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- PDB-8f6u: Crystal Structure of Nanobody VHH113 Bound to Its Antigen PA14 Cif -

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Basic information

Entry
Database: PDB / ID: 8f6u
TitleCrystal Structure of Nanobody VHH113 Bound to Its Antigen PA14 Cif
Components
  • CFTR inhibitory factor
  • Nanobody VHH113
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 / hydrolase activity / CFTR inhibitory factor
Function and homology information
Biological speciesPseudomonas aeruginosa PA14 (bacteria)
Vicugna pacos (alpaca)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 1.7 Å
AuthorsSimard, A.R. / Taher, N.M. / Mishra, A.K. / 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 VHH113 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
DepositionNov 17, 2022Deposition site: RCSB / Processing site: RCSB
Revision 1.0Mar 13, 2024Provider: repository / Type: Initial release
Revision 1.1Nov 6, 2024Group: Structure summary / Category: pdbx_entry_details / pdbx_modification_feature

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
D: CFTR inhibitory factor
B: Nanobody VHH113
C: CFTR inhibitory factor
A: Nanobody VHH113


Theoretical massNumber of molelcules
Total (without water)101,0114
Polymers101,0114
Non-polymers00
Water14,124784
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: gel filtration, Cif is an obligate homodimer. The biological assembly contains one Cif dimer and two nanobody VHH113 molecules.
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area7020 Å2
ΔGint-38 kcal/mol
Surface area29830 Å2
MethodPISA
Unit cell
Length a, b, c (Å)115.185, 185.941, 106.753
Angle α, β, γ (deg.)90.000, 90.000, 90.000
Int Tables number20
Space group name H-MC2221
Space group name HallC2c2
Symmetry operation#1: x,y,z
#2: x,-y,-z
#3: -x,y,-z+1/2
#4: -x,-y,z+1/2
#5: x+1/2,y+1/2,z
#6: x+1/2,-y+1/2,-z
#7: -x+1/2,y+1/2,-z+1/2
#8: -x+1/2,-y+1/2,z+1/2
Components on special symmetry positions
IDModelComponents
11C-402-

HOH

21A-271-

HOH

31A-329-

HOH

41A-331-

HOH

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Components

#1: Protein CFTR inhibitory factor


Mass: 34164.699 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Pseudomonas aeruginosa PA14 (bacteria) / Gene: PA2394 / Plasmid: pDPM73 / Details (production host): C-terminal 6X-His / Production host: Escherichia coli (E. coli) / Strain (production host): TOP10 / References: UniProt: A0A0M3KL26
#2: Antibody Nanobody VHH113


Mass: 16340.880 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Vicugna pacos (alpaca) / Plasmid: pComb3X / Details (production host): C-terminal 6XHis and HA tag / Production host: Escherichia coli BL21 (bacteria) / Variant (production host): DE3 RIL
#3: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 784 / Source method: isolated from a natural source / Formula: H2O
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: 2.83 Å3/Da / Density % sol: 56.63 %
Crystal growTemperature: 292.8 K / Method: vapor diffusion, sitting drop / pH: 5
Details: 200 mM potassium bromide, 200 mM potassium thiocyanate, 100 mM sodium acetate pH 5, 3% (w/v) gamma-PGA (Na+ form, LM), 10% (w/v) PEG 2000 MME

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: NSLS-II / Beamline: 17-ID-2 / Wavelength: 0.979339 Å
DetectorType: DECTRIS EIGER X 16M / Detector: PIXEL / Date: Aug 14, 2018
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.979339 Å / Relative weight: 1
ReflectionResolution: 1.7→46.49 Å / Num. obs: 124313 / % possible obs: 99.06 % / Redundancy: 6.7 % / Biso Wilson estimate: 26.22 Å2 / CC1/2: 0.998 / CC star: 1 / Rmerge(I) obs: 0.1067 / Rpim(I) all: 0.04459 / Rrim(I) all: 0.1158 / Net I/σ(I): 11.8
Reflection shellResolution: 1.7→1.761 Å / Redundancy: 6.6 % / Rmerge(I) obs: 1.646 / Mean I/σ(I) obs: 1.26 / Num. unique obs: 12262 / CC1/2: 0.439 / CC star: 0.781 / Rpim(I) all: 0.6909 / Rrim(I) all: 1.788 / % possible all: 98.74

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Processing

Software
NameVersionClassification
PHENIX1.20.1_4487refinement
XDSdata reduction
XSCALEdata scaling
PHASERphasing
Cootmodel building
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 3KD2, 8E1C

3kd2

8e1c


Resolution: 1.7→46.49 Å / SU ML: 0.1918 / Cross valid method: FREE R-VALUE / σ(F): 1.33 / Phase error: 20.4094
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
Rfree0.1988 6228 5.01 %
Rwork0.1761 118069 -
obs0.1772 124297 99.07 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.1 Å / Solvent model: FLAT BULK SOLVENT MODEL
Displacement parametersBiso mean: 29.61 Å2
Refinement stepCycle: LAST / Resolution: 1.7→46.49 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms6527 0 0 784 7311
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.00636812
X-RAY DIFFRACTIONf_angle_d0.83899259
X-RAY DIFFRACTIONf_chiral_restr0.0546965
X-RAY DIFFRACTIONf_plane_restr0.0081221
X-RAY DIFFRACTIONf_dihedral_angle_d12.86772439
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
1.7-1.720.34081810.31423880X-RAY DIFFRACTION98.38
1.72-1.740.37632200.30183897X-RAY DIFFRACTION98.92
1.74-1.760.27121920.28443891X-RAY DIFFRACTION98.93
1.76-1.780.30572180.26483845X-RAY DIFFRACTION98.45
1.78-1.810.28712090.25483912X-RAY DIFFRACTION98.78
1.81-1.830.29852100.2583894X-RAY DIFFRACTION99.01
1.83-1.860.2712030.25733895X-RAY DIFFRACTION98.89
1.86-1.890.29712200.2463891X-RAY DIFFRACTION98.82
1.89-1.910.26412090.22693911X-RAY DIFFRACTION98.85
1.91-1.950.23131660.21243919X-RAY DIFFRACTION98.86
1.95-1.980.23592380.19523871X-RAY DIFFRACTION98.94
1.98-2.020.20511890.19453930X-RAY DIFFRACTION98.94
2.02-2.050.21042100.19193901X-RAY DIFFRACTION98.89
2.05-2.10.20982220.18643915X-RAY DIFFRACTION99.11
2.1-2.140.23561800.1813944X-RAY DIFFRACTION99.16
2.14-2.190.19342240.1763884X-RAY DIFFRACTION99.25
2.19-2.250.17992110.17223932X-RAY DIFFRACTION99.11
2.25-2.310.19352040.17123917X-RAY DIFFRACTION99.11
2.31-2.380.20942050.17083948X-RAY DIFFRACTION98.98
2.38-2.450.18852080.17873929X-RAY DIFFRACTION99.33
2.45-2.540.23332190.18123929X-RAY DIFFRACTION99.14
2.54-2.640.19612090.17713935X-RAY DIFFRACTION99.45
2.64-2.760.20141800.17743986X-RAY DIFFRACTION99.24
2.76-2.910.21222170.18183921X-RAY DIFFRACTION99.11
2.91-3.090.19962610.18363912X-RAY DIFFRACTION99.45
3.09-3.330.1689970.16664117X-RAY DIFFRACTION99.43
3.33-3.660.16832880.1563920X-RAY DIFFRACTION99.57
3.66-4.190.16122960.14713927X-RAY DIFFRACTION99.41
4.19-5.280.1852250.13444228X-RAY DIFFRACTION99.16
5.28-46.490.18113170.17744088X-RAY DIFFRACTION99.5

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