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- PDB-8gjr: Crystal Structure of Nanobody VHH114 Bound to Its Antigen PA14 Cif -

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Basic information

Entry
Database: PDB / ID: 8gjr
TitleCrystal Structure of Nanobody VHH114 Bound to Its Antigen PA14 Cif
Components
  • CFTR inhibitory factor
  • Nanobody VHH114
KeywordsIMMUNE SYSTEM / Pseudomonas aeruginosa / nanobody VHH immunoglobulin domain / CFTR inhibitory factor (Cif)
Function / homologyAlpha/beta hydrolase family / Alpha/beta hydrolase fold-1 / Alpha/Beta hydrolase fold / CITRATE ANION / CFTR inhibitory factor
Function and homology information
Biological speciesPseudomonas aeruginosa PA14 (bacteria)
Vicugna pacos (alpaca)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 1.85 Å
AuthorsSimard, A.R. / Madden, D.R.
Funding support United States, 5items
OrganizationGrant numberCountry
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)R01-AI091699 United States
National Institutes of Health/National Institute of Diabetes and Digestive and Kidney Disease (NIH/NIDDK)P30-DK117469 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)P20-GM113132 United States
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)T32AI007519 United States
Cystic Fibrosis FoundationSTANTO19R0 United States
Citation
Journal: To Be Published
Title: Crystal Structure of Nanobody VHH114 Bound to Its Antigen PA14 Cif
Authors: Simard, A.R. / Madden, D.R.
#1: Journal: Anal Chim Acta / Year: 2019
Title: Nanobody-based binding assay for the discovery of potent inhibitors of CFTR inhibitory factor (Cif).
Authors: Vasylieva, N. / Kitamura, S. / Dong, J. / Barnych, B. / Hvorecny, K.L. / Madden, D.R. / Gee, S.J. / Wolan, D.W. / Morisseau, C. / Hammock, B.D.
#2: Journal: Acta Crystallogr D Biol Crystallogr / Year: 2012
Title: Towards automated crystallographic structure refinement with phenix.refine.
Authors: Afonine, P.V. / Grosse-Kunstleve, R.W. / Echols, N. / Headd, J.J. / Moriarty, N.W. / Mustyakimov, M. / Terwilliger, T.C. / Urzhumtsev, A. / Zwart, P.H. / Adams, P.D.
History
DepositionMar 16, 2023Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jun 26, 2024Provider: repository / Type: Initial release
Revision 1.1Nov 6, 2024Group: Structure summary / Category: pdbx_entry_details / pdbx_modification_feature / Item: _pdbx_entry_details.has_protein_modification

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
C: CFTR inhibitory factor
A: Nanobody VHH114
hetero molecules


Theoretical massNumber of molelcules
Total (without water)47,2394
Polymers46,8612
Non-polymers3782
Water6,035335
1
C: CFTR inhibitory factor
A: Nanobody VHH114
hetero molecules

C: CFTR inhibitory factor
A: Nanobody VHH114
hetero molecules


Theoretical massNumber of molelcules
Total (without water)94,4798
Polymers93,7224
Non-polymers7564
Water724
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
crystal symmetry operation12_564x,x-y+1,-z-1/21
Buried area7860 Å2
ΔGint-37 kcal/mol
Surface area30060 Å2
MethodPISA
Unit cell
Length a, b, c (Å)163.487, 163.487, 74.959
Angle α, β, γ (deg.)90.000, 90.000, 120.000
Int Tables number182
Space group name H-MP6322
Space group name HallP6c2c
Symmetry operation#1: x,y,z
#2: x-y,x,z+1/2
#3: y,-x+y,z+1/2
#4: -y,x-y,z
#5: -x+y,-x,z
#6: x-y,-y,-z
#7: -x,-x+y,-z
#8: -x,-y,z+1/2
#9: y,x,-z
#10: -y,-x,-z+1/2
#11: -x+y,y,-z+1/2
#12: x,x-y,-z+1/2
Components on special symmetry positions
IDModelComponents
11C-527-

HOH

21C-576-

HOH

31C-620-

HOH

41C-704-

HOH

51C-716-

HOH

61A-233-

HOH

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Components

#1: Protein CFTR inhibitory factor


Mass: 33335.828 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Pseudomonas aeruginosa PA14 (bacteria) / Gene: PA14_26090 / Plasmid: pET16b / Details (production host): N-terminal 10XHis-SUMO fusion / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: A0A0M3KL26
#2: Antibody Nanobody VHH114


Mass: 13525.262 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Vicugna pacos (alpaca) / Plasmid: pET16b / Details (production host): 10XHis-SUMO fusion / Production host: Escherichia coli BL21(DE3) (bacteria)
#3: Chemical ChemComp-FLC / CITRATE ANION


Mass: 189.100 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C6H5O7
#4: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 335 / 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: 3.09 Å3/Da / Density % sol: 60.21 % / Description: hexagonal rods
Crystal growTemperature: 292.8 K / Method: vapor diffusion, hanging drop / pH: 3.5
Details: 13.5 % (w/v) PEG6K, 2.5 % (v/v) ethylene glycol, 100 mM sodium citrate pH 3.5

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: NSLS-II / Beamline: 17-ID-1 / Wavelength: 0.920112 Å
DetectorType: DECTRIS EIGER X 9M / Detector: PIXEL / Date: Feb 12, 2023
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.920112 Å / Relative weight: 1
ReflectionResolution: 1.85→27.62 Å / Num. obs: 50643 / % possible obs: 99.91 % / Redundancy: 20.2 % / Biso Wilson estimate: 32 Å2 / CC1/2: 0.999 / CC star: 1 / Rmerge(I) obs: 0.139 / Rpim(I) all: 0.03181 / Rrim(I) all: 0.1427 / Net I/σ(I): 16.31
Reflection shellResolution: 1.85→1.916 Å / Redundancy: 20.8 % / Rmerge(I) obs: 2.404 / Mean I/σ(I) obs: 1.23 / Num. unique obs: 5007 / CC1/2: 0.618 / CC star: 0.874 / Rpim(I) all: 0.539 / Rrim(I) all: 2.465 / % possible all: 99.96

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Processing

Software
NameVersionClassification
XDSdata reduction
XSCALEdata scaling
PHASERphasing
Cootmodel building
PHENIX1.20.1_4487refinement
RefinementMethod to determine structure: MOLECULAR REPLACEMENT / Resolution: 1.85→27.62 Å / SU ML: 0.2244 / Cross valid method: FREE R-VALUE / σ(F): 1.36 / Phase error: 20.157
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Details: Atoms modeled with zero occupancy could not be placed with confidence and were selected for zero-occupancy flagging after manual inspection of the 2Fo-Fc map at a 0.5-sigma cutoff.
RfactorNum. reflection% reflection
Rfree0.1917 2540 5.02 %
Rwork0.1699 48090 -
obs0.171 50630 99.96 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.1 Å / Solvent model: FLAT BULK SOLVENT MODEL
Displacement parametersBiso mean: 33.57 Å2
Refinement stepCycle: LAST / Resolution: 1.85→27.62 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms3223 0 26 335 3584
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.00793350
X-RAY DIFFRACTIONf_angle_d0.86284547
X-RAY DIFFRACTIONf_chiral_restr0.0567481
X-RAY DIFFRACTIONf_plane_restr0.007595
X-RAY DIFFRACTIONf_dihedral_angle_d14.52811224
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
1.85-1.890.40281270.31122650X-RAY DIFFRACTION99.96
1.89-1.920.29851270.27392642X-RAY DIFFRACTION99.96
1.92-1.970.26691270.25022642X-RAY DIFFRACTION99.89
1.97-2.010.27751270.23222616X-RAY DIFFRACTION99.89
2.01-2.060.24511270.22262642X-RAY DIFFRACTION99.93
2.06-2.120.26332540.21492528X-RAY DIFFRACTION100
2.12-2.180.24481270.20472650X-RAY DIFFRACTION100
2.18-2.250.24531270.19042653X-RAY DIFFRACTION100
2.25-2.330.22581270.18232653X-RAY DIFFRACTION99.96
2.33-2.420.19521270.18412677X-RAY DIFFRACTION100
2.42-2.530.2011270.18142658X-RAY DIFFRACTION100
2.53-2.670.19551270.17682684X-RAY DIFFRACTION99.96
2.67-2.830.17911270.17372676X-RAY DIFFRACTION100
2.83-3.050.21231270.18382701X-RAY DIFFRACTION99.96
3.05-3.360.17712170.16822620X-RAY DIFFRACTION99.96
3.36-3.840.16671640.15152688X-RAY DIFFRACTION99.89
3.85-4.840.13571270.12352774X-RAY DIFFRACTION100
4.84-27.620.18581270.15642936X-RAY DIFFRACTION99.9

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