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- PDB-8f6v: Crystal Structure of Nanobody VHH108 Bound to Its Antigen PA14 Cif -

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Basic information

Entry
Database: PDB / ID: 8f6v
TitleCrystal Structure of Nanobody VHH108 Bound to Its Antigen PA14 Cif
Components
  • CFTR inhibitory factor
  • Nanobody VHH108
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 speciesVicugna pacos (alpaca)
Pseudomonas aeruginosa PA14 (bacteria)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 2.3 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 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 1.1Oct 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: Nanobody VHH108
D: CFTR inhibitory factor
B: Nanobody VHH108
C: CFTR inhibitory factor


Theoretical massNumber of molelcules
Total (without water)96,6594
Polymers96,6594
Non-polymers00
Water4,810267
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration, Cif is an obligate homodimer. The biological assembly contains one Cif dimer and two nanobody VHH108 molecules.
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Unit cell
Length a, b, c (Å)98.995, 98.995, 196.987
Angle α, β, γ (deg.)90.000, 90.000, 120.000
Int Tables number169
Space group name H-MP61
Space group name HallP61
Symmetry operation#1: x,y,z
#2: x-y,x,z+1/6
#3: y,-x+y,z+5/6
#4: -y,x-y,z+1/3
#5: -x+y,-x,z+2/3
#6: -x,-y,z+1/2

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Components

#1: Antibody Nanobody VHH108


Mass: 14164.782 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 10X-His-SUMO fusion / Production host: Escherichia coli BL21 (bacteria) / Variant (production host): DE3 RIL
#2: 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
#3: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 267 / 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: 2.89 Å3/Da / Density % sol: 57.41 %
Crystal growTemperature: 292.8 K / Method: vapor diffusion / pH: 6.5
Details: Morpheus I Condition G2 (composition by Molecular Dimensions stock solutions with catalogue numbers listed at end) 20% (v/v) ethylene glycol, 10% (w/v) PEG8000, 20 mM sodium formate, 20 mM ...Details: Morpheus I Condition G2 (composition by Molecular Dimensions stock solutions with catalogue numbers listed at end) 20% (v/v) ethylene glycol, 10% (w/v) PEG8000, 20 mM sodium formate, 20 mM ammonium acetate, 20 mM sodium citrate tribasic dihydrate, 20 mM potassium sodium tartrate tetrahydrate, 20 mM sodium oxamate, 100 mM imidazole/MES monohydrate (acid) pH 6.5 Stock mixtures listed below with catalogue number in brackets. 30% (v/v) Precipitant Mix 2 [MD2-100-82], 100 mM carboxylic acids mix [MD2-100-76], 100 mM Buffer System 1 pH 6.5 [MD2-100-100]

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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: 0.979312 Å
DetectorType: DECTRIS EIGER X 16M / Detector: PIXEL / Date: Dec 7, 2020
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.979312 Å / Relative weight: 1
ReflectionResolution: 2.3→44.23 Å / Num. obs: 48028 / % possible obs: 99.26 % / Redundancy: 2.9 % / Biso Wilson estimate: 49.93 Å2 / CC1/2: 0.991 / CC star: 0.998 / Rmerge(I) obs: 0.08607 / Rpim(I) all: 0.05634 / Rrim(I) all: 0.1034 / Net I/σ(I): 7.36
Reflection shellResolution: 2.3→2.382 Å / Redundancy: 2.4 % / Rmerge(I) obs: 0.7833 / Mean I/σ(I) obs: 1.28 / Num. unique obs: 4792 / CC1/2: 0.425 / CC star: 0.772 / Rpim(I) all: 0.6205 / Rrim(I) all: 1.004 / % possible all: 99.79

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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, 8E1B

3kd2

8e1b


Resolution: 2.3→44.23 Å / SU ML: 0.2789 / Cross valid method: FREE R-VALUE / σ(F): 1.35 / Phase error: 24.5748
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
Rfree0.2098 2417 5.03 %
Rwork0.1813 45603 -
obs0.1827 48020 99.27 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.1 Å / Solvent model: FLAT BULK SOLVENT MODEL
Displacement parametersBiso mean: 57.77 Å2
Refinement stepCycle: LAST / Resolution: 2.3→44.23 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms6554 0 0 267 6821
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.00916740
X-RAY DIFFRACTIONf_angle_d0.97689141
X-RAY DIFFRACTIONf_chiral_restr0.0595960
X-RAY DIFFRACTIONf_plane_restr0.0081204
X-RAY DIFFRACTIONf_dihedral_angle_d14.12382449
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
2.3-2.350.3461270.29062716X-RAY DIFFRACTION99.86
2.35-2.40.33951220.28512697X-RAY DIFFRACTION99.68
2.4-2.450.30591260.26632682X-RAY DIFFRACTION99.43
2.45-2.520.29382390.25532587X-RAY DIFFRACTION99.12
2.52-2.580.26351220.25092679X-RAY DIFFRACTION99.26
2.58-2.660.24211190.24322703X-RAY DIFFRACTION98.84
2.66-2.740.2591220.23412657X-RAY DIFFRACTION97.58
2.75-2.840.27521190.21562677X-RAY DIFFRACTION98.76
2.84-2.960.31091230.22212656X-RAY DIFFRACTION98.02
2.96-3.090.25462500.2192604X-RAY DIFFRACTION99.27
3.09-3.250.23381220.20342679X-RAY DIFFRACTION99.79
3.25-3.460.1991240.1982731X-RAY DIFFRACTION99.9
3.46-3.730.21771220.1742739X-RAY DIFFRACTION99.93
3.73-4.10.19361210.16272703X-RAY DIFFRACTION99.68
4.1-4.690.1561190.13152735X-RAY DIFFRACTION99.79
4.69-5.910.16962320.14272606X-RAY DIFFRACTION99.51
5.91-44.230.15991080.15252752X-RAY DIFFRACTION99.24
Refinement TLS params.

Method: refined / Refine-ID: X-RAY DIFFRACTION

IDL11 (°2)L12 (°2)L13 (°2)L22 (°2)L23 (°2)L33 (°2)S11 (Å °)S12 (Å °)S13 (Å °)S21 (Å °)S22 (Å °)S23 (Å °)S31 (Å °)S32 (Å °)S33 (Å °)T11 (Å2)T12 (Å2)T13 (Å2)T22 (Å2)T23 (Å2)T33 (Å2)Origin x (Å)Origin y (Å)Origin z (Å)
13.293794008950.373258936276-0.09642419893262.16740285846-0.3522196818752.102816358410.0286132417895-0.0587122395740.620763131714-0.1595941608530.0108041224455-0.302707237939-0.4206950318460.473608040148-0.04480495513970.438028600785-0.05024278643670.04178513411540.467752408017-0.07724140977370.48683896984215.1284861984-21.7277140229-9.99865133698
22.400122557240.854371900735-0.101078246842.27200516645-0.8275250934481.8720666388-0.0542431725804-0.309907158390.1225868410370.2640483843560.0161797496191-0.554950717188-0.5093613902460.2874166040330.0284911177850.662834520940.006616759209780.0002129696942010.491547371628-0.1107208955850.563268177076-13.679376892322.934710120610.7185533583
31.73066921262-0.521183830168-0.3400005118311.620777607790.1216331248431.250698896180.06324899081280.057802083841-0.378583345428-0.06476048548320.007162415958990.2734315965020.21136668464-0.134930200101-0.06632029594070.397537445278-0.0228104901082-0.08804689096080.3538773818040.003351455221350.440237779514-19.1964176059-35.3218746971-7.60524300705
41.5385590412-0.3334994529140.005534983171142.142461587690.3312517771971.05120347898-0.0253228420846-0.179062950369-0.06104365169320.1904920140730.08077260045430.454490232966-0.11033244407-0.366245498521-0.05748428336980.3668969586220.05971311191240.05649416879390.4823003907410.0701192716470.389229321184-40.1209260472-2.461200925138.1645534288
Refinement TLS group

Refine-ID: X-RAY DIFFRACTION

IDRefine TLS-IDSelection detailsAuth asym-IDLabel asym-IDAuth seq-IDLabel seq-ID
11Chain AAA4 - 1261 - 123
22Chain BBC4 - 1251 - 122
33Chain CCD26 - 3191 - 294
44Chain DDB26 - 3191 - 294

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