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- PDB-8eln: Crystal Structure of Nanobody VHH222 Bound to Its Antigen PA14 Cif -

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Basic information

Entry
Database: PDB / ID: 8eln
TitleCrystal Structure of Nanobody VHH222 Bound to Its Antigen PA14 Cif
Components
  • CFTR inhibitory factor
  • Nanobody VHH222
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 / CFTR inhibitory factor
Function and homology information
Biological speciesPseudomonas aeruginosa PA14 (bacteria)
Vicugna pacos (alpaca)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 2.4 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
CitationJournal: To Be Published
Title: Crystal Structure of Nanobody VHH222 Bound to Its Antigen PA14 Cif
Authors: Simard, A.R. / Madden, D.R.
History
DepositionSep 26, 2022Deposition site: RCSB / Processing site: RCSB
Revision 1.0Oct 18, 2023Provider: repository / Type: Initial release
Revision 1.1Oct 23, 2024Group: Structure summary / Category: pdbx_entry_details / pdbx_modification_feature

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: CFTR inhibitory factor
B: CFTR inhibitory factor
C: Nanobody VHH222
D: Nanobody VHH222
E: CFTR inhibitory factor
F: CFTR inhibitory factor
G: Nanobody VHH222
H: Nanobody VHH222
I: CFTR inhibitory factor
J: CFTR inhibitory factor
K: Nanobody VHH222
L: Nanobody VHH222


Theoretical massNumber of molelcules
Total (without water)286,17512
Polymers286,17512
Non-polymers00
Water11,097616
1
A: CFTR inhibitory factor
B: CFTR inhibitory factor
C: Nanobody VHH222
D: Nanobody VHH222


Theoretical massNumber of molelcules
Total (without water)95,3924
Polymers95,3924
Non-polymers00
Water724
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area7200 Å2
ΔGint-46 kcal/mol
Surface area29890 Å2
MethodPISA
2
E: CFTR inhibitory factor
F: CFTR inhibitory factor
G: Nanobody VHH222
H: Nanobody VHH222


Theoretical massNumber of molelcules
Total (without water)95,3924
Polymers95,3924
Non-polymers00
Water724
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area6900 Å2
ΔGint-48 kcal/mol
Surface area30280 Å2
MethodPISA
3
I: CFTR inhibitory factor
J: CFTR inhibitory factor
K: Nanobody VHH222
L: Nanobody VHH222


Theoretical massNumber of molelcules
Total (without water)95,3924
Polymers95,3924
Non-polymers00
Water724
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area7260 Å2
ΔGint-46 kcal/mol
Surface area30430 Å2
MethodPISA
Unit cell
Length a, b, c (Å)76.222, 164.326, 107.291
Angle α, β, γ (deg.)90.000, 97.540, 90.000
Int Tables number4
Space group name H-MP1211
Space group name HallP2yb
Symmetry operation#1: x,y,z
#2: -x,y+1/2,-z

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Components

#1: Protein
CFTR inhibitory factor


Mass: 34164.699 Da / Num. of mol.: 6
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 VHH222


Mass: 13531.143 Da / Num. of mol.: 6
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Vicugna pacos (alpaca) / Plasmid: pET16b / Details (production host): N-terminal 10X-His SUMO fusion / Production host: Escherichia coli BL21 (bacteria) / Variant (production host): DE3 RIL
#3: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 616 / 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: 2.33 Å3/Da / Density % sol: 47.25 %
Crystal growTemperature: 292.8 K / Method: vapor diffusion, sitting drop / pH: 5
Details: 20% (w/v) PEG4000, 200 mM ammonium acetate, 100 mM sodium acetate, pH 5

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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: 0.97933 Å
DetectorType: DECTRIS EIGER X 16M / Detector: PIXEL / Date: Oct 31, 2020
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.97933 Å / Relative weight: 1
ReflectionResolution: 2.4→47.44 Å / Num. obs: 101735 / % possible obs: 99.68 % / Redundancy: 3.5 % / Biso Wilson estimate: 38.39 Å2 / CC1/2: 0.988 / CC star: 0.997 / Rmerge(I) obs: 0.1614 / Rpim(I) all: 0.1002 / Rrim(I) all: 0.1904 / Net I/σ(I): 6.88
Reflection shellResolution: 2.4→2.486 Å / Redundancy: 3.6 % / Rmerge(I) obs: 1.013 / Mean I/σ(I) obs: 1.38 / Num. unique obs: 10117 / CC1/2: 0.486 / CC star: 0.809 / Rpim(I) all: 0.6169 / Rrim(I) all: 1.188 / % possible all: 99.6

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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: 2.4→47.44 Å / SU ML: 0.3079 / Cross valid method: FREE R-VALUE / σ(F): 1.36 / Phase error: 24.3397
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Details: Authors state that the 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- ...Details: Authors state that the 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. Although the correlation is low, residues listed as RSRZ outliers have reasonable concordance with the 2Fo-Fc map, are not rotamer outliers, and are devoid of clashes with neighboring atoms.
RfactorNum. reflection% reflection
Rfree0.2289 5080 4.99 %
Rwork0.1965 96625 -
obs0.1981 101705 99.7 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.1 Å / Solvent model: FLAT BULK SOLVENT MODEL
Displacement parametersBiso mean: 42.27 Å2
Refinement stepCycle: LAST / Resolution: 2.4→47.44 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms19544 0 0 616 20160
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.006720127
X-RAY DIFFRACTIONf_angle_d0.849427295
X-RAY DIFFRACTIONf_chiral_restr0.06042863
X-RAY DIFFRACTIONf_plane_restr0.00623573
X-RAY DIFFRACTIONf_dihedral_angle_d14.18447297
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
2.4-2.430.32612540.2883093X-RAY DIFFRACTION99.52
2.43-2.4600.29233439X-RAY DIFFRACTION99.54
2.46-2.490.34392540.2823077X-RAY DIFFRACTION99.73
2.49-2.520.30732540.27743120X-RAY DIFFRACTION99.59
2.52-2.5500.26853423X-RAY DIFFRACTION99.71
2.55-2.590.30352540.26593084X-RAY DIFFRACTION99.76
2.59-2.620.30192540.26833185X-RAY DIFFRACTION99.8
2.62-2.6600.25823336X-RAY DIFFRACTION99.76
2.66-2.70.30572540.2523141X-RAY DIFFRACTION99.76
2.7-2.750.27962540.24513108X-RAY DIFFRACTION99.61
2.75-2.7900.24833399X-RAY DIFFRACTION99.79
2.79-2.850.29832540.2513117X-RAY DIFFRACTION99.41
2.85-2.90.29672540.2553129X-RAY DIFFRACTION99.73
2.9-2.9600.24723366X-RAY DIFFRACTION99.76
2.96-3.020.28512540.24273152X-RAY DIFFRACTION99.62
3.02-3.090.25972540.23573101X-RAY DIFFRACTION99.67
3.09-3.1700.21723454X-RAY DIFFRACTION99.8
3.17-3.260.23852540.21593090X-RAY DIFFRACTION99.64
3.26-3.350.23322540.19873150X-RAY DIFFRACTION99.88
3.35-3.4600.20243382X-RAY DIFFRACTION99.68
3.46-3.580.24322540.19493139X-RAY DIFFRACTION99.88
3.58-3.730.21972540.18583113X-RAY DIFFRACTION99.76
3.73-3.900.17713422X-RAY DIFFRACTION99.94
3.9-4.10.20282540.16683134X-RAY DIFFRACTION99.88
4.1-4.360.18732540.15293155X-RAY DIFFRACTION99.8
4.36-4.700.14213412X-RAY DIFFRACTION99.85
4.7-5.170.1552540.14723156X-RAY DIFFRACTION99.77
5.17-5.920.21052540.16813135X-RAY DIFFRACTION99.5
5.92-7.4500.16973419X-RAY DIFFRACTION99.85
7.45-47.440.15432540.14173194X-RAY DIFFRACTION98.91

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