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- PDB-8ppm: IL-12Rb1 neutralizing Fab4, crystal kappa variant -

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Basic information

Entry
Database: PDB / ID: 8ppm
TitleIL-12Rb1 neutralizing Fab4, crystal kappa variant
Components
  • Fab4 Light chain, crystal kappa variant
  • Fab4 heavy chain
KeywordsIMMUNOSUPPRESSANT / antibody / Fab fragment / designed crystal contact
Function / homology:
Function and homology information
Biological speciesHomo sapiens x Mus musculus hybrid cell line (mammal)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 2.03 Å
AuthorsBloch, Y. / Savvides, S.N.
Funding support Belgium, 2items
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
DepositionJul 7, 2023Deposition site: PDBE / Processing site: PDBE
Revision 1.0Feb 7, 2024Provider: repository / Type: Initial release
Revision 1.1May 1, 2024Group: Database references / Category: citation / citation_author
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation_author.identifier_ORCID
Revision 1.2Nov 13, 2024Group: Structure summary / Category: pdbx_entry_details / pdbx_modification_feature / Item: _pdbx_entry_details.has_protein_modification

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
H: Fab4 heavy chain
L: Fab4 Light chain, crystal kappa variant
hetero molecules


Theoretical massNumber of molelcules
Total (without water)48,1054
Polymers48,0262
Non-polymers782
Water3,333185
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: homology, This is the way for antibodies.
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area3810 Å2
ΔGint-21 kcal/mol
Surface area19470 Å2
MethodPISA
Unit cell
Length a, b, c (Å)72.069, 72.069, 163.833
Angle α, β, γ (deg.)90.000, 90.000, 120.000
Int Tables number154
Space group name H-MP3221
Space group name HallP322"
Symmetry operation#1: x,y,z
#2: -y,x-y,z+2/3
#3: -x+y,-x,z+1/3
#4: x-y,-y,-z+1/3
#5: -x,-x+y,-z+2/3
#6: y,x,-z

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Components

#1: Antibody Fab4 heavy chain


Mass: 24786.586 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Details: resideus [225-] (GHHHHHH) encode cloning scar and purification tag. Tag was not removed prior to crystallization.
Source: (gene. exp.) Homo sapiens x Mus musculus hybrid cell line (mammal)
Cell line: HEK293S MGAT-/- / Plasmid: pHLsec / Production host: Homo sapiens (human)
#2: Antibody Fab4 Light chain, crystal kappa variant


Mass: 23239.732 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens x Mus musculus hybrid cell line (mammal)
Plasmid: pHLsec / Cell line (production host): HEK293S MGAT-/- / Production host: Homo sapiens (human)
#3: Chemical ChemComp-K / POTASSIUM ION


Mass: 39.098 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: K
#4: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 185 / Source method: isolated from a natural source / Formula: H2O
Has ligand of interestN
Has protein modificationY

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Experimental details

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Experiment

ExperimentMethod: X-RAY DIFFRACTION / Number of used crystals: 1

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Sample preparation

CrystalDensity Matthews: 2.63 Å3/Da / Density % sol: 53.3 %
Crystal growTemperature: 287 K / Method: vapor diffusion, sitting drop
Details: Fab4 dissociated from target in crystallization drop.

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: PETRA III, EMBL c/o DESY / Beamline: P14 (MX2) / Wavelength: 0.9763 Å
DetectorType: DECTRIS EIGER2 X CdTe 16M / Detector: PIXEL / Date: Apr 24, 2023
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.9763 Å / Relative weight: 1
ReflectionResolution: 2.03→163.83 Å / Num. obs: 31735 / % possible obs: 97 % / Redundancy: 18.71 % / Biso Wilson estimate: 39.95 Å2 / CC1/2: 0.999 / Rrim(I) all: 0.11 / Net I/σ(I): 18.38
Reflection shell

Diffraction-ID: 1

Resolution (Å)Redundancy (%)Mean I/σ(I) obsNum. unique obsCC1/2Rrim(I) all% possible all
6.06-163.8317.9759.4513630.9990.04599.9
2.15-2.319.43.5948710.8930.87499.9
2.03-2.1511.252.0542500.6881.10481.5

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Processing

Software
NameVersionClassification
PHENIX1.20.1_4487refinement
XDS20220820data reduction
XDS20220820data scaling
PHASER2.8.3phasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT / Resolution: 2.03→62.41 Å / SU ML: 0.2227 / Cross valid method: FREE R-VALUE / σ(F): 1.34 / Phase error: 23.6753
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
RfactorNum. reflection% reflection
Rfree0.2204 2005 6.32 %
Rwork0.1928 29730 -
obs0.1946 31735 97.04 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.1 Å / Solvent model: FLAT BULK SOLVENT MODEL
Displacement parametersBiso mean: 46.63 Å2
Refinement stepCycle: LAST / Resolution: 2.03→62.41 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms3281 0 2 185 3468
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.00163366
X-RAY DIFFRACTIONf_angle_d0.4854581
X-RAY DIFFRACTIONf_chiral_restr0.042513
X-RAY DIFFRACTIONf_plane_restr0.0044586
X-RAY DIFFRACTIONf_dihedral_angle_d11.01791197
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
2.03-2.080.3707880.28361322X-RAY DIFFRACTION60.93
2.08-2.140.27341440.24692077X-RAY DIFFRACTION97.71
2.14-2.20.29431430.22152132X-RAY DIFFRACTION99.65
2.2-2.270.24831470.22242186X-RAY DIFFRACTION100
2.27-2.350.25161480.21962146X-RAY DIFFRACTION100
2.35-2.450.26721450.23832179X-RAY DIFFRACTION100
2.45-2.560.27221440.23562138X-RAY DIFFRACTION100
2.56-2.690.28331460.22032171X-RAY DIFFRACTION100
2.69-2.860.25341460.21712188X-RAY DIFFRACTION100
2.86-3.080.26261420.21692180X-RAY DIFFRACTION99.96
3.08-3.390.24021490.20692190X-RAY DIFFRACTION100
3.39-3.880.18891540.18432225X-RAY DIFFRACTION100
3.88-4.890.15681530.14682226X-RAY DIFFRACTION100
4.89-62.410.20921560.17622370X-RAY DIFFRACTION99.88
Refinement TLS params.

Method: refined / Refine-ID: X-RAY DIFFRACTION

IDL11 (°2)L12 (°2)L13 (°2)L22 (°2)L23 (°2)L33 (°2)S11 (Å °)S12 (Å °)S13 (Å °)S21 (Å °)S22 (Å °)S23 (Å °)S31 (Å °)S32 (Å °)S33 (Å °)T11 (Å2)T12 (Å2)T13 (Å2)T22 (Å2)T23 (Å2)T33 (Å2)Origin x (Å)Origin y (Å)Origin z (Å)
12.870890227810.290825489966-0.7856698462654.693060054250.8486095175392.664358776260.119115501947-0.206558441108-0.0325500166118-0.00686556231012-0.06676568880990.02868505091850.1252577789090.264145903919-0.05135024642530.3196450868760.01774672167720.04896805048920.324587569521-0.00347886820480.25905061846632.26709075932.56365765775.80577563511
23.504135268452.92941973474-0.7497238512884.10049473318-1.251223412624.804045752690.200265555966-0.2725810302870.664638747461-0.01445511340240.170597787853-0.148871937393-0.225954655116-0.0424013301605-0.5036239729520.229006150937-0.0328232633029-0.0231180571320.270728018975-0.01445790928110.34315582303420.793598893630.359039172926.0082893403
32.659936700060.0868035395204-1.608469379891.847341738260.5481291261363.925132292060.00646795172862-0.0916762990169-0.00108849343793-0.02064610541050.0282875920017-0.209653635113-0.06326334224440.1210225739-0.1005391680250.260669126557-0.0163564611201-0.003413991278980.275022816781-0.0542234571850.33802546556621.578723341424.887566496629.2472203443
44.49716636762-0.76568962705-1.319030406813.862356827260.2480407743853.06162176757-0.006215095010210.147488443686-0.294537519491-0.122913628613-0.1159047784240.4604969146310.351534518974-0.4006621849780.08952897020830.42203979383-0.110255883334-0.01049081419530.384099197119-0.07147612930440.3919983643859.82535906765-1.664333680157.81498408976
51.57591471662-0.164736841882-0.06334203656353.109490093151.890342232741.92323711190.005658405342960.03789235494670.0886517023185-0.218050311147-0.1172629895170.164211633133-0.00299932958562-0.06365609893740.09190102420320.2958840818540.0164401456206-0.03243023267740.2978895580070.03837583797310.23764740119513.311427556117.833110105414.3903830584
63.222653104261.27071511811-0.1287852297236.169086937520.1333924884832.756905541780.04404981237660.1107778740070.0791753953849-0.351409332218-0.186807159781-0.100084891695-0.0537313434148-0.1726707435330.1219797916250.2452319046480.01726475404860.02629634127680.367537245752-0.04518654553990.2918861075149.4874592684530.229385086618.5177092458
77.482370840726.37679894310.4596689745748.99252229055-0.306782849042.220681065880.460977968965-0.3847152488630.8836908251910.116729193174-0.6842387950720.226015685579-0.0980555004127-0.05027937007020.2613702065430.3118030691830.04305491606730.008302830504480.39943676932-0.1153770637130.5904315778424.4568270871639.59978884821.5034292582
Refinement TLS group

Refine-ID: X-RAY DIFFRACTION

IDRefine TLS-IDSelection detailsAuth asym-IDLabel asym-IDAuth seq-IDLabel seq-ID
11chain 'H' and (resid 1 through 119 )HA1 - 1191 - 119
22chain 'H' and (resid 120 through 153 )HA120 - 153120 - 151
33chain 'H' and (resid 154 through 222 )HA154 - 222152 - 220
44chain 'L' and (resid 1 through 90)LB1 - 901 - 90
55chain 'L' and (resid 91 through 128 )LB91 - 12891 - 128
66chain 'L' and (resid 129 through 187 )LB129 - 187129 - 187
77chain 'L' and (resid 188 through 211 )LB188 - 211188 - 211

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