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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 / inhibitory synapse / response to caloric restriction ...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 / inhibitory synapse / response to caloric restriction / glutamate binding / response to dexamethasone / cellular response to brain-derived neurotrophic factor stimulus / response to progesterone / GABA-ergic synapse / response to cocaine / synaptic vesicle membrane / pyridoxal phosphate binding / presynaptic membrane / chemical synaptic transmission / cellular response to hypoxia / response to xenobiotic stimulus / axon / Golgi membrane / neuronal cell body / protein-containing complex binding / perinuclear region of cytoplasm / 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.
CitationJournal: 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 1.1Nov 13, 2024Group: Data collection / Category: em_admin / Item: _em_admin.last_update

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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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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