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- PDB-8zo4: Crystal structure of Tfh TCR ectodomain thermostable mutant -

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Basic information

Entry
Database: PDB / ID: 8zo4
TitleCrystal structure of Tfh TCR ectodomain thermostable mutant
Components
  • TCR stable mutant Alpha chain
  • TCR stable mutant Beta chian
KeywordsIMMUNE SYSTEM / T cell receptor / ecotodomain / thermostable mutant
Biological speciesHomo sapiens (human)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 2.6 Å
AuthorsMori, S. / Nagae, M. / Yamasaki, S.
Funding support Japan, 1items
OrganizationGrant numberCountry
Japan Society for the Promotion of Science (JSPS) Japan
CitationJournal: J Clin Invest / Year: 2024
Title: A conserved human CD4+ T cell subset recognizing the mycobacterial adjuvant trehalose monomycolate.
Authors: Yuki Sakai / Minori Asa / Mika Hirose / Wakana Kusuhara / Nagatoshi Fujiwara / Hiroto Tamashima / Takahiro Ikazaki / Shiori Oka / Kota Kuraba / Kentaro Tanaka / Takashi Yoshiyama / Masamichi ...Authors: Yuki Sakai / Minori Asa / Mika Hirose / Wakana Kusuhara / Nagatoshi Fujiwara / Hiroto Tamashima / Takahiro Ikazaki / Shiori Oka / Kota Kuraba / Kentaro Tanaka / Takashi Yoshiyama / Masamichi Nagae / Yoshihiko Hoshino / Daisuke Motooka / Ildiko Van Rhijn / Xiuyuan Lu / Eri Ishikawa / D Branch Moody / Takayuki Kato / Shinsuke Inuki / Go Hirai / Sho Yamasaki /
Abstract: Mycobacterium tuberculosis causes human tuberculosis (TB). As mycobacteria are protected by a thick lipid cell wall, humans have developed immune responses against diverse mycobacterial lipids. Most ...Mycobacterium tuberculosis causes human tuberculosis (TB). As mycobacteria are protected by a thick lipid cell wall, humans have developed immune responses against diverse mycobacterial lipids. Most of these immunostimulatory lipids are known as adjuvants acting through innate immune receptors, such as C-type lectin receptors. Although a few mycobacterial lipid antigens activate unconventional T cells, the antigenicity of most adjuvantic lipids is unknown. Here, we identified that trehalose monomycolate (TMM), an abundant mycobacterial adjuvant, activated human T cells bearing a unique αβ T cell receptor (αβTCR). This recognition was restricted by CD1b, a monomorphic antigen-presenting molecule conserved in primates but not mice. Single-cell TCR-RNA-Seq using newly established CD1b-TMM tetramers revealed that TMM-specific T cells were present as CD4+ effector memory T cells in the periphery of uninfected donors but expressed IFN-γ, TNF, and anti-mycobacterial effectors upon TMM stimulation. TMM-specific T cells were detected in cord blood and PBMCs of donors without bacillus Calmette-Guérin vaccination but were expanded in patients with active TB. A cryo-electron microscopy study of CD1b-TMM-TCR complexes revealed unique antigen recognition by conserved features of TCRs, positively charged CDR3α, and long CDR3β regions. These results indicate that humans have a commonly shared and preformed CD4+ T cell subset recognizing a typical mycobacterial adjuvant as an antigen. Furthermore, the dual role of TMM justifies reconsideration of the mechanism of action of adjuvants.
History
DepositionMay 28, 2024Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Jan 1, 2025Provider: repository / Type: Initial release
Revision 1.1Mar 26, 2025Group: Database references / Category: citation / Item: _citation.journal_volume

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: TCR stable mutant Alpha chain
B: TCR stable mutant Beta chian
C: TCR stable mutant Alpha chain
D: TCR stable mutant Beta chian
hetero molecules


Theoretical massNumber of molelcules
Total (without water)111,54310
Polymers110,8424
Non-polymers7026
Water93752
1
A: TCR stable mutant Alpha chain
B: TCR stable mutant Beta chian
hetero molecules


Theoretical massNumber of molelcules
Total (without water)56,1228
Polymers55,4212
Non-polymers7026
Water362
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area5150 Å2
ΔGint-73 kcal/mol
Surface area20480 Å2
MethodPISA
2
C: TCR stable mutant Alpha chain
D: TCR stable mutant Beta chian


Theoretical massNumber of molelcules
Total (without water)55,4212
Polymers55,4212
Non-polymers00
Water362
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area4540 Å2
ΔGint-35 kcal/mol
Surface area20610 Å2
MethodPISA
Unit cell
Length a, b, c (Å)89.156, 89.156, 208.683
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: Protein TCR stable mutant Alpha chain


Mass: 28089.523 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Production host: Homo sapiens (human)
#2: Protein TCR stable mutant Beta chian


Mass: 27331.410 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Production host: Homo sapiens (human)
#3: Sugar ChemComp-NAG / 2-acetamido-2-deoxy-beta-D-glucopyranose / N-acetyl-beta-D-glucosamine / 2-acetamido-2-deoxy-beta-D-glucose / 2-acetamido-2-deoxy-D-glucose / 2-acetamido-2-deoxy-glucose / N-ACETYL-D-GLUCOSAMINE


Type: D-saccharide, beta linking / Mass: 221.208 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C8H15NO6
IdentifierTypeProgram
DGlcpNAcbCONDENSED IUPAC CARBOHYDRATE SYMBOLGMML 1.0
N-acetyl-b-D-glucopyranosamineCOMMON NAMEGMML 1.0
b-D-GlcpNAcIUPAC CARBOHYDRATE SYMBOLPDB-CARE 1.0
GlcNAcSNFG CARBOHYDRATE SYMBOLGMML 1.0
#4: Chemical
ChemComp-SO4 / SULFATE ION


Mass: 96.063 Da / Num. of mol.: 5 / Source method: obtained synthetically / Formula: SO4
#5: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 52 / 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: 2.28 Å3/Da / Density % sol: 46.02 %
Crystal growTemperature: 293 K / Method: vapor diffusion, sitting drop / pH: 5.5
Details: 0.1M Bis-tris (pH 5.5), 0.2M Potassium sulfate, 25% PEG 3350

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Data collection

DiffractionMean temperature: 95 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: Photon Factory / Beamline: BL-1A / Wavelength: 1.052 Å
DetectorType: DECTRIS EIGER R 4M / Detector: PIXEL / Date: May 15, 2022
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 1.052 Å / Relative weight: 1
ReflectionResolution: 2.6→44.58 Å / Num. obs: 303548 / % possible obs: 99.94 % / Redundancy: 10 % / Biso Wilson estimate: 56.11 Å2 / CC1/2: 0.998 / Rmerge(I) obs: 0.108 / Rpim(I) all: 0.035 / Net I/σ(I): 17.95
Reflection shellResolution: 2.6→2.693 Å / Redundancy: 10 % / Rmerge(I) obs: 1.054 / Num. unique obs: 30201 / CC1/2: 0.771 / Rpim(I) all: 0.3489 / % possible all: 99.97

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Processing

Software
NameVersionClassification
REFMAC1.20.1_4487refinement
XDSdata reduction
Aimlessdata scaling
MOLREPphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT / Resolution: 2.6→44.58 Å / SU ML: 0.3236 / Cross valid method: FREE R-VALUE / σ(F): 1.36 / Phase error: 29.9781
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
RfactorNum. reflection% reflection
Rfree0.2756 1448 4.77 %
Rwork0.226 28936 -
obs0.2285 30384 99.97 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.1 Å / Solvent model: FLAT BULK SOLVENT MODEL
Displacement parametersBiso mean: 58.76 Å2
Refinement stepCycle: LAST / Resolution: 2.6→44.58 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms6840 0 39 52 6931
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.00287038
X-RAY DIFFRACTIONf_angle_d0.62099555
X-RAY DIFFRACTIONf_chiral_restr0.04531023
X-RAY DIFFRACTIONf_plane_restr0.00541259
X-RAY DIFFRACTIONf_dihedral_angle_d5.19943
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
2.6-2.690.38741390.31692870X-RAY DIFFRACTION99.97
2.69-2.80.38141240.29462860X-RAY DIFFRACTION100
2.8-2.930.3831350.32742837X-RAY DIFFRACTION100
2.93-3.080.33261680.28162848X-RAY DIFFRACTION100
3.08-3.280.32281410.26592853X-RAY DIFFRACTION100
3.28-3.530.30351310.24932873X-RAY DIFFRACTION100
3.53-3.880.27211590.22292885X-RAY DIFFRACTION100
3.88-4.440.2521160.17732935X-RAY DIFFRACTION100
4.45-5.60.2321760.17992903X-RAY DIFFRACTION100
5.6-44.580.2371590.21883072X-RAY DIFFRACTION99.75

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