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- PDB-6t9d: Crystal structure of a bispecific DutaFab in complex with human V... -

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Basic information

Entry
Database: PDB / ID: 6t9d
TitleCrystal structure of a bispecific DutaFab in complex with human VEGF121
Components
  • VP mat DutaFab VH chain
  • VP mat DutaFab VL chain
  • Vascular endothelial growth factor A
KeywordsIMMUNE SYSTEM / monoclonal antibody / bispecific antibody / Fab fragment / VEGF
Function / homology
Function and homology information


basophil chemotaxis / positive regulation of endothelial cell chemotaxis by VEGF-activated vascular endothelial growth factor receptor signaling pathway / cellular stress response to acid chemical / VEGF-A complex / Signaling by VEGF / lymph vessel morphogenesis / positive regulation of lymphangiogenesis / negative regulation of adherens junction organization / vascular endothelial growth factor receptor 1 binding / negative regulation of establishment of endothelial barrier ...basophil chemotaxis / positive regulation of endothelial cell chemotaxis by VEGF-activated vascular endothelial growth factor receptor signaling pathway / cellular stress response to acid chemical / VEGF-A complex / Signaling by VEGF / lymph vessel morphogenesis / positive regulation of lymphangiogenesis / negative regulation of adherens junction organization / vascular endothelial growth factor receptor 1 binding / negative regulation of establishment of endothelial barrier / VEGF ligand-receptor interactions / vascular endothelial growth factor receptor binding / positive regulation of mast cell chemotaxis / post-embryonic camera-type eye development / primitive erythrocyte differentiation / positive regulation of protein kinase C signaling / positive regulation of cell proliferation by VEGF-activated platelet derived growth factor receptor signaling pathway / negative regulation of blood-brain barrier permeability / VEGF-activated neuropilin signaling pathway / bone trabecula formation / positive regulation of vascular endothelial growth factor signaling pathway / coronary vein morphogenesis / cardiac vascular smooth muscle cell development / lung vasculature development / lymphangiogenesis / eye photoreceptor cell development / endothelial cell chemotaxis / motor neuron migration / positive regulation of trophoblast cell migration / positive regulation of epithelial tube formation / vascular endothelial growth factor receptor-2 signaling pathway / VEGF binds to VEGFR leading to receptor dimerization / regulation of nitric oxide mediated signal transduction / positive regulation of axon extension involved in axon guidance / vascular wound healing / positive regulation of protein localization to early endosome / regulation of hematopoietic progenitor cell differentiation / positive regulation of blood vessel endothelial cell proliferation involved in sprouting angiogenesis / positive regulation of branching involved in ureteric bud morphogenesis / camera-type eye morphogenesis / neuropilin binding / induction of positive chemotaxis / coronary artery morphogenesis / negative regulation of cell-cell adhesion mediated by cadherin / vascular endothelial growth factor receptor 2 binding / tube formation / positive regulation of vascular permeability / dopaminergic neuron differentiation / commissural neuron axon guidance / platelet-derived growth factor receptor binding / surfactant homeostasis / negative regulation of epithelial to mesenchymal transition / extracellular matrix binding / cell migration involved in sprouting angiogenesis / cardiac muscle cell development / epithelial cell maturation / sprouting angiogenesis / positive regulation of positive chemotaxis / endothelial cell proliferation / Regulation of gene expression by Hypoxia-inducible Factor / positive regulation of leukocyte migration / vascular endothelial growth factor signaling pathway / positive regulation of p38MAPK cascade / positive regulation of endothelial cell chemotaxis / artery morphogenesis / positive regulation of cell migration involved in sprouting angiogenesis / branching involved in blood vessel morphogenesis / positive regulation of DNA biosynthetic process / retinal ganglion cell axon guidance / positive regulation of neuroblast proliferation / positive chemotaxis / negative regulation of fat cell differentiation / transmembrane receptor protein tyrosine kinase activator activity / positive regulation of sprouting angiogenesis / chemoattractant activity / outflow tract morphogenesis / positive regulation of focal adhesion assembly / mesoderm development / monocyte differentiation / positive regulation of receptor internalization / macrophage differentiation / fibronectin binding / positive regulation of cell division / positive regulation of cell adhesion / neuroblast proliferation / positive regulation of blood vessel endothelial cell migration / cellular response to vascular endothelial growth factor stimulus / mammary gland alveolus development / vasculogenesis / positive regulation of osteoblast differentiation / vascular endothelial growth factor receptor signaling pathway / heart morphogenesis / ovarian follicle development / cell maturation / homeostasis of number of cells within a tissue / positive regulation of protein autophosphorylation / positive regulation of endothelial cell proliferation / epithelial cell differentiation / lactation / TFAP2 (AP-2) family regulates transcription of growth factors and their receptors
Similarity search - Function
Vascular endothelial growth factor, heparin-binding domain / Vascular endothelial growth factor, heparin-binding domain superfamily / VEGF heparin-binding domain / : / PDGF/VEGF domain / Platelet-derived growth factor, conserved site / PDGF/VEGF domain / Platelet-derived growth factor (PDGF) family signature. / Platelet-derived growth factor (PDGF) family profile. / Platelet-derived and vascular endothelial growth factors (PDGF, VEGF) family / Cystine-knot cytokine
Similarity search - Domain/homology
Vascular endothelial growth factor A, long form
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 2.905 Å
AuthorsKimbung, R. / Logan, D.T. / Beckmann, R. / Jensen, K. / Speck, J. / Fenn, S. / Kettenberger, H.
CitationJournal: Nat Commun / Year: 2021
Title: DutaFabs are engineered therapeutic Fab fragments that can bind two targets simultaneously.
Authors: Beckmann, R. / Jensen, K. / Fenn, S. / Speck, J. / Krause, K. / Meier, A. / Roth, M. / Fauser, S. / Kimbung, R. / Logan, D.T. / Steegmaier, M. / Kettenberger, H.
History
DepositionOct 28, 2019Deposition site: PDBE / Processing site: PDBE
Revision 1.0Dec 16, 2020Provider: repository / Type: Initial release
Revision 1.1Feb 10, 2021Group: Database references / Category: citation / citation_author
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year / _citation_author.identifier_ORCID / _citation_author.name
Revision 1.2Jan 24, 2024Group: Data collection / Database references ...Data collection / Database references / Derived calculations / Refinement description
Category: atom_type / chem_comp_atom ...atom_type / chem_comp_atom / chem_comp_bond / database_2 / pdbx_initial_refinement_model
Item: _atom_type.pdbx_N_electrons / _atom_type.pdbx_scat_Z ..._atom_type.pdbx_N_electrons / _atom_type.pdbx_scat_Z / _database_2.pdbx_DOI / _database_2.pdbx_database_accession

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
AAA: VP mat DutaFab VH chain
BBB: VP mat DutaFab VL chain
CCC: Vascular endothelial growth factor A
DDD: Vascular endothelial growth factor A
HHH: VP mat DutaFab VH chain
LLL: VP mat DutaFab VL chain


Theoretical massNumber of molelcules
Total (without water)122,8016
Polymers122,8016
Non-polymers00
Water3,837213
1


  • Idetical with deposited unit
  • defined by author&software
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area14340 Å2
ΔGint-92 kcal/mol
Surface area44690 Å2
MethodPISA
Unit cell
Length a, b, c (Å)70.080, 107.875, 87.369
Angle α, β, γ (deg.)90.000, 95.340, 90.000
Int Tables number4
Space group name H-MP1211
Noncrystallographic symmetry (NCS)NCS domain:
IDEns-IDDetails
11Chains A H
22Chains B L
33Chains C D

NCS ensembles :
ID
3
1
2

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Components

#1: Antibody VP mat DutaFab VH chain


Mass: 23502.230 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Details: VH region of DutaFab bispecific mAb / Source: (gene. exp.) Homo sapiens (human) / Cell line (production host): HEK293 / Production host: Homo sapiens (human)
#2: Antibody VP mat DutaFab VL chain


Mass: 23820.379 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Details: VL region of DutaFab bispecific FAb / Source: (gene. exp.) Homo sapiens (human) / Cell line (production host): HEK293 / Production host: Homo sapiens (human)
#3: Protein Vascular endothelial growth factor A / VEGF-A / Vascular permeability factor / VPF


Mass: 14078.085 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: VEGFA, VEGF / Production host: Escherichia coli (E. coli) / References: UniProt: P15692
#4: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 213 / Source method: isolated from a natural source / Formula: H2O

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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: 2.68 Å3/Da / Density % sol: 54.06 % / Description: Needles
Crystal growTemperature: 293 K / Method: vapor diffusion, hanging drop / pH: 6.5
Details: VEGFA-121 from Peprotech, catalogue number 100-20A, was mixed at a 1:1 molar ratio to VEGF monomer. The complex was concentrated to 11 mg/ml. Crystallization involved hanging drop vapour ...Details: VEGFA-121 from Peprotech, catalogue number 100-20A, was mixed at a 1:1 molar ratio to VEGF monomer. The complex was concentrated to 11 mg/ml. Crystallization involved hanging drop vapour diffusion against 0.1 M MES pH 6.5 and 1.6 M magnesium sulphate. Crystals grew in about 120 days and were frozen in liquid nitrogen with 20 % glycerol as cryoprotectant.

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: MAX II / Beamline: I911-3 / Wavelength: 1 Å
DetectorType: MARMOSAIC 225 mm CCD / Detector: CCD / Date: Nov 11, 2014 / Details: KB MIRRORS
RadiationMonochromator: DOUBLE CRYSTAL / Protocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 1 Å / Relative weight: 1
ReflectionResolution: 2.9→30 Å / Num. obs: 28318 / % possible obs: 98.9 % / Redundancy: 3 % / Biso Wilson estimate: 37.5 Å2 / Rmerge(I) obs: 0.212 / Net I/σ(I): 6.7
Reflection shellResolution: 2.9→3.1 Å / Redundancy: 3 % / Rmerge(I) obs: 0.842 / Mean I/σ(I) obs: 1.5 / Num. unique obs: 4494 / % possible all: 98.2

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Processing

Software
NameVersionClassification
REFMAC5.8.0257refinement
XDSdata reduction
XDSdata scaling
PHASERphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 1BJ1, 1JPS

1bj1

1jps


Resolution: 2.905→29.294 Å / Cor.coef. Fo:Fc: 0.901 / Cor.coef. Fo:Fc free: 0.808 / SU B: 24.612 / SU ML: 0.432 / Cross valid method: FREE R-VALUE / ESU R Free: 0.461
Details: Hydrogens have been added in their riding positions
RfactorNum. reflection% reflection
Rfree0.2801 1416 5.001 %
Rwork0.2124 --
all0.216 --
obs-28317 98.879 %
Solvent computationIon probe radii: 0.8 Å / Shrinkage radii: 0.8 Å / VDW probe radii: 1.2 Å
Displacement parametersBiso mean: 35.023 Å2
Baniso -1Baniso -2Baniso -3
1--1.396 Å20 Å2-0.886 Å2
2--0.703 Å20 Å2
3---0.843 Å2
Refinement stepCycle: LAST / Resolution: 2.905→29.294 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms8047 0 0 213 8260
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
X-RAY DIFFRACTIONr_bond_refined_d0.0070.0138295
X-RAY DIFFRACTIONr_bond_other_d0.0010.0177323
X-RAY DIFFRACTIONr_angle_refined_deg1.5251.64711276
X-RAY DIFFRACTIONr_angle_other_deg1.1691.57317124
X-RAY DIFFRACTIONr_dihedral_angle_1_deg21.6285.461077
X-RAY DIFFRACTIONr_dihedral_angle_2_deg36.36823.047384
X-RAY DIFFRACTIONr_dihedral_angle_3_deg18.883151313
X-RAY DIFFRACTIONr_dihedral_angle_4_deg17.5181533
X-RAY DIFFRACTIONr_chiral_restr0.0550.21069
X-RAY DIFFRACTIONr_gen_planes_refined0.0050.0210461
X-RAY DIFFRACTIONr_gen_planes_other0.0010.021713
X-RAY DIFFRACTIONr_nbd_refined0.1850.21408
X-RAY DIFFRACTIONr_symmetry_nbd_other0.1840.26705
X-RAY DIFFRACTIONr_nbtor_refined0.1680.23685
X-RAY DIFFRACTIONr_symmetry_nbtor_other0.0750.23895
X-RAY DIFFRACTIONr_xyhbond_nbd_refined0.1840.2265
X-RAY DIFFRACTIONr_symmetry_xyhbond_nbd_other0.0140.21
X-RAY DIFFRACTIONr_symmetry_nbd_refined0.3270.211
X-RAY DIFFRACTIONr_nbd_other0.2480.255
X-RAY DIFFRACTIONr_symmetry_xyhbond_nbd_refined0.0430.21
X-RAY DIFFRACTIONr_mcbond_it2.3723.7524137
X-RAY DIFFRACTIONr_mcbond_other2.3723.7524136
X-RAY DIFFRACTIONr_mcangle_it3.9255.6185160
X-RAY DIFFRACTIONr_mcangle_other3.9255.6185161
X-RAY DIFFRACTIONr_scbond_it2.1923.8924158
X-RAY DIFFRACTIONr_scbond_other2.1923.8934159
X-RAY DIFFRACTIONr_scangle_it3.6155.7486114
X-RAY DIFFRACTIONr_scangle_other3.6155.7496115
X-RAY DIFFRACTIONr_lrange_it6.61241.2698635
X-RAY DIFFRACTIONr_lrange_other6.6141.3088623
X-RAY DIFFRACTIONr_ncsr_local_group_10.0810.056162
X-RAY DIFFRACTIONr_ncsr_local_group_20.0680.056477
X-RAY DIFFRACTIONr_ncsr_local_group_30.0860.052599
LS refinement shell

Refine-ID: X-RAY DIFFRACTION / Total num. of bins used: 20

Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRfactor allNum. reflection allFsc freeFsc work% reflection obs (%)WRfactor Rwork
2.905-2.980.3921000.37618980.37720650.5290.56896.75540.366
2.98-3.0610.3911020.31919410.32320510.7010.73699.60990.304
3.061-3.1480.337990.29818780.319870.720.76899.49670.27
3.148-3.2440.375960.26118290.26719290.770.80899.79260.233
3.244-3.3490.299920.25717580.25918580.8230.8399.56940.229
3.349-3.4650.299900.23717120.2418070.8510.87199.72330.208
3.465-3.5940.285870.2216390.22417360.8910.89699.4240.195
3.594-3.7380.289820.2215720.22416620.850.89199.51870.196
3.738-3.9020.319820.21515560.22116490.8490.89799.33290.19
3.902-4.0890.24770.19214490.19515350.8940.91699.41370.166
4.089-4.3050.272730.17613970.1814790.8950.93399.39150.157
4.305-4.560.227680.15412980.15713760.9260.94499.27330.138
4.56-4.8670.225660.14312420.14713160.9280.9599.39210.128
4.867-5.2440.2610.14611580.14812290.9480.95699.18630.13
5.244-5.7270.267560.17610700.18111410.9150.94198.68540.153
5.727-6.3720.212520.2259790.22410460.9160.90298.5660.197
6.372-7.3010.252450.1788560.1829150.9060.93398.46990.153
7.301-8.8060.229390.1387410.1437950.9450.95798.11320.123
8.806-11.9260.202300.1535780.1556230.9420.96297.59230.146
11.926-29.2940.447190.2973500.3034070.7940.88490.66340.284

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