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- PDB-8evd: Crystal Structure of Nanobody VHH101 Bound to Its Antigen PA14 Cif -

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Basic information

Entry
Database: PDB / ID: 8evd
TitleCrystal Structure of Nanobody VHH101 Bound to Its Antigen PA14 Cif
Components
  • CFTR inhibitory factor
  • Nanobody VHH101
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 Å
AuthorsSimard, A.R. / Taher, N.M. / Beauchemin, K.S. / 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 VHH101 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.
History
DepositionOct 20, 2022Deposition site: RCSB / Processing site: RCSB
Revision 1.0Mar 13, 2024Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Nanobody VHH101
B: Nanobody VHH101
C: CFTR inhibitory factor
D: CFTR inhibitory factor
E: Nanobody VHH101
F: Nanobody VHH101
G: CFTR inhibitory factor
H: CFTR inhibitory factor
I: Nanobody VHH101
J: Nanobody VHH101
K: CFTR inhibitory factor
L: CFTR inhibitory factor


Theoretical massNumber of molelcules
Total (without water)302,22912
Polymers302,22912
Non-polymers00
Water16,610922
1
A: Nanobody VHH101
B: Nanobody VHH101
C: CFTR inhibitory factor
D: CFTR inhibitory factor


Theoretical massNumber of molelcules
Total (without water)100,7434
Polymers100,7434
Non-polymers00
Water724
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
2
E: Nanobody VHH101
F: Nanobody VHH101
G: CFTR inhibitory factor
H: CFTR inhibitory factor


Theoretical massNumber of molelcules
Total (without water)100,7434
Polymers100,7434
Non-polymers00
Water724
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
3
I: Nanobody VHH101
J: Nanobody VHH101
K: CFTR inhibitory factor
L: CFTR inhibitory factor


Theoretical massNumber of molelcules
Total (without water)100,7434
Polymers100,7434
Non-polymers00
Water724
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Unit cell
Length a, b, c (Å)188.960, 91.990, 151.215
Angle α, β, γ (deg.)90.000, 92.540, 90.000
Int Tables number5
Space group name H-MC121
Space group name HallC2y
Symmetry operation#1: x,y,z
#2: -x,y,-z
#3: x+1/2,y+1/2,z
#4: -x+1/2,y+1/2,-z
Components on special symmetry positions
IDModelComponents
11E-208-

HOH

21E-240-

HOH

Noncrystallographic symmetry (NCS)NCS domain:
IDEns-IDDetails
d_1ens_1(chain "A" and resid 4 through 127)
d_2ens_1chain "B"
d_3ens_1(chain "E" and resid 4 through 127)
d_4ens_1(chain "F" and resid 4 through 127)
d_5ens_1chain "I"
d_6ens_1(chain "J" and resid 4 through 127)
d_1ens_2(chain "C" and resid 26 through 317)
d_2ens_2(chain "D" and resid 26 through 317)
d_3ens_2chain "G"
d_4ens_2(chain "H" and resid 26 through 317)
d_5ens_2chain "K"
d_6ens_2(chain "L" and resid 26 through 317)

NCS domain segments:
Dom-IDComponent-IDEns-IDBeg label comp-IDEnd label comp-IDLabel asym-IDLabel seq-ID
d_11ens_1VALVALA1 - 124
d_21ens_1VALVALB1 - 124
d_31ens_1VALVALE2 - 125
d_41ens_1VALVALF2 - 125
d_51ens_1VALVALI1 - 124
d_61ens_1VALVALJ1 - 124
d_11ens_2GLUARGC1 - 292
d_21ens_2GLUARGD1 - 292
d_31ens_2GLUARGG1 - 292
d_41ens_2GLUARGH1 - 292
d_51ens_2GLUARGK1 - 292
d_61ens_2GLUARGL1 - 292

NCS ensembles :
ID
ens_1
ens_2

NCS oper:
IDCodeMatrixVector
1given(0.283713831033, -0.0358337709437, 0.958239220101), (-0.0675027950659, -0.997568767321, -0.0173184041803), (0.956530101324, -0.0597703549012, -0.285442936392)-55.8715551593, 40.1145195548, 77.1741214836
2given(-0.455935865267, -0.0192700154849, 0.889803997106), (0.00241073192437, 0.999735174694, 0.0228859968142), (-0.890009368004, 0.0125796256622, -0.455768667071)-71.8950023756, 30.7974269829, 33.0320820044
3given(0.695209523572, -0.00552328784966, -0.718785928929), (-0.0173170876721, -0.999808941548, -0.00906635957805), (-0.718598522688, 0.0187502984715, -0.695172345176)20.3959417449, 74.1096350774, 46.971463886
4given(-0.992574761016, 0.0286539908216, -0.118212912175), (-0.0189436874329, -0.996414050545, -0.0824631831921), (-0.120151905942, -0.0796114858907, 0.989558250338)-47.0134502217, 103.228340815, -1.93145202213
5given(-0.422283016996, 0.00100792299745, -0.906463478386), (-0.027406434431, 0.999528020313, 0.0138789034472), (0.906049634903, 0.0307037571047, -0.422056084415)-0.681099047854, 58.186229307, 77.9390136687
6given(0.287714327077, -0.0239093439697, 0.957417781987), (-0.019640784608, -0.999625386537, -0.019061116708), (0.957514859193, -0.0133202800689, -0.288076143692)-55.8609823009, 42.6962391465, 75.9069191859
7given(-0.482816460296, 0.00375457010857, 0.875713519863), (-0.0117733046681, 0.999872603932, -0.0107779963), (-0.875642424146, -0.015513836094, -0.482710747679)-73.4224323436, 31.1834728289, 35.3148876884
8given(0.711517243638, 0.00146026710139, -0.702667118645), (-0.0118626276449, -0.999830362523, -0.0140898631042), (-0.702568494932, 0.018360658946, -0.711379221043)20.4960341136, 74.5009679587, 48.2583556314
9given(-0.99090816391, 0.00567648711253, -0.134420192646), (0.00415802782021, -0.997340160151, -0.0727689202506), (-0.134475728299, -0.0726662400558, 0.988248903897)-45.7816055397, 104.289043974, -2.76023742463
10given(-0.401335403345, 0.0127329931668, -0.91584265292), (-0.0583306613512, 0.997518330443, 0.0394298665507), (0.91407189431, 0.0692463090344, -0.39959669758)0.427665300692, 55.4964876049, 76.5156421172

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Components

#1: Antibody
Nanobody VHH101


Mass: 16206.850 Da / Num. of mol.: 6
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Vicugna pacos (alpaca) / Plasmid: pComb3X / Details (production host): C-terminal 6X-His and HA tag / Production host: Escherichia coli BL21 (bacteria) / Variant (production host): DE3 RIL
#2: 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
#3: Water ChemComp-HOH / water / Water


Mass: 18.015 Da / Num. of mol.: 922 / Source method: isolated from a natural source / Formula: H2O

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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: 2.18 Å3/Da / Density % sol: 43.72 % / Description: Two crystals growing together
Crystal growTemperature: 292.8 K / Method: vapor diffusion, sitting drop / pH: 8.5
Details: 15% (v/v) isopropanol, 1 M ammonium citrate/ammonium hydroxide pH 8.5

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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: 0.979339 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: 2→47.19 Å / Num. obs: 165194 / % possible obs: 94.53 % / Redundancy: 3.6 % / Biso Wilson estimate: 35.12 Å2 / CC1/2: 0.997 / CC star: 0.999 / Rmerge(I) obs: 0.08194 / Rpim(I) all: 0.04854 / Rrim(I) all: 0.09597 / Net I/σ(I): 10.57
Reflection shellResolution: 2→2.071 Å / Redundancy: 3.6 % / Rmerge(I) obs: 0.9025 / Mean I/σ(I) obs: 2.02 / Num. unique obs: 16964 / CC1/2: 0.539 / CC star: 0.837 / Rpim(I) all: 0.5297 / Rrim(I) all: 1.054 / % possible all: 97.76

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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: 3KD2, 8E2N

3kd2

8e2n


Resolution: 2→47.19 Å / SU ML: 0.2232 / Cross valid method: FREE R-VALUE / σ(F): 1.34 / Phase error: 21.309
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
Rfree0.2061 8270 5.01 %
Rwork0.1844 156862 -
obs0.1855 165132 94.54 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.1 Å / Solvent model: FLAT BULK SOLVENT MODEL
Displacement parametersBiso mean: 40.94 Å2
Refinement stepCycle: LAST / Resolution: 2→47.19 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms19650 0 0 922 20572
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.008920184
X-RAY DIFFRACTIONf_angle_d0.965927376
X-RAY DIFFRACTIONf_chiral_restr0.06192868
X-RAY DIFFRACTIONf_plane_restr0.00693580
X-RAY DIFFRACTIONf_dihedral_angle_d13.80277218
Refine LS restraints NCS
Ens-IDDom-IDAuth asym-IDRefine-IDTypeRms dev position (Å)
ens_1d_2AX-RAY DIFFRACTIONTorsion NCS0.551149538015
ens_1d_3AX-RAY DIFFRACTIONTorsion NCS0.640424790101
ens_1d_4AX-RAY DIFFRACTIONTorsion NCS0.548036115083
ens_1d_5AX-RAY DIFFRACTIONTorsion NCS0.550343369998
ens_1d_6AX-RAY DIFFRACTIONTorsion NCS0.496557465483
ens_2d_2CX-RAY DIFFRACTIONTorsion NCS0.575369916819
ens_2d_3CX-RAY DIFFRACTIONTorsion NCS0.663678017136
ens_2d_4CX-RAY DIFFRACTIONTorsion NCS0.57949942877
ens_2d_5CX-RAY DIFFRACTIONTorsion NCS0.692809768736
ens_2d_6CX-RAY DIFFRACTIONTorsion NCS0.646015703174
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
2-2.020.30242480.27925446X-RAY DIFFRACTION98.05
2.02-2.050.29923050.26385339X-RAY DIFFRACTION97.94
2.05-2.070.30212440.25675379X-RAY DIFFRACTION97.3
2.07-2.10.26853390.24985384X-RAY DIFFRACTION97.65
2.1-2.130.26542760.23795348X-RAY DIFFRACTION97.47
2.13-2.150.24982580.23485375X-RAY DIFFRACTION97.31
2.15-2.190.25343050.22735340X-RAY DIFFRACTION97.08
2.19-2.220.26612670.23085330X-RAY DIFFRACTION97.05
2.22-2.250.25772800.22325318X-RAY DIFFRACTION96.75
2.25-2.290.23952980.21995362X-RAY DIFFRACTION96.72
2.29-2.330.25492730.22075315X-RAY DIFFRACTION96.73
2.33-2.370.24532810.21465272X-RAY DIFFRACTION96.24
2.37-2.420.23122450.21025322X-RAY DIFFRACTION95.65
2.42-2.470.25392860.21895240X-RAY DIFFRACTION95.57
2.47-2.520.25043010.21555260X-RAY DIFFRACTION95.71
2.52-2.580.23182690.20795277X-RAY DIFFRACTION95.55
2.58-2.640.25662680.21075234X-RAY DIFFRACTION94.33
2.64-2.710.25472660.20165225X-RAY DIFFRACTION94.53
2.71-2.790.20823400.19875252X-RAY DIFFRACTION95.95
2.79-2.880.22072010.19475330X-RAY DIFFRACTION95.38
2.88-2.990.22612830.19555265X-RAY DIFFRACTION95.2
2.99-3.110.21193710.19375145X-RAY DIFFRACTION94.74
3.11-3.250.1861450.1725261X-RAY DIFFRACTION93.42
3.25-3.420.19623240.1715113X-RAY DIFFRACTION92.56
3.42-3.630.16843880.16364959X-RAY DIFFRACTION91.42
3.63-3.910.219330.15475240X-RAY DIFFRACTION90.48
3.91-4.310.17493900.15234788X-RAY DIFFRACTION88.29
4.31-4.930.15523900.1434726X-RAY DIFFRACTION87.07
4.93-6.2100.17065146X-RAY DIFFRACTION87.25
6.21-47.190.19273960.1684871X-RAY DIFFRACTION87.55
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)
11.818754120370.0456202920873-0.5475984679532.01422836224-0.1759098264222.12321253402-0.0219398191002-0.247821435106-0.1149460068730.3249121676610.05635858388830.03354235870630.298841468714-0.131759288373-0.02826086475550.279410830508-0.0152487912932-0.002633564444440.246614417730.009053205627890.1719585231-56.388622414419.945356326850.4021464031
21.878668722640.005212132620650.09901459886662.62700769425-0.198219443972.22503103850.1015727880010.2040798401690.36324068171-0.342102638564-0.0189697759358-0.573842817699-0.2487554775480.323337675242-0.07994318071030.2832210409050.00453252090110.1053891455730.2543833026990.01458378933450.370767168596-24.354438618823.03031009057.6967042967
31.05601284903-0.132022136276-0.03791933650341.851925318670.03232609977071.271453375560.02173982731690.02955021050720.160178313878-0.0296259293482-0.0249882308588-0.0428407899093-0.1818535060070.01543919328380.001026585838710.2484438164990.00195168327216-0.006237105933520.2423278291680.005480306770140.21949047382-50.361666220142.647325702821.7162098427
41.36068275063-0.206628346412-0.05144185071731.910584571810.07874787443860.840416329579-0.00838558069129-0.0506430928101-0.2292502417280.0918412543769-0.05271314231640.14029022070.18940005973-0.05996834630090.06498514588570.253523219454-0.01456753016680.0211742634180.229362823858-0.002180697939340.252384307782-50.57789295810.64185759311920.8613867996
53.08812123378-0.7184061573030.2220659316472.89826815803-0.004468335718092.76803490160.05881264288770.292616614932-0.375976552485-0.298541526556-0.120843945947-0.438758023490.2339995818560.4684905656880.02991120584820.2528301669660.01681720649380.05340981801630.3689820281920.0221688638250.350232177332-1.6914240134251.912176563560.5753319624
62.94564063527-0.0215538013623-0.1085223422173.17275433826-0.1786953555532.6717506751-0.08176763481670.1021666237110.320453631892-0.27812901553-0.07001249436790.544312656214-0.396790423104-0.4892123222610.1342810088080.3221235667920.0479261154102-0.07475796339110.322851344687-0.07701842756280.301354318438-55.264054307954.666108641752.8342362842
72.616817218840.00486972246247-0.3237973297682.7814726857-0.02561497183491.411045605440.0614301671592-0.1154926168240.6907315125250.104968040807-0.0441106081394-0.0413582866389-0.311472466878-0.0124317403986-0.02017656524760.2568876016310.001111877795490.006013274727150.261736627094-0.04652054292620.350075900779-29.974629704774.057821641168.3414187057
81.265849479540.4058514739840.2587765061012.433345217240.1932828688781.258952810340.0523865760165-0.120570201517-0.2749263200040.158357745441-0.0324534959141-0.3061624045030.203432682650.107361460597-0.017422672160.2636293747710.00387885463705-0.02839384387270.274671717570.009814148905730.300734385118-30.533869859432.153512537568.9733880903
92.464827320190.1085137157890.3103508406633.06673516372-0.3485390565111.784067954110.103637815766-0.597870170790.170089862320.878721857372-0.197719293054-0.0985192112202-0.4726803878330.09266358572090.1151845258210.529008865518-0.0878516311883-0.02004394515740.54285948980.03622097231790.3068294494343.5575001353580.152134554153.0455106209
101.787036386580.6375964209730.8147232852582.629724979710.386235955282.327067762070.043386275538-0.0146795600245-0.103113815064-0.03759170791110.0588478478728-0.027356900780.0899384377101-0.0992259569016-0.08380308808140.1549846024450.006376214374970.008619836703950.226634142714-0.002098439320150.168411100746-22.605954430780.23348905616.14696635607
111.55441666328-0.311421131539-0.01064567939492.119873906050.2407777901521.88356001402-0.0452041436114-0.099779050418-0.4178654631340.0825108990376-0.0548888022381-0.008729875601820.4119772260110.01138058411410.09532430065110.2900882657560.008185598223330.02933376927420.2988498432280.05385470205870.3479036932261.4939194311160.098417478822.317960058
121.52904082842-0.576585773315-0.05814583542972.44726170581-0.2346706885191.236297004040.0557940954034-0.2660757276150.4021791199880.300564078357-0.0599615113211-0.342668613056-0.2622657402550.239702030596-0.003717119811380.266546176892-0.0687246281344-0.02649468833850.321309424537-0.04136337421660.3323785320631.29406662914101.83081874624.7573412931
Refinement TLS group

Refine-ID: X-RAY DIFFRACTION

IDRefine TLS-IDSelection detailsAuth asym-IDLabel asym-IDAuth seq-IDLabel seq-ID
11Chain 'A'AA4 - 1281 - 125
22Chain 'B'BB4 - 1271 - 124
33Chain 'C'CC26 - 3191 - 294
44Chain 'D'DD26 - 3191 - 294
55Chain 'E'EE3 - 1281 - 126
66Chain 'F'FF3 - 1291 - 127
77Chain 'G'GG26 - 3171 - 292
88Chain 'H'HH26 - 3191 - 294
99Chain 'I'II4 - 1271 - 124
1010Chain 'J'JJ4 - 1291 - 126
1111Chain 'K'KK26 - 3171 - 292
1212Chain 'L'LL26 - 3191 - 294

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