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- PDB-9d7y: Crystal structure of scFv corresponding to human autoantibody b96.11 -

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Basic information

Entry
Database: PDB / ID: 9d7y
TitleCrystal structure of scFv corresponding to human autoantibody b96.11
ComponentsscFv corresponding to human autoantibody b96.11
KeywordsIMMUNE SYSTEM / Autoantibody
Biological speciesHomo sapiens (human)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 2.6 Å
AuthorsBuckle, A.M. / McGowan, S.
Funding support1items
OrganizationGrant numberCountry
Not funded
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
DepositionAug 18, 2024Deposition site: RCSB / Processing site: RCSB
Revision 1.0Nov 6, 2024Provider: repository / Type: Initial release
Revision 1.1Mar 19, 2025Group: Database references / Category: citation / citation_author
Revision 1.2Jul 16, 2025Group: Database references / Category: pdbx_database_related

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: scFv corresponding to human autoantibody b96.11
B: scFv corresponding to human autoantibody b96.11
C: scFv corresponding to human autoantibody b96.11
hetero molecules


Theoretical massNumber of molelcules
Total (without water)84,3027
Polymers83,9183
Non-polymers3844
Water2,828157
1
A: scFv corresponding to human autoantibody b96.11
hetero molecules


Theoretical massNumber of molelcules
Total (without water)28,1653
Polymers27,9731
Non-polymers1922
Water181
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
2
B: scFv corresponding to human autoantibody b96.11
hetero molecules


Theoretical massNumber of molelcules
Total (without water)28,0692
Polymers27,9731
Non-polymers961
Water181
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
3
C: scFv corresponding to human autoantibody b96.11
hetero molecules


Theoretical massNumber of molelcules
Total (without water)28,0692
Polymers27,9731
Non-polymers961
Water181
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Unit cell
Length a, b, c (Å)96.544, 103.963, 85.088
Angle α, β, γ (deg.)90.000, 90.000, 90.000
Int Tables number19
Space group name H-MP212121
Space group name HallP2ac2ab
Symmetry operation#1: x,y,z
#2: x+1/2,-y+1/2,-z
#3: -x,y+1/2,-z+1/2
#4: -x+1/2,-y,z+1/2
Noncrystallographic symmetry (NCS)NCS domain:
IDEns-IDDetails
d_1ens_1(chain "A" and (resid 1 through 2 or (resid 3...
d_2ens_1(chain "B" and ((resid 1 and (name N or name...
d_3ens_1(chain "C" and ((resid 1 and (name N or name...

NCS domain segments:

Ens-ID: ens_1

Dom-IDComponent-IDBeg auth comp-IDBeg label comp-IDEnd auth comp-IDEnd label comp-IDAuth asym-IDLabel asym-IDAuth seq-IDLabel seq-ID
d_11GLUGLUVALVALAA1 - 2572 - 258
d_21GLUGLUVALVALBB1 - 2572 - 258
d_31GLUGLUSERSERCC1 - 1272 - 128
d_32ALAALAVALVALCC150 - 257151 - 258

NCS oper:
IDCodeMatrixVector
1given(0.529310793158, 0.0173995231744, -0.848249574618), (0.0216716666279, 0.999186199809, 0.0340187739016), (0.848151179397, -0.0363894861945, 0.528502963268)75.9491220777, 13.2296283517, -52.4041533939
2given(-0.473016962854, 0.0268311128504, -0.880644675357), (0.0346147338096, 0.999330418807, 0.0118547142535), (0.880373087421, -0.0248758000867, -0.473628991421)163.77929511, 31.5160042877, 73.0556116284

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Components

#1: Antibody scFv corresponding to human autoantibody b96.11


Mass: 27972.594 Da / Num. of mol.: 3
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Plasmid: pcDNA3.1(-) / Cell line (production host): expi293 / Organ (production host): Kidney / Production host: Homo sapiens (human)
#2: Chemical
ChemComp-SO4 / SULFATE ION


Mass: 96.063 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: SO4
#3: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 157 / Source method: isolated from a natural source / Formula: H2O
Has ligand of interestN
Has protein modificationY

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Experimental details

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Experiment

ExperimentMethod: X-RAY DIFFRACTION / Number of used crystals: 1

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Sample preparation

CrystalDensity Matthews: 2.55 Å3/Da / Density % sol: 51.84 %
Crystal growTemperature: 293 K / Method: vapor diffusion, hanging drop / pH: 6.5
Details: The hanging drop contained 1 microliter of mother liquor combined with 1 microliter of 20 mg/mL b96.11 scFv protein solution. Crystals formed as shards within 24 hours at 293 K, with final ...Details: The hanging drop contained 1 microliter of mother liquor combined with 1 microliter of 20 mg/mL b96.11 scFv protein solution. Crystals formed as shards within 24 hours at 293 K, with final crystals being selected from well conditions containing 0.1M Bis-Tris pH 6.5, 2.5M (NH4)2SO4

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: Australian Synchrotron / Beamline: MX2 / Wavelength: 0.9537 Å
DetectorType: DECTRIS EIGER X 16M / Detector: PIXEL / Date: Aug 15, 2019
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.9537 Å / Relative weight: 1
ReflectionResolution: 2.6→45.77 Å / Num. obs: 26242 / % possible obs: 85.66 % / Redundancy: 1.9 % / Biso Wilson estimate: 32.73 Å2 / CC1/2: 0.968 / CC star: 0.992 / Rmerge(I) obs: 0.1372 / Net I/σ(I): 3.92
Reflection shellResolution: 2.6→2.69 Å / Redundancy: 1.9 % / Mean I/σ(I) obs: 0.68 / Num. unique obs: 2526 / CC1/2: 0.362 / CC star: 0.729 / Rpim(I) all: 0.94 / % possible all: 70.06

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Processing

Software
NameVersionClassification
Blu-Icedata collection
PHENIX1.20.1-4487refinement
XDSdata reduction
Aimlessdata scaling
PHENIXphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT / Resolution: 2.6→45.77 Å / SU ML: 0.4245 / Cross valid method: FREE R-VALUE / σ(F): 0 / Phase error: 32.2931
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
RfactorNum. reflection% reflection
Rfree0.3008 1806 7.73 %
Rwork0.2271 21571 -
obs0.2329 23377 86.67 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL
Displacement parametersBiso mean: 34.1 Å2
Refinement stepCycle: LAST / Resolution: 2.6→45.77 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms5343 0 20 157 5520
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.00955500
X-RAY DIFFRACTIONf_angle_d1.31597473
X-RAY DIFFRACTIONf_chiral_restr0.0728790
X-RAY DIFFRACTIONf_plane_restr0.0092959
X-RAY DIFFRACTIONf_dihedral_angle_d17.009778
Refine LS restraints NCS
Ens-IDDom-IDAsym-IDAuth asym-IDRefine-IDTypeRms dev position (Å)
ens_1d_2AAX-RAY DIFFRACTIONTorsion NCS0.88154554133
ens_1d_3AAX-RAY DIFFRACTIONTorsion NCS0.902326567525
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
2.6-2.670.38781100.32981307X-RAY DIFFRACTION68.79
2.67-2.750.42311210.33211433X-RAY DIFFRACTION76.51
2.75-2.840.42571240.31841510X-RAY DIFFRACTION79.94
2.84-2.940.39191230.30181526X-RAY DIFFRACTION81.07
2.94-3.060.3591330.27861547X-RAY DIFFRACTION81.71
3.06-3.20.36731300.2591617X-RAY DIFFRACTION86.36
3.2-3.360.31681490.24611679X-RAY DIFFRACTION87.93
3.37-3.580.30781350.22061805X-RAY DIFFRACTION93.99
3.58-3.850.261550.21151776X-RAY DIFFRACTION93.47
3.85-4.240.27721580.19221817X-RAY DIFFRACTION95.5
4.24-4.850.22381480.1661814X-RAY DIFFRACTION94.28
4.85-6.110.25241550.19661858X-RAY DIFFRACTION93.76
6.11-45.770.28511650.21531882X-RAY DIFFRACTION92.46

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