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- PDB-7lz6: The Cryo-EM structure of a complex between GAD65 and b96.11 Fab -

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Basic information

Entry
Database: PDB / ID: 7lz6
TitleThe Cryo-EM structure of a complex between GAD65 and b96.11 Fab
Components
  • Glutamate decarboxylase 2
  • b96.11 Fab heavy chain
  • b96.11 Fab light chain
KeywordsIMMUNE SYSTEM / Neurotransmitter biosynthesis / Autoantibody / autoantigen / type 1 diabetes / stiff person syndrome / GABA
Function / homology
Function and homology information


GABA synthesis / gamma-aminobutyrate shunt / MECP2 regulates transcription of genes involved in GABA signaling / glutamate decarboxylase / glutamate decarboxylase activity / gamma-aminobutyric acid biosynthetic process / clathrin-sculpted gamma-aminobutyric acid transport vesicle membrane / GABA synthesis, release, reuptake and degradation / pyridoxal phosphate binding / presynaptic membrane ...GABA synthesis / gamma-aminobutyrate shunt / MECP2 regulates transcription of genes involved in GABA signaling / glutamate decarboxylase / glutamate decarboxylase activity / gamma-aminobutyric acid biosynthetic process / clathrin-sculpted gamma-aminobutyric acid transport vesicle membrane / GABA synthesis, release, reuptake and degradation / pyridoxal phosphate binding / presynaptic membrane / chemical synaptic transmission / Golgi membrane / axon / plasma membrane / cytosol / cytoplasm
Similarity search - Function
Pyridoxal-phosphate binding site / DDC / GAD / HDC / TyrDC pyridoxal-phosphate attachment site. / Pyridoxal phosphate-dependent decarboxylase / Pyridoxal-dependent decarboxylase conserved domain / Pyridoxal phosphate-dependent transferase, major domain / Pyridoxal phosphate-dependent transferase
Similarity search - Domain/homology
Glutamate decarboxylase 2
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 7.3 Å
AuthorsReboul, C.F. / Le, S.N. / Williams, D.E. / Buckle, A.M.
Citation
Journal: Nat Commun / Year: 2025
Title: Structure and dynamics of GAD65 in complex with an autoimmune polyendocrine syndrome type 2-associated autoantibody.
Authors: Susanne H D Ständer / Cyril F Reboul / Sarah N Le / Daniel E Williams / Peter G Chandler / Mauricio G S Costa / David E Hoke / John D T Jimma / James Fodor / Gustavo Fenalti / Stuart I ...Authors: Susanne H D Ständer / Cyril F Reboul / Sarah N Le / Daniel E Williams / Peter G Chandler / Mauricio G S Costa / David E Hoke / John D T Jimma / James Fodor / Gustavo Fenalti / Stuart I Mannering / Benjamin T Porebski / Peter Schofield / Daniel Christ / Malcolm Buckle / Sheena McGowan / Dominika Elmlund / Kasper D Rand / Ashley M Buckle /
Abstract: The enzyme glutamate decarboxylase (GAD) produces the neurotransmitter GABA, using pyridoxal-5'-phosphate (PLP). GAD exists as two isoforms, GAD65 and GAD67. Only GAD65 acts as a major autoantigen, ...The enzyme glutamate decarboxylase (GAD) produces the neurotransmitter GABA, using pyridoxal-5'-phosphate (PLP). GAD exists as two isoforms, GAD65 and GAD67. Only GAD65 acts as a major autoantigen, frequently implicated in type 1 diabetes and other autoimmune diseases. Here we characterize the structure and dynamics of GAD65 and its interaction with the autoimmune polyendocrine syndrome type 2-associated autoantibody b96.11. Using hydrogen-deuterium exchange mass spectrometry (HDX), X-ray crystallography, cryo-electron microscopy, and computational approaches, we examine the conformational dynamics of apo- and holoGAD65 and the GAD65-autoantibody complex. HDX reveals local dynamics accompanying autoinactivation, with the catalytic loop promoting collective motions at the CTD-PLP domain interface. In the GAD65-b96.11 complex, heavy chain CDRs dominate the interaction, with a long CDRH3 bridging the GAD65 dimer via electrostatic interactions with the PEVKEKmotif. This bridging links structural elements controlling GAD65's conformational flexibility to its autoantigenicity. Thus, intrinsic dynamics, rather than sequence differences within epitopes, appear to be responsible for the contrasting autoantigenicities of GAD65 and GAD67. Our findings elucidate the structural and dynamic factors that govern the varying autoantibody reactivities of GAD65 and GAD67, offering a revised rationale for the autoimmune response to GAD65.
#1: Journal: Biorxiv / Year: 2024
Title: Structure and dynamics of the autoantigen GAD65 in complex with the human autoimmune polyendocrine syndrome type 2-associated autoantibody b96.11
Authors: Stander, S.H.D. / Reboul, C.F. / Le, S.N. / Williams, D.E. / Chandler, P.G. / Costa, M.G.S. / Hoke, D.E. / Jimma, J.D.T. / Fodor, J. / Fenalti, G. / Mannering, S.I. / Porebski, B.T. / ...Authors: Stander, S.H.D. / Reboul, C.F. / Le, S.N. / Williams, D.E. / Chandler, P.G. / Costa, M.G.S. / Hoke, D.E. / Jimma, J.D.T. / Fodor, J. / Fenalti, G. / Mannering, S.I. / Porebski, B.T. / Schofield, P. / Christ, D. / Buckle, M. / McGowan, S. / Elmlund, D. / Rand, K.D. / Buckle, A.M.
History
DepositionMar 9, 2021Deposition site: RCSB / Processing site: RCSB
Revision 1.0Oct 30, 2024Provider: repository / Type: Initial release
Revision 1.0Oct 30, 2024Data content type: EM metadata / Data content type: EM metadata / Provider: repository / Type: Initial release
Revision 1.0Oct 30, 2024Data content type: FSC / Data content type: FSC / Provider: repository / Type: Initial release
Revision 1.0Oct 30, 2024Data content type: Image / Data content type: Image / Provider: repository / Type: Initial release
Revision 1.0Oct 30, 2024Data content type: Primary map / Data content type: Primary map / Provider: repository / Type: Initial release
Revision 1.0Oct 30, 2024Data content type: FSC / Data content type: FSC / Provider: repository / Type: Initial release
Revision 1.0Oct 30, 2024Data content type: Image / Data content type: Image / Provider: repository / Type: Initial release
Revision 1.0Oct 30, 2024Data content type: Primary map / Data content type: Primary map / Provider: repository / Type: Initial release
Revision 1.1Nov 13, 2024Group: Data collection / Category: em_admin / Item: _em_admin.last_update
Revision 1.2Jul 16, 2025Group: Data collection / Database references
Category: citation / citation_author ...citation / citation_author / em_admin / pdbx_database_related
Item: _em_admin.last_update
Revision 1.1Jul 16, 2025Data content type: EM metadata / Data content type: EM metadata / EM metadata / Group: Database references / Experimental summary
Data content type: EM metadata / EM metadata ...EM metadata / EM metadata / EM metadata / EM metadata
Category: citation / citation_author ...citation / citation_author / em_admin / pdbx_database_related
Data content type: EM metadata / Item: _em_admin.last_update

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Glutamate decarboxylase 2
B: Glutamate decarboxylase 2
C: b96.11 Fab heavy chain
D: b96.11 Fab light chain
E: b96.11 Fab heavy chain
F: b96.11 Fab light chain


Theoretical massNumber of molelcules
Total (without water)208,3366
Polymers208,3366
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: mass spectrometry, Hydrogen-deuterium exchange mass spectrometry (HDX-MS), surface plasmon resonance, The binding affinity of b96.11 to GAD65 was measured using surface plasmon resonance (BIAcore T-100)
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein Glutamate decarboxylase 2 / 65 kDa glutamic acid decarboxylase / GAD-65 / Glutamate decarboxylase 65 kDa isoform


Mass: 56554.215 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: GAD2, GAD65 / Production host: Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: Q05329, glutamate decarboxylase
#2: Antibody b96.11 Fab heavy chain


Mass: 24553.436 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Plasmid: pcDNA3 / Cell line (production host): HEK293F / Production host: Homo sapiens (human)
#3: Antibody b96.11 Fab light chain


Mass: 23060.504 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Plasmid: pcDNA3 / Cell line (production host): HEK293F / Production host: Homo sapiens (human)
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: GAD65-b96.11 complex / Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Saccharomyces cerevisiae (brewer's yeast)
Buffer solutionpH: 7.5
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: Constant temperature and 100% humidity during the blotting process
Grid material: COPPER / Grid mesh size: 200 divisions/in. / Grid type: PELCO Ultrathin Carbon with Lacey Carbon
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 %

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD
Image recordingElectron dose: 63.63 e/Å2 / Film or detector model: FEI FALCON II (4k x 4k)

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Processing

EM softwareName: EMAN2 / Category: image acquisition / Details: Automatically picked with the Gaussian picker
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C2 (2 fold cyclic)
3D reconstructionResolution: 7.3 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 25700 / Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT / Space: REAL

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