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- PDB-8e2n: Crystal Structure of Nanobody VHH113 Specific for PA14 Cif -

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Basic information

Entry
Database: PDB / ID: 8e2n
TitleCrystal Structure of Nanobody VHH113 Specific for PA14 Cif
ComponentsNanobody VHH113
KeywordsIMMUNE SYSTEM / Pseudomonas aeruginosa / nanobody VHH / immunoglobulin domain / CFTR inhibitory factor (Cif)
Biological speciesVicugna pacos (alpaca)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 1.1 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 VHH113 Specific for PA14 Cif
Authors: Simard, A.R. / Madden, D.R.
#1: 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.
#2: 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.
History
DepositionAug 15, 2022Deposition site: RCSB / Processing site: RCSB
Revision 1.0Oct 4, 2023Provider: repository / Type: Initial release
Revision 1.1Nov 20, 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
B: Nanobody VHH113
A: Nanobody VHH113


Theoretical massNumber of molelcules
Total (without water)28,1372
Polymers28,1372
Non-polymers00
Water5,765320
1
B: Nanobody VHH113


Theoretical massNumber of molelcules
Total (without water)14,0691
Polymers14,0691
Non-polymers00
Water181
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
2
A: Nanobody VHH113


Theoretical massNumber of molelcules
Total (without water)14,0691
Polymers14,0691
Non-polymers00
Water181
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Unit cell
Length a, b, c (Å)51.083, 51.607, 75.991
Angle α, β, γ (deg.)90.000, 90.000, 90.000
Int Tables number19
Space group name H-MP212121
Space group name HallP2ac2ab
Symmetry operation#1: x,y,z
#2: x+1/2,-y+1/2,-z
#3: -x,y+1/2,-z+1/2
#4: -x+1/2,-y,z+1/2

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Components

#1: Antibody Nanobody VHH113


Mass: 14068.520 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Vicugna pacos (alpaca) / Plasmid: pET16b / Details (production host): N-terminal SUMO fusion / Production host: Escherichia coli BL21 (bacteria) / Variant (production host): DE3 RIL
#2: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 320 / 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: 1.78 Å3/Da / Density % sol: 31 %
Description: Crystals were first observed 104 days after setting drop. Drop appeared to to have partially evaporated and concentrations of crystallization buffer are likely higher than starting conditions.
Crystal growTemperature: 292.8 K / Method: vapor diffusion, sitting drop / Details: 17% (w/v) PEG3350, 150 mM magnesium formate

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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: 0.979106 Å
DetectorType: DECTRIS EIGER X 16M / Detector: PIXEL / Date: Apr 5, 2022
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.979106 Å / Relative weight: 1
ReflectionResolution: 1.1→42.39 Å / Num. obs: 81669 / % possible obs: 99.23 % / Redundancy: 6.1 % / Biso Wilson estimate: 10.71 Å2 / CC1/2: 0.999 / CC star: 1 / Rmerge(I) obs: 0.09307 / Rpim(I) all: 0.04051 / Rrim(I) all: 0.1017 / Net I/σ(I): 8.89
Reflection shellResolution: 1.1→1.139 Å / Redundancy: 3.5 % / Rmerge(I) obs: 1.343 / Mean I/σ(I) obs: 0.78 / Num. unique obs: 7655 / CC1/2: 0.524 / CC star: 0.829 / Rpim(I) all: 0.8083 / Rrim(I) all: 1.578 / % possible all: 94.23

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Processing

Software
NameVersionClassification
XDSdata reduction
XSCALEdata scaling
PHASERphasing
Cootmodel building
PHENIX1.20.1_4487refinement
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: Structure of nanobody VHH113 complexed with it's antigen Cif (to be deposited)

Resolution: 1.1→42.39 Å / SU ML: 0.1479 / Cross valid method: FREE R-VALUE / σ(F): 1.34 / Phase error: 26.583
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Details: Although the correlation is low, residue 1 of Chain B has reasonable concordance with the 2Fo-Fc map, is not a rotamer outlier, and is devoid of clashes with neighboring atoms. Residues 129: ...Details: Although the correlation is low, residue 1 of Chain B has reasonable concordance with the 2Fo-Fc map, is not a rotamer outlier, and is devoid of clashes with neighboring atoms. Residues 129:131 of Chain A 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 at which alternative conformations of the main-chain are observed.
RfactorNum. reflection% reflectionSelection details
Rfree0.2126 4114 5.05 %rfree reflections assigned in thin resolution shells
Rwork0.1928 77418 --
obs0.1938 81532 99.23 %-
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL
Displacement parametersBiso mean: 15.34 Å2
Refinement stepCycle: LAST / Resolution: 1.1→42.39 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms1957 0 0 320 2277
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.00592089
X-RAY DIFFRACTIONf_angle_d0.85552845
X-RAY DIFFRACTIONf_chiral_restr0.0799304
X-RAY DIFFRACTIONf_plane_restr0.008380
X-RAY DIFFRACTIONf_dihedral_angle_d18.4948734
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
1.1-1.110.43611850.41942385X-RAY DIFFRACTION91.82
1.11-1.130.3583720.38192533X-RAY DIFFRACTION93.84
1.13-1.140.39211310.35382615X-RAY DIFFRACTION97.51
1.14-1.160.34392050.32422586X-RAY DIFFRACTION99.36
1.16-1.170.2981910.29932697X-RAY DIFFRACTION99.57
1.17-1.190.31451160.27332688X-RAY DIFFRACTION99.72
1.19-1.210.28762110.27492582X-RAY DIFFRACTION99.71
1.21-1.220.23161070.25492693X-RAY DIFFRACTION99.64
1.22-1.250.32581030.24012676X-RAY DIFFRACTION99.68
1.25-1.270.26862070.24122572X-RAY DIFFRACTION99.68
1.27-1.290.25711250.22822691X-RAY DIFFRACTION99.79
1.29-1.310.305830.22012733X-RAY DIFFRACTION99.79
1.31-1.340.24662090.21662577X-RAY DIFFRACTION99.75
1.34-1.370.20631550.20152658X-RAY DIFFRACTION99.96
1.37-1.40.3435540.20022758X-RAY DIFFRACTION99.82
1.4-1.440.23172050.18962618X-RAY DIFFRACTION99.93
1.44-1.480.20281950.1742628X-RAY DIFFRACTION99.96
1.48-1.520.2964110.1632789X-RAY DIFFRACTION99.96
1.52-1.570.21822120.16742611X-RAY DIFFRACTION99.89
1.57-1.620.18592060.16342621X-RAY DIFFRACTION99.82
1.62-1.6900.16442826X-RAY DIFFRACTION99.61
1.69-1.770.2082070.16562644X-RAY DIFFRACTION99.86
1.77-1.860.1912080.16592614X-RAY DIFFRACTION99.86
1.86-1.9800.16112847X-RAY DIFFRACTION99.86
1.98-2.130.20712050.16522660X-RAY DIFFRACTION99.79
2.13-2.340.20392060.1722665X-RAY DIFFRACTION99.93
2.34-2.680.18061350.19472754X-RAY DIFFRACTION99.83
2.68-3.380.2158710.18192842X-RAY DIFFRACTION99.83
3.38-42.390.16541990.16652855X-RAY DIFFRACTION99.77

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