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- PDB-9iaz: CryoEM structure of the Themis:Grb2 complex with bound ProMacrobo... -

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Basic information

Entry
Database: PDB / ID: 9iaz
TitleCryoEM structure of the Themis:Grb2 complex with bound ProMacrobody 256, local refinement
Components
  • Growth factor receptor-bound protein 2
  • ProMacrobody 256
  • Protein THEMIS
KeywordsSIGNALING PROTEIN / adapter protein / CABIT / TCR / T cell development
Function / homology
Function and homology information


positive T cell selection / negative T cell selection / guanyl-nucleotide exchange factor adaptor activity / Grb2-EGFR complex / branching involved in labyrinthine layer morphogenesis / STAT5 Activation / Co-inhibition by BTLA / neurotrophin TRKA receptor binding / COP9 signalosome / Activated NTRK2 signals through PI3K ...positive T cell selection / negative T cell selection / guanyl-nucleotide exchange factor adaptor activity / Grb2-EGFR complex / branching involved in labyrinthine layer morphogenesis / STAT5 Activation / Co-inhibition by BTLA / neurotrophin TRKA receptor binding / COP9 signalosome / Activated NTRK2 signals through PI3K / MET receptor recycling / transmembrane receptor protein tyrosine kinase adaptor activity / Interleukin-15 signaling / negative regulation of natural killer cell mediated cytotoxicity / Signaling by cytosolic FGFR1 fusion mutants / MET activates PTPN11 / MET activates RAP1 and RAC1 / vesicle membrane / CD28 dependent Vav1 pathway / Signaling by LTK / Signal regulatory protein family interactions / MET activates PI3K/AKT signaling / Regulation of KIT signaling / epidermal growth factor receptor binding / natural killer cell mediated cytotoxicity / STAT5 activation downstream of FLT3 ITD mutants / PI-3K cascade:FGFR3 / PI-3K cascade:FGFR2 / PI-3K cascade:FGFR4 / PI-3K cascade:FGFR1 / GRB2:SOS provides linkage to MAPK signaling for Integrins / endodermal cell differentiation / positive regulation of actin filament polymerization / RHOU GTPase cycle / RET signaling / regulation of MAPK cascade / Interleukin-3, Interleukin-5 and GM-CSF signaling / negative regulation of epidermal growth factor receptor signaling pathway / insulin receptor substrate binding / PI3K events in ERBB2 signaling / fibroblast growth factor receptor signaling pathway / PI3K Cascade / Role of LAT2/NTAL/LAB on calcium mobilization / Interleukin receptor SHC signaling / SOS-mediated signalling / Activated NTRK3 signals through RAS / carbohydrate transmembrane transporter activity / Signal attenuation / Activated NTRK2 signals through RAS / maltose binding / GAB1 signalosome / SHC1 events in ERBB4 signaling / RHO GTPases Activate WASPs and WAVEs / maltose transport / maltodextrin transmembrane transport / Signalling to RAS / Schwann cell development / SHC-related events triggered by IGF1R / Activated NTRK2 signals through FRS2 and FRS3 / positive regulation of Rac protein signal transduction / SHC-mediated cascade:FGFR3 / MET activates RAS signaling / ephrin receptor binding / SHC-mediated cascade:FGFR2 / SHC-mediated cascade:FGFR4 / Signaling by PDGFRA transmembrane, juxtamembrane and kinase domain mutants / Signaling by PDGFRA extracellular domain mutants / Erythropoietin activates RAS / SHC-mediated cascade:FGFR1 / Signaling by FGFR4 in disease / Signaling by CSF3 (G-CSF) / ATP-binding cassette (ABC) transporter complex, substrate-binding subunit-containing / FRS-mediated FGFR3 signaling / Signaling by FLT3 ITD and TKD mutants / FRS-mediated FGFR2 signaling / FRS-mediated FGFR4 signaling / phosphotyrosine residue binding / signal transduction in response to DNA damage / FRS-mediated FGFR1 signaling / Signaling by FGFR3 in disease / Tie2 Signaling / myelination / Signaling by FGFR2 in disease / Signaling by FLT3 fusion proteins / GRB2 events in EGFR signaling / SHC1 events in EGFR signaling / FLT3 Signaling / Signaling by FGFR1 in disease / FCERI mediated Ca+2 mobilization / EGFR Transactivation by Gastrin / NCAM signaling for neurite out-growth / insulin-like growth factor receptor signaling pathway / Downstream signal transduction / GRB2 events in ERBB2 signaling / Insulin receptor signalling cascade / SHC1 events in ERBB2 signaling / Antigen activates B Cell Receptor (BCR) leading to generation of second messengers / Constitutive Signaling by Overexpressed ERBB2 / Signaling by phosphorylated juxtamembrane, extracellular and kinase domain KIT mutants / T cell activation
Similarity search - Function
Protein THEMIS / CABIT domain / Cell-cycle sustaining, positive selection, / GRB2, N-terminal SH3 domain / GRB2, C-terminal SH3 domain / Grb2-like / Maltose/Cyclodextrin ABC transporter, substrate-binding protein / Solute-binding family 1, conserved site / Bacterial extracellular solute-binding proteins, family 1 signature. / Bacterial extracellular solute-binding protein ...Protein THEMIS / CABIT domain / Cell-cycle sustaining, positive selection, / GRB2, N-terminal SH3 domain / GRB2, C-terminal SH3 domain / Grb2-like / Maltose/Cyclodextrin ABC transporter, substrate-binding protein / Solute-binding family 1, conserved site / Bacterial extracellular solute-binding proteins, family 1 signature. / Bacterial extracellular solute-binding protein / Bacterial extracellular solute-binding protein / SH3 domain / SH2 domain / Src homology 2 (SH2) domain profile. / Src homology 2 domains / SH2 domain / Src homology 3 domains / SH2 domain superfamily / SH3-like domain superfamily / Src homology 3 (SH3) domain profile. / SH3 domain
Similarity search - Domain/homology
Maltose/maltodextrin-binding periplasmic protein / Growth factor receptor-bound protein 2 / Protein THEMIS
Similarity search - Component
Biological speciesHomo sapiens (human)
synthetic construct (others)
Escherichia coli (E. coli)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.2 Å
AuthorsClancy, D.M. / Felix, J. / Bloch, Y. / Savvides, S.N.
Funding support Belgium, 2items
OrganizationGrant numberCountry
Research Foundation - Flanders (FWO)1219923N Belgium
Research Foundation - Flanders (FWO)G049820N Belgium
CitationJournal: Nat Commun / Year: 2026
Title: Structural and mechanistic insights into the constitutive Themis-Grb2 complex in T cell signalling.
Authors: Danielle M Clancy / Alba Sanz-Sanjuan / Elisabeth Gilis / Peter Tougaard / Imke Velghe / Yana Van Droogenbroeck / Jan Felix / Yehudi Bloch / Álvaro Furones Cuadrado / Romain Merceron / ...Authors: Danielle M Clancy / Alba Sanz-Sanjuan / Elisabeth Gilis / Peter Tougaard / Imke Velghe / Yana Van Droogenbroeck / Jan Felix / Yehudi Bloch / Álvaro Furones Cuadrado / Romain Merceron / Stephan Schenck / Peter Vandenabeele / Janine D Brunner / Tom Taghon / Dirk Elewaut / Savvas N Savvides /
Abstract: Thymocyte selection is essential for establishing the T cell repertoire, maintaining self-tolerance and preventing autoimmunity. Themis, the archetypal member of a metazoan protein family defined by ...Thymocyte selection is essential for establishing the T cell repertoire, maintaining self-tolerance and preventing autoimmunity. Themis, the archetypal member of a metazoan protein family defined by CABIT domains, centrally regulates this process by integrating T cell receptor (TCR) signalling. Themis has been proposed to constitutively partner with the multifunctional adaptor Grb2, yet the structural and mechanistic basis of this assembly has remained enigmatic. Here, we use Cryo-EM to reveal how the tandem CABIT domains and proline-rich sequence of Themis cooperatively engulf the C-terminal SH3 domain of Grb2, while the unbound domains of Grb2 remain poised to recruit additional binding partners. Furthermore, we uncover inherent interdomain flexibility in unbound Themis that resolves upon Grb2 binding. Structure-guided mutations abrogate the Themis-Grb2 interaction and fail to regulate the tyrosine phosphatase SHP-1 after TCR stimulation, recapitulating the phenotype of Themis-deficient cells. Our findings define the Themis-Grb2 complex as a dynamic structural hub in T cell signalling.
History
DepositionFeb 11, 2025Deposition site: PDBE / Processing site: PDBE
Revision 1.0Apr 29, 2026Provider: repository / Type: Initial release
Revision 1.0Apr 29, 2026Data content type: EM metadata / Data content type: EM metadata / Provider: repository / Type: Initial release
Revision 1.0Apr 29, 2026Data content type: Additional map / Part number: 1 / Data content type: Additional map / Provider: repository / Type: Initial release
Revision 1.0Apr 29, 2026Data content type: FSC / Data content type: FSC / Provider: repository / Type: Initial release
Revision 1.0Apr 29, 2026Data content type: Half map / Part number: 1 / Data content type: Half map / Provider: repository / Type: Initial release
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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Growth factor receptor-bound protein 2
B: Protein THEMIS
C: ProMacrobody 256


Theoretical massNumber of molelcules
Total (without water)152,2653
Polymers152,2653
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

#1: Protein Growth factor receptor-bound protein 2 / Adapter protein GRB2 / Protein Ash / SH2/SH3 adapter GRB2


Mass: 26198.406 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Details: Human Growth factor-receptor bound protein 2 (Grb2) residues 1-217 with C-terminal His tag.
Source: (gene. exp.) Homo sapiens (human) / Gene: GRB2, ASH / Production host: Homo sapiens (human) / References: UniProt: P62993
#2: Protein Protein THEMIS / Thymocyte-expressed molecule involved in selection


Mass: 73550.250 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Details: Human Themis residues 1-563 / Source: (gene. exp.) Homo sapiens (human) / Gene: THEMIS, C6orf190, C6orf207 / Production host: Homo sapiens (human) / References: UniProt: Q8N1K5
#3: Antibody ProMacrobody 256 / MMBP / Maltodextrin-binding protein / Maltose-binding protein / MBP


Mass: 52516.008 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Details: ProMacrobody 256 (Themis-specific Nanobody 256 fused via rigid proline linker to MBP),ProMacrobody 256 (Themis-specific Nanobody 256 fused via rigid proline linker to MBP)
Source: (gene. exp.) synthetic construct (others), (gene. exp.) Escherichia coli (E. coli)
Gene: malE, Z5632, ECs5017 / Production host: Escherichia coli (E. coli) / References: UniProt: P0AEY0
Has protein modificationY

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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: Themis-Grb2 complex bound to ProMacrobody 256 / Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Molecular weightValue: 0.142 MDa / Experimental value: YES
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Homo sapiens (human)
Buffer solutionpH: 8
Details: 25 mM Tris pH8, 100 mM NaCl, 2 mM DTT with added 8 mM CHAPSO for cryogrid sample preparation
Buffer component
IDConc.NameFormulaBuffer-ID
1100 mMSodium chlorideNaCl1
225 mMTris(hydroxymethyl)aminomethaneTris1
32 mMDithiothreitolDTT1
48 mMCHAPSOCHAPSO1
SpecimenConc.: 2.6 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R2/1
VitrificationInstrument: LEICA EM GP / Cryogen name: ETHANE / Humidity: 95 % / Details: Leica EM GP2, 5 s blotting time

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Electron microscopy imaging

MicroscopyModel: JEOL CRYO ARM 300
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 60000 X / Nominal defocus max: 1900 nm / Nominal defocus min: 1400 nm
Specimen holderCryogen: NITROGEN
Image recordingAverage exposure time: 3.37 sec. / Electron dose: 61.8 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) / Num. of real images: 27698

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Processing

EM software
IDNameVersionCategory
1cryoSPARCv4.5.1particle selection
2crYOLOparticle selection
3SerialEMimage acquisition
5cryoSPARCv4.5.1CTF correction
8UCSF ChimeraX1.8model fitting
9NAMDmodel fitting
11PHENIX1.21.1_5286:model refinement
12cryoSPARCv4.5.1initial Euler assignment
13cryoSPARCv4.5.1final Euler assignment
14cryoSPARCv4.5.1classification
15cryoSPARC3D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3.2 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 601216 / Details: Local refinement map / Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT
Atomic model building

3D fitting-ID: 1

IDPDB-IDDetails (eV)Source nameTypeAccession codeInitial refinement model-ID
1AlphaFold2 multimer predicted model for Themis-Grb2 complexAlphaFoldin silico model
2ESMFold predicted model for Nb256Otherin silico model
37OMT

7omt

Maltose-bound MBP used to model ProMbPDBexperimental model7OMT3
RefinementHighest resolution: 3.2 Å
Stereochemistry target values: REAL-SPACE (WEIGHTED MAP SUM AT ATOM CENTERS)
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0025790
ELECTRON MICROSCOPYf_angle_d0.4637845
ELECTRON MICROSCOPYf_dihedral_angle_d3.861760
ELECTRON MICROSCOPYf_chiral_restr0.041865
ELECTRON MICROSCOPYf_plane_restr0.0031013

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