MacromoleculeName: Murine Interleukin-12 Receptor beta 2 (domains 1 to 6) with C-terminal Strep-II tag.
type: protein_or_peptide / ID: 4 / Enantiomer: LEVO
Source (natural)Organism: Mus musculus (house mouse)
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: NIDVCKLGTV TVQPAPVIPL GSAANISCSL NPKQGCSHYP SSNELILLKF VNDVLVENLH GKKVHDHTGH SSTFQVTNLS LGMTLFVCKL NCSNSQKKPP VPVCGVEISV GVAPEPPQNI SCVQEGENGT VACSWNSGKV TYLKTNYTLQ LSGPNNLTCQ KQCFSDNRQN ...String:
NIDVCKLGTV TVQPAPVIPL GSAANISCSL NPKQGCSHYP SSNELILLKF VNDVLVENLH GKKVHDHTGH SSTFQVTNLS LGMTLFVCKL NCSNSQKKPP VPVCGVEISV GVAPEPPQNI SCVQEGENGT VACSWNSGKV TYLKTNYTLQ LSGPNNLTCQ KQCFSDNRQN CNRLDLGINL SPDLAESRFI VRVTAINDLG NSSSLPHTFT FLDIVIPLPP WDIRINFLNA SGSRGTLQWE DEGQVVLNQL RYQPLNSTSW NMVNATNAKG KYDLRDLRPF TEYEFQISSK LHLSGGSWSN WSESLRTRTP EEEPVGILDI WYMKQDIDYD RQQISLFWKS LNPSEARGKI LHYQVTLQEV TKKTTLQNTT RHTSWTRVIP RTGAWTASVS AANSKGASAP THINIVDLCG TGLLAPHQVS AKSENMDNIL VTWQPPKKAD SAVREYIVEW RALQPGSITK FPPHWLRIPP DNMSALISEN IKPYICYEIR VHALSESQGG CSSIRGDSKH KAPVSGPHIT AITEKKERLF ISWTHIPFPE QRGCILHYRI YWKERDSTAQ PELCEIQYRR SQNSHPISSL QPRVTYVLWM TAVTAAGESP QGNEREFCPQ GKANGTWSHP QFEK

UniProtKB: Interleukin-12 receptor subunit beta-2

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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

BufferpH: 7.4
Component:
ConcentrationFormulaName
150.0 mMNaClsodium chloride
25.0 mMC8H18N2O4SHEPES

Details: HEPES-buffered saline (HBS): 25 mM HEPES, pH 7.4, 150 mM NaCl.
GridModel: Quantifoil R2/1 / Material: COPPER / Mesh: 300 / Pretreatment - Type: GLOW DISCHARGE
VitrificationCryogen name: ETHANE / Chamber humidity: 95 % / Instrument: LEICA PLUNGER / Details: Leica EM GP2, 4 s. blotting time..

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Electron microscopy

MicroscopeJEOL CRYO ARM 300
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Number real images: 7174 / Average exposure time: 3.37 sec. / Average electron dose: 61.8 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.5 µm / Nominal defocus min: 1.0 µm / Nominal magnification: 60000
Sample stageCooling holder cryogen: NITROGEN

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Image processing

Particle selectionNumber selected: 538128
Startup modelType of model: NONE
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 5.6 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC / Number images used: 66809
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC
Final 3D classificationNumber classes: 2 / Software - Name: cryoSPARC
FSC plot (resolution estimation)

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