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- EMDB-52603: CryoEM structure of the Themis:Grb2 complex with bound ProMacrobo... -

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Entry
Database: EMDB / ID: EMD-52603
TitleCryoEM structure of the Themis:Grb2 complex with bound ProMacrobody 256
Map dataSharpened map of the human Themis(1-563):Grb2:ProMb complex, used for model refinement in Phenix.
Sample
  • Complex: Themis-Grb2 complex bound to ProMacrobody 256
    • Protein or peptide: Growth factor receptor-bound protein 2
    • Protein or peptide: Protein THEMIS
    • Protein or peptide: ProMacrobody 256
Keywordsadapter protein / CABIT / TCR / T cell development / SIGNALING PROTEIN
Function / homology
Function and homology information


positive T cell selection / negative T cell selection / guanyl-nucleotide exchange factor adaptor activity / Grb2-EGFR complex / STAT5 Activation / Co-inhibition by BTLA / neurotrophin TRKA receptor binding / COP9 signalosome / Activated NTRK2 signals through PI3K / MET receptor recycling ...positive T cell selection / negative T cell selection / guanyl-nucleotide exchange factor adaptor activity / Grb2-EGFR complex / 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 / natural killer cell mediated cytotoxicity / Regulation of KIT signaling / epidermal growth factor receptor binding / PI-3K cascade:FGFR3 / STAT5 activation downstream of FLT3 ITD mutants / PI-3K cascade:FGFR2 / PI-3K cascade:FGFR4 / PI-3K cascade:FGFR1 / GRB2:SOS provides linkage to MAPK signaling for Integrins / RHOU GTPase cycle / RET signaling / Interleukin-3, Interleukin-5 and GM-CSF signaling / PI3K events in ERBB2 signaling / regulation of MAPK cascade / insulin receptor substrate binding / PI3K Cascade / Role of LAT2/NTAL/LAB on calcium mobilization / Interleukin receptor SHC signaling / Signal attenuation / SOS-mediated signalling / Activated NTRK3 signals through RAS / carbohydrate transmembrane transporter activity / Activated NTRK2 signals through RAS / GAB1 signalosome / RHO GTPases Activate WASPs and WAVEs / maltose binding / SHC1 events in ERBB4 signaling / maltose transport / Schwann cell development / maltodextrin transmembrane transport / Signalling to RAS / SHC-related events triggered by IGF1R / Activated NTRK2 signals through FRS2 and FRS3 / positive regulation of Rac protein signal transduction / ephrin receptor binding / SHC-mediated cascade:FGFR3 / ATP-binding cassette (ABC) transporter complex, substrate-binding subunit-containing / MET activates RAS signaling / 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 / myelination / Signaling by FGFR4 in disease / Signaling by CSF3 (G-CSF) / FRS-mediated FGFR3 signaling / signal transduction in response to DNA damage / Signaling by FLT3 ITD and TKD mutants / phosphotyrosine residue binding / FRS-mediated FGFR2 signaling / FRS-mediated FGFR4 signaling / Signaling by FGFR3 in disease / FRS-mediated FGFR1 signaling / Tie2 Signaling / insulin-like growth factor receptor signaling pathway / 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 / EGFR Transactivation by Gastrin / FCERI mediated Ca+2 mobilization / NCAM signaling for neurite out-growth / Downstream signal transduction / GRB2 events in ERBB2 signaling / Insulin receptor signalling cascade / SHC1 events in ERBB2 signaling / Constitutive Signaling by Overexpressed ERBB2 / Antigen activates B Cell Receptor (BCR) leading to generation of second messengers / Signaling by phosphorylated juxtamembrane, extracellular and kinase domain KIT mutants / B cell receptor signaling pathway / cellular response to ionizing radiation / InlB-mediated entry of Listeria monocytogenes into host cell / T cell activation / FCGR3A-mediated phagocytosis / FCERI mediated MAPK 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) / Escherichia coli (E. coli)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.3 Å
AuthorsClancy DM / Felix J / Bloch Y / Savvides SN
Funding support Belgium, 2 items
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
DepositionJan 23, 2025-
Header (metadata) releaseApr 29, 2026-
Map releaseApr 29, 2026-
UpdateJul 29, 2026-
Current statusJul 29, 2026Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_52603.png

Downloads & links

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Map

FileDownload / File: emd_52603.map.gz / Format: CCP4 / Size: 274.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationSharpened map of the human Themis(1-563):Grb2:ProMb complex, used for model refinement in Phenix.
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.76 Å/pix.
x 416 pix.
= 314.08 Å		0.76 Å/pix.
x 416 pix.
= 314.08 Å		0.76 Å/pix.
x 416 pix.
= 314.08 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.755 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.12
Minimum - Maximum-1.2323908 - 1.8572114
Average (Standard dev.)0.00031070635 (±0.018471986)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions416416416
Spacing416416416
CellA=B=C: 314.08 Å
α=β=γ: 90.0 °

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Supplemental data

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Additional map: 3D EM map of the human Themis(1-563):Grb2:ProMb256 sharpened...

Fileemd_52603_additional_1.map
Annotation3D EM map of the human Themis(1-563):Grb2:ProMb256 sharpened using DeepEMhancer
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map A

Fileemd_52603_half_map_1.map
AnnotationHalf map_A
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map B

Fileemd_52603_half_map_2.map
AnnotationHalf map_B
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Themis-Grb2 complex bound to ProMacrobody 256

EntireName: Themis-Grb2 complex bound to ProMacrobody 256
Components
  • Complex: Themis-Grb2 complex bound to ProMacrobody 256
    • Protein or peptide: Growth factor receptor-bound protein 2
    • Protein or peptide: Protein THEMIS
    • Protein or peptide: ProMacrobody 256

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Supramolecule #1: Themis-Grb2 complex bound to ProMacrobody 256

SupramoleculeName: Themis-Grb2 complex bound to ProMacrobody 256 / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 142 KDa

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Macromolecule #1: Growth factor receptor-bound protein 2

MacromoleculeName: Growth factor receptor-bound protein 2 / type: protein_or_peptide / ID: 1
Details: Human Growth factor-receptor bound protein 2 (Grb2) residues 1-217 with C-terminal His tag.
Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 26.198406 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: MEAIAKYDFK ATADDELSFK RGDILKVLNE ECDQNWYKAE LNGKDGFIPK NYIEMKPHPW FFGKIPRAKA EEMLSKQRHD GAFLIRESE SAPGDFSLSV KFGNDVQHFK VLRDGAGKYF LWVVKFNSLN ELVDYHRSTS VSRNQQIFLR DIEQVPQQPT Y VQALFDFD ...String:
MEAIAKYDFK ATADDELSFK RGDILKVLNE ECDQNWYKAE LNGKDGFIPK NYIEMKPHPW FFGKIPRAKA EEMLSKQRHD GAFLIRESE SAPGDFSLSV KFGNDVQHFK VLRDGAGKYF LWVVKFNSLN ELVDYHRSTS VSRNQQIFLR DIEQVPQQPT Y VQALFDFD PQEDGELGFR RGDFIHVMDN SDPNWWKGAC HGQTGMFPRN YVTPVNRNVK HHHHHH

UniProtKB: Growth factor receptor-bound protein 2

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Macromolecule #2: Protein THEMIS

MacromoleculeName: Protein THEMIS / type: protein_or_peptide / ID: 2 / Details: Human Themis residues 1-563 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 64.72327 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: MALSLEEFVH SLDLRTLPRV LEIQAGIYLE GSIYEMFGNE CCFSTGEVIK ITGLKVKKII AEICEQIEGC ESLQPFELPM NFPGLFKIV ADKTPYLTME EITRTIHIGP SRLGHPCFYH QKDIKLENLI IKQGEQIMLN SVEEIDGEIM VSCAVARNHQ T HSFNLPLS ...String:
MALSLEEFVH SLDLRTLPRV LEIQAGIYLE GSIYEMFGNE CCFSTGEVIK ITGLKVKKII AEICEQIEGC ESLQPFELPM NFPGLFKIV ADKTPYLTME EITRTIHIGP SRLGHPCFYH QKDIKLENLI IKQGEQIMLN SVEEIDGEIM VSCAVARNHQ T HSFNLPLS QEGEFYECED ERIYTLKEIV EWKIPKNRTR TVNLTDFSNK WDSTNPFPKD FYGTLILKPV YEIQGVMKFR KD IIRILPS LDVEVKDITD SYDANWFLQL LSTEDLFEMT SKEFPIVTEV IEAPEGNHLP QSILQPGKTI VIHKKYQASR ILA SEIRSN FPKRHFLIPT SYKGKFKRRP REFPTAYDLE IAKSEKEPLH VVATKAFHSP HDKLSSVSVG DQFLVHQSET TEVL CEGIK KVVNVLACEK ILKKSYEAAL LPLYMEGGFV EVIHDKKQYP ISELCKQFRL PFNVKVSVRD LSIEEDVLAA TPGLQ LEED ITDSYLLISD FANPTECWEI PVGRLNMTVQ LVSNFSRDAE PFLVRTLVEE ITEEQYYMMR RYESSASHPP PRPPKH PSV

UniProtKB: Protein THEMIS

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Macromolecule #3: ProMacrobody 256

MacromoleculeName: ProMacrobody 256 / type: protein_or_peptide / ID: 3
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)
Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Escherichia coli (E. coli)
Molecular weightTheoretical: 52.516008 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: QVQLQESGGG LVQAGGSLRL SCAASGRTFS TYAMGWFRQA PGKEREFVAA ILWSDGSTYY ADSVKGRFTI SRDNAKNTVY LQMNSLKPE DTAVYYCAAD YTGVRYDYWG QGTQVTVPPL VIWINGDKGY NGLAEVGKKF EKDTGIKVTV EHPDKLEEKF P QVAATGDG ...String:
QVQLQESGGG LVQAGGSLRL SCAASGRTFS TYAMGWFRQA PGKEREFVAA ILWSDGSTYY ADSVKGRFTI SRDNAKNTVY LQMNSLKPE DTAVYYCAAD YTGVRYDYWG QGTQVTVPPL VIWINGDKGY NGLAEVGKKF EKDTGIKVTV EHPDKLEEKF P QVAATGDG PDIIFWAHDR FGGYAQSGLL AEITPDKAFQ DKLYPFTWDA VRYNGKLIAY PIAVEALSLI YNKDLLPNPP KT WEEIPAL DKELKAKGKS ALMFNLQEPY FTWPLIAADG GYAFKYENGK YDIKDVGVDN AGAKAGLTFL VDLIKNKHMN ADT DYSIAE AAFNKGETAM TINGPWAWSN IDTSKVNYGV TVLPTFKGQP SKPFVGVLSA GINAASPNKE LAKEFLENYL LTDE GLEAV NKDKPLGAVA LKSYEEELAK DPRIAATMEN AQKGEIMPNI PQMSAFWYAV RTAVINAASG RQTVDEALKD AQT

UniProtKB: Maltose/maltodextrin-binding periplasmic protein

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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

Concentration2.6 mg/mL
BufferpH: 8
Component:
ConcentrationFormulaName
100.0 mMNaClSodium chloride
25.0 mMTrisTris(hydroxymethyl)aminomethane
2.0 mMDTTDithiothreitol
8.0 mMCHAPSOCHAPSO

Details: 25 mM Tris pH8, 100 mM NaCl, 2 mM DTT with added 8 mM CHAPSO for cryogrid sample preparation
GridModel: Quantifoil R2/1 / Material: COPPER / Mesh: 300 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE
VitrificationCryogen name: ETHANE / Chamber humidity: 95 % / Instrument: LEICA EM GP / Details: Leica EM GP2, 5 s blotting time.

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Electron microscopy

MicroscopeJEOL CRYO ARM 300
SoftwareName: SerialEM
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Number real images: 27698 / 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: 1.9000000000000001 µm / Nominal defocus min: 1.4000000000000001 µm / Nominal magnification: 60000
Sample stageCooling holder cryogen: NITROGEN

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Image processing

CTF correctionSoftware - Name: cryoSPARC (ver. v4.5.1) / Type: PHASE FLIPPING AND AMPLITUDE CORRECTION
Startup modelType of model: NONE
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 3.3 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC / Number images used: 601216
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. v4.5.1)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. v4.5.1)
Final 3D classificationNumber classes: 10 / Software - Name: cryoSPARC (ver. v4.5.1)
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial model
ChainDetailsPDB ID
source_name: AlphaFold, initial_model_type: in silico modelAlphaFold2 multimer predicted model for Themis-Grb2 complex
source_name: Other, initial_model_type: in silico modelESMFold predicted model for Nb256
source_name: PDB, initial_model_type: experimental modelMaltose-bound MBP used to model ProMb

7omt

SoftwareName: NAMD
RefinementProtocol: FLEXIBLE FIT
Output model

PDB-9i3p:
CryoEM structure of the Themis:Grb2 complex with bound ProMacrobody 256

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