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- PDB-6t9e: Crystal structure of a bispecific DutaFab in complex with human PDGF -

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Basic information

Entry
Database: PDB / ID: 6t9e
TitleCrystal structure of a bispecific DutaFab in complex with human PDGF
Components
  • DutaFab mat VH chain
  • DutaFab mat VL chain
  • Platelet-derived growth factor subunit B
KeywordsIMMUNE SYSTEM / monoclonal antibody / bispecific antibody / Fab fragment / PDGF
Function / homology
Function and homology information


metanephric glomerular mesangial cell development / positive regulation of vascular associated smooth muscle cell dedifferentiation / platelet-derived growth factor complex / positive regulation of metanephric mesenchymal cell migration / negative regulation of phosphatidylinositol biosynthetic process / positive regulation of glomerular filtration / positive regulation of metanephric mesenchymal cell migration by platelet-derived growth factor receptor-beta signaling pathway / cellular response to mycophenolic acid / negative regulation of vascular associated smooth muscle cell differentiation / superoxide-generating NADPH oxidase activator activity ...metanephric glomerular mesangial cell development / positive regulation of vascular associated smooth muscle cell dedifferentiation / platelet-derived growth factor complex / positive regulation of metanephric mesenchymal cell migration / negative regulation of phosphatidylinositol biosynthetic process / positive regulation of glomerular filtration / positive regulation of metanephric mesenchymal cell migration by platelet-derived growth factor receptor-beta signaling pathway / cellular response to mycophenolic acid / negative regulation of vascular associated smooth muscle cell differentiation / superoxide-generating NADPH oxidase activator activity / protein kinase C signaling / positive regulation of hyaluronan biosynthetic process / platelet-derived growth factor binding / positive regulation of glomerular mesangial cell proliferation / interleukin-18-mediated signaling pathway / positive regulation of chemotaxis / Signaling by PDGF / paracrine signaling / platelet-derived growth factor receptor binding / positive regulation of vascular associated smooth muscle cell migration / positive regulation of cell-substrate adhesion / positive regulation of DNA biosynthetic process / cellular response to platelet-derived growth factor stimulus / positive regulation of smooth muscle cell migration / positive regulation of calcium ion import / chemoattractant activity / monocyte chemotaxis / positive regulation of cell division / platelet-derived growth factor receptor signaling pathway / Non-integrin membrane-ECM interactions / negative regulation of platelet activation / embryonic placenta development / positive regulation of blood vessel endothelial cell migration / positive regulation of protein autophosphorylation / positive regulation of vascular associated smooth muscle cell proliferation / positive regulation of endothelial cell proliferation / collagen binding / reactive oxygen species metabolic process / Downstream signal transduction / positive regulation of mitotic nuclear division / cell chemotaxis / negative regulation of miRNA transcription / platelet alpha granule lumen / negative regulation of protein binding / positive regulation of smooth muscle cell proliferation / growth factor activity / positive regulation of MAP kinase activity / cellular response to growth factor stimulus / positive regulation of miRNA transcription / response to wounding / Golgi lumen / peptidyl-tyrosine phosphorylation / Constitutive Signaling by Aberrant PI3K in Cancer / positive regulation of reactive oxygen species metabolic process / positive regulation of fibroblast proliferation / Platelet degranulation / PIP3 activates AKT signaling / heart development / gene expression / PI5P, PP2A and IER3 Regulate PI3K/AKT Signaling / RAF/MAP kinase cascade / basolateral plasma membrane / collagen-containing extracellular matrix / positive regulation of MAPK cascade / positive regulation of phosphatidylinositol 3-kinase/protein kinase B signal transduction / positive regulation of ERK1 and ERK2 cascade / positive regulation of cell migration / protein heterodimerization activity / endoplasmic reticulum lumen / Golgi membrane / negative regulation of gene expression / protein phosphorylation / negative regulation of DNA-templated transcription / positive regulation of cell population proliferation / positive regulation of gene expression / positive regulation of DNA-templated transcription / cell surface / protein homodimerization activity / extracellular space / extracellular region / identical protein binding / cytoplasm
Similarity search - Function
Platelet-derived growth factor, N-terminal / Platelet-derived growth factor, N terminal region / 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
Platelet-derived growth factor subunit B
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 2.989 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: DutaFab mat VH chain
BBB: DutaFab mat VL chain
CCC: Platelet-derived growth factor subunit B
DDD: Platelet-derived growth factor subunit B
HHH: DutaFab mat VH chain
LLL: DutaFab mat VL chain


Theoretical massNumber of molelcules
Total (without water)119,4796
Polymers119,4796
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author&software
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area12190 Å2
ΔGint-79 kcal/mol
Surface area46940 Å2
MethodPISA
Unit cell
Length a, b, c (Å)89.301, 75.474, 116.505
Angle α, β, γ (deg.)90.000, 110.584, 90.000
Int Tables number4
Space group name H-MP1211
Noncrystallographic symmetry (NCS)NCS domain:
IDEns-IDDetails
11Chains A H
21Chains B L
31Chains C D

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Components

#1: Antibody DutaFab mat VH chain


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


Mass: 23923.521 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Cell line (production host): HEK293 / Production host: Homo sapiens (human)
#3: Protein Platelet-derived growth factor subunit B / PDGF subunit B / PDGF-2 / Platelet-derived growth factor B chain / Platelet-derived growth factor ...PDGF subunit B / PDGF-2 / Platelet-derived growth factor B chain / Platelet-derived growth factor beta polypeptide / Proto-oncogene c-Sis


Mass: 12313.501 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Details: Human platelet-derived growth factor subunit B purchased from Peprotech
Source: (gene. exp.) Homo sapiens (human) / Gene: PDGFB, PDGF2, SIS / Production host: Escherichia coli (E. coli) / References: UniProt: P01127

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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: 2.18 Å3/Da / Density % sol: 43.5 % / Description: Plates
Crystal growTemperature: 293 K / Method: vapor diffusion, hanging drop / pH: 7.5
Details: DutaFab and PDGF-BB (Peprotech, catalogue number 100-13A) were mixed at a 1:1 molar ratio in relation to PDGF monomer. The complex was concentrated to 12 mg/ml and crystallization was ...Details: DutaFab and PDGF-BB (Peprotech, catalogue number 100-13A) were mixed at a 1:1 molar ratio in relation to PDGF monomer. The complex was concentrated to 12 mg/ml and crystallization was performed by hanging drop vapour diffusion against 0.1 M Tris pH 7.5, 42 % 2-methyl-2,4-pentanediol (MPD) at 20 C. Plate-shaped crystals grew within a few weeks and were frozen in liquid nitrogen with 20% glycerol as cryo-protectant.

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: Diamond / Beamline: I03 / Wavelength: 0.976 Å
DetectorType: DECTRIS PILATUS3 6M / Detector: PIXEL / Date: May 3, 2015
RadiationMonochromator: DOUBLE CRYSTAL / Protocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.976 Å / Relative weight: 1
ReflectionResolution: 2.989→30 Å / Num. obs: 29016 / % possible obs: 97.7 % / Redundancy: 3 % / Biso Wilson estimate: 43.9 Å2 / Rmerge(I) obs: 0.162 / Net I/σ(I): 7.3
Reflection shellResolution: 2.989→3.2 Å / Redundancy: 3 % / Rmerge(I) obs: 0.773 / Mean I/σ(I) obs: 1.8 / Num. unique obs: 4371 / % possible all: 92.8

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Processing

Software
NameVersionClassification
REFMAC5.8.0257refinement
XDSdata reduction
XDSdata scaling
PHASERphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 4QCI

4qci


Resolution: 2.989→29.735 Å / Cor.coef. Fo:Fc: 0.92 / Cor.coef. Fo:Fc free: 0.875 / SU B: 49.23 / SU ML: 0.743 / Cross valid method: FREE R-VALUE / ESU R Free: 0.5
Details: Hydrogens have been added in their riding positions
RfactorNum. reflection% reflection
Rfree0.3044 1451 5.001 %
Rwork0.2531 --
all0.256 --
obs-29016 97.832 %
Solvent computationIon probe radii: 0.8 Å / Shrinkage radii: 0.8 Å / VDW probe radii: 1.2 Å
Displacement parametersBiso mean: 62.916 Å2
Baniso -1Baniso -2Baniso -3
1-6.669 Å20 Å2-6.372 Å2
2---3.405 Å20 Å2
3---1.187 Å2
Refinement stepCycle: LAST / Resolution: 2.989→29.735 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms7935 0 0 0 7935
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
X-RAY DIFFRACTIONr_bond_refined_d0.0060.0128136
X-RAY DIFFRACTIONr_angle_refined_deg1.4481.64111059
X-RAY DIFFRACTIONr_dihedral_angle_1_deg8.15151013
X-RAY DIFFRACTIONr_dihedral_angle_2_deg35.01522.312385
X-RAY DIFFRACTIONr_dihedral_angle_3_deg19.121151326
X-RAY DIFFRACTIONr_dihedral_angle_4_deg16.8361544
X-RAY DIFFRACTIONr_chiral_restr0.1080.21066
X-RAY DIFFRACTIONr_gen_planes_refined0.0060.026174
X-RAY DIFFRACTIONr_nbd_refined0.2610.23568
X-RAY DIFFRACTIONr_nbtor_refined0.3120.25423
X-RAY DIFFRACTIONr_xyhbond_nbd_refined0.1830.2285
X-RAY DIFFRACTIONr_symmetry_nbd_refined0.1890.239
X-RAY DIFFRACTIONr_symmetry_xyhbond_nbd_refined0.20.25
X-RAY DIFFRACTIONr_mcbond_it5.4896.294085
X-RAY DIFFRACTIONr_mcangle_it8.9089.4255087
X-RAY DIFFRACTIONr_scbond_it5.3386.2314051
X-RAY DIFFRACTIONr_scangle_it8.8369.2885972
X-RAY DIFFRACTIONr_lrange_it14.03182.54112005
X-RAY DIFFRACTIONr_ncsr_local_group_10.0660.056412
X-RAY DIFFRACTIONr_ncsr_local_group_20.0690.056551
X-RAY DIFFRACTIONr_ncsr_local_group_30.1120.052611
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
2.989-3.0660.525910.4617320.46321360.0410.04985.34640.41
3.066-3.1490.3951050.42620030.42421200.1490.15699.4340.366
3.149-3.2390.4191010.40619120.40720300.3750.3499.16260.355
3.239-3.3380.384990.37418820.37419890.3150.35699.59780.32
3.338-3.4460.369970.3418440.34119500.4490.48999.53850.294
3.446-3.5650.303920.30317430.30318420.6360.6499.620.25
3.565-3.6980.372890.30416960.30717960.560.6599.38750.255
3.698-3.8460.311860.24616400.24917400.7830.80699.19540.194
3.846-4.0140.271830.21415780.21716820.8460.85698.75150.177
4.014-4.2060.27800.20515120.20816070.8520.89299.06660.175
4.206-4.4290.269750.21614220.21915200.880.88998.48680.186
4.429-4.6910.22720.18713680.18914530.8990.999.10530.166
4.691-5.0060.249670.1812700.18313540.8840.92998.74450.158
5.006-5.3940.269620.19811830.20212640.8860.91398.49680.168
5.394-5.890.286590.22611130.22911920.8730.89698.32210.192
5.89-6.5530.43520.2439970.25210650.8190.88398.49770.208
6.553-7.5060.273470.2128970.2159610.8970.91798.2310.183
7.506-9.050.218400.1617650.1648260.9420.94897.45760.153
9.05-12.2440.255320.1646060.1686570.9210.95397.10810.163
12.244-29.7350.26220.2554020.2554400.9280.93296.36360.257

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