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- EMDB-23603: The Cryo-EM structure of a complex between GAD65 and b96.11 Fab -

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Basic information

Entry
Database: EMDB / ID: EMD-23603
TitleThe Cryo-EM structure of a complex between GAD65 and b96.11 Fab
Map data
Sample
  • Complex: GAD65-b96.11 complex
    • Protein or peptide: Glutamate decarboxylase 2
    • Protein or peptide: b96.11 Fab heavy chain
    • Protein or peptide: b96.11 Fab light chain
KeywordsNeurotransmitter biosynthesis / Autoantibody / autoantigen / type 1 diabetes / stiff person syndrome / GABA / IMMUNE SYSTEM
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)
Methodsingle particle reconstruction / cryo EM / Resolution: 7.3 Å
AuthorsReboul CF / Le SN
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 SHD / Reboul CF / Le SN / Williams DE / Chandler PG / Costa MGS / Hoke DE / Jimma JDT / Fodor J / Fenalti G / Mannering SI / Porebski BT / Schofield P / Christ D / Buckle M / McGowan ...Authors: Stander SHD / Reboul CF / Le SN / Williams DE / Chandler PG / Costa MGS / Hoke DE / Jimma JDT / Fodor J / Fenalti G / Mannering SI / Porebski BT / Schofield P / Christ D / Buckle M / McGowan S / Elmlund D / Rand KD / Buckle AM
History
DepositionMar 9, 2021-
Header (metadata) releaseOct 30, 2024-
Map releaseOct 30, 2024-
UpdateJul 16, 2025-
Current statusJul 16, 2025Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_23603.png

Downloads & links

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Map

FileDownload / File: emd_23603.map.gz / Format: CCP4 / Size: 91.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.06 Å/pix.
x 288 pix.
= 305.28 Å		1.06 Å/pix.
x 288 pix.
= 305.28 Å		1.06 Å/pix.
x 288 pix.
= 305.28 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.06 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.011
Minimum - Maximum-0.008236222 - 0.032226175
Average (Standard dev.)0.00005394105 (±0.0013490632)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions288288288
Spacing288288288
CellA=B=C: 305.27997 Å
α=β=γ: 90.0 °

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Supplemental data

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Sample components

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Entire : GAD65-b96.11 complex

EntireName: GAD65-b96.11 complex
Components
  • Complex: GAD65-b96.11 complex
    • Protein or peptide: Glutamate decarboxylase 2
    • Protein or peptide: b96.11 Fab heavy chain
    • Protein or peptide: b96.11 Fab light chain

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Supramolecule #1: GAD65-b96.11 complex

SupramoleculeName: GAD65-b96.11 complex / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Homo sapiens (human)

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Macromolecule #1: Glutamate decarboxylase 2

MacromoleculeName: Glutamate decarboxylase 2 / type: protein_or_peptide / ID: 1 / Number of copies: 2 / Enantiomer: LEVO / EC number: glutamate decarboxylase
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 56.554215 KDa
Recombinant expressionOrganism: Saccharomyces cerevisiae (brewer's yeast)
SequenceString: NYAFLHATDL LPACDGERPT LAFLQDVMNI LLQYVVKSFD RSTKVIDFHY PNELLQEYNW ELADQPQNLE EILMHCQTTL KYAIKTGHP RYFNQLSTGL DMVGLAADWL TSTANTNMFT YEIAPVFVLL EYVTLKKMRE IIGWPGGSGD GIFSPGGAIS N MYAMMIAR ...String:
NYAFLHATDL LPACDGERPT LAFLQDVMNI LLQYVVKSFD RSTKVIDFHY PNELLQEYNW ELADQPQNLE EILMHCQTTL KYAIKTGHP RYFNQLSTGL DMVGLAADWL TSTANTNMFT YEIAPVFVLL EYVTLKKMRE IIGWPGGSGD GIFSPGGAIS N MYAMMIAR FKMFPEVKEK GMAALPRLIA FTSEHSHFSL KKGAAALGIG TDSVILIKCD ERGKMIPSDL ERRILEAKQK GF VPFLVSA TAGTTVYGAF DPLLAVADIC KKYKIWMHVD AAWGGGLLMS RKHKWKLSGV ERANSVTWNP HKMMGVPLQC SAL LVREEG LMQNCNQMHA SYLFQQDKHY DLSYDTGDKA LQCGRHVDVF KLWLMWRAKG TTGFEAHVDK CLELAEYLYN IIKN REGYE MVFDGKPQHT NVCFWYIPPS LRTLEDNEER MSRLSKVAPV IKARMMEYGT TMVSYQPLGD KVNFFRMVIS NPAAT HQDI DFLIEEIERL GQD

UniProtKB: Glutamate decarboxylase 2

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Macromolecule #2: b96.11 Fab heavy chain

MacromoleculeName: b96.11 Fab heavy chain / type: protein_or_peptide / ID: 2 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 24.553436 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: EVQLVESGGG LVQPGRSLRL SCSASGFTFG DYAMSWFRLA PGKGLEWVGL IKSRAIDGTP QYAASVKGRF TISRDDSNSI AYLQMNSLT TEDTAIYYCA RDFYDFWNEF SHRTFDFWGQ GTLVTVSSAS TKGPSVFPLA PSSKSTSGGT AALGCLVKDY F PEPVTVSW ...String:
EVQLVESGGG LVQPGRSLRL SCSASGFTFG DYAMSWFRLA PGKGLEWVGL IKSRAIDGTP QYAASVKGRF TISRDDSNSI AYLQMNSLT TEDTAIYYCA RDFYDFWNEF SHRTFDFWGQ GTLVTVSSAS TKGPSVFPLA PSSKSTSGGT AALGCLVKDY F PEPVTVSW NSGALTSGVH TFPAVLQSSG LYSLSSVVTV PSSSLGTQTY ICNVNHKPSN TKVDKRVEPK

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Macromolecule #3: b96.11 Fab light chain

MacromoleculeName: b96.11 Fab light chain / type: protein_or_peptide / ID: 3 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 23.060504 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: QSALTQPASA SGSPGQSVTI TCTGSSSDVG GYKYVSWYQH HPGKAPKLMI YEVSKRPSGV PDRFSGSKSG NMASLTVSGL QAEDEADYY CSSYAGSYNF YVFGNGTKVT VLGQPKANPT VTLFPPSSEE LQANKATLVC LISDFYPGAV TVAWKADGSP V KAGVETTK ...String:
QSALTQPASA SGSPGQSVTI TCTGSSSDVG GYKYVSWYQH HPGKAPKLMI YEVSKRPSGV PDRFSGSKSG NMASLTVSGL QAEDEADYY CSSYAGSYNF YVFGNGTKVT VLGQPKANPT VTLFPPSSEE LQANKATLVC LISDFYPGAV TVAWKADGSP V KAGVETTK PSKQSNNKYA ASSYLSLTPE QWKSHRSYSC QVTHEGSTVE KTVAPTECS

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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

BufferpH: 7.5
GridModel: PELCO Ultrathin Carbon with Lacey Carbon / Material: COPPER / Mesh: 200 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE
Details: Constant temperature and 100% humidity during the blotting process
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: FEI FALCON II (4k x 4k) / Average electron dose: 63.63 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Startup modelType of model: NONE
Final reconstructionApplied symmetry - Point group: C2 (2 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 7.3 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 25700
Initial angle assignmentType: RANDOM ASSIGNMENT
Final angle assignmentType: MAXIMUM LIKELIHOOD
FSC plot (resolution estimation)

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Atomic model buiding 1

RefinementSpace: REAL / Protocol: FLEXIBLE FIT
Output model

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

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