PDB-8ee2: Crystal Structure of Nanobody VHH219 Bound to Its Antigen PA14 Cif

ID or keywords:

Entry

Database: PDB / ID: 8ee2

Title

Crystal Structure of Nanobody VHH219 Bound to Its Antigen PA14 Cif

Components

CFTR inhibitory factor
Nanobody VHH219

Keywords

IMMUNE SYSTEM /

Pseudomonas aeruginosa / nanobody VHH /

immunoglobulin domain / CFTR inhibitory factor (Cif)

Function / homology

Alpha/beta hydrolase family /

Alpha/beta hydrolase fold-1 /

Alpha/Beta hydrolase fold /

CFTR inhibitory factor

Function and homology information

Biological species

Pseudomonas aeruginosa PA14 (bacteria)

Vicugna pacos (alpaca)

Method

X-RAY DIFFRACTION /

SYNCHROTRON /

MOLECULAR REPLACEMENT / Resolution: 2.4 Å

Authors

Simard, A.R. / Madden, D.R.

Funding support

United States, 5items

Organization	Grant number	Country
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 Foundation	STANTO19R0	United States

Citation

Journal: To Be Published
Title: Crystal Structure of Nanobody VHH219 Bound to Its Antigen PA14 Cif
Authors: Simard, A.R. / Madden, D.R.

History

Deposition	Sep 6, 2022	Deposition site: RCSB / Processing site: RCSB
Revision 1.0	Sep 13, 2023	Provider: repository / Type: Initial release

Structure viewer	Molecule: MolmilJmol/JSmol

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Download

PDBx/mmCIF format	8ee2.cif.gz	1 MB	Display	PDBx/mmCIF format
PDB format	pdb8ee2.ent.gz	799.7 KB	Display	PDB format
PDBx/mmJSON format	8ee2.json.gz		Tree view	PDBx/mmJSON format
Others	Other downloads

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Validation report

Arichive directory	https://data.pdbj.org/pub/pdb/validation_reports/ee/8ee2 ftp://data.pdbj.org/pub/pdb/validation_reports/ee/8ee2	HTTPS FTP

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Related structure data

Related structure data	3kd2S 8e1cS S: Starting model for refinement
Similar structure data	Similarity search - Function & homologyF&H Search Similarity search - HomologyF&H Search

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Links

PDB pages	PDBj / wwPDB / NCBI
Related items in Molecule of the Month	#21 - Sep 2001 Antibodies similarity (1) #256 - Apr 2021 SARS-CoV-2 Spike and Antibodies similarity (1) #165 - Sep 2013 Designed Protein Cages similarity (1) #220 - Apr 2018 Dehalogenases similarity (1)

Deposited unit

A: CFTR inhibitory factor

B: CFTR inhibitory factor

C: Nanobody VHH219

D: Nanobody VHH219

E: CFTR inhibitory factor

F: CFTR inhibitory factor

G: Nanobody VHH219

H: Nanobody VHH219

I: CFTR inhibitory factor

J: CFTR inhibitory factor

K: Nanobody VHH219

L: Nanobody VHH219

287 kDa, 12 polymers
Search similar-shape structures of this assembly by Omokage search (details)

1

A: CFTR inhibitory factor

B: CFTR inhibitory factor

C: Nanobody VHH219

D: Nanobody VHH219

defined by author&software
Evidence: gel filtration, Cif is an obligate homodimer. The biological assembly contains one Cif dimer and two nanobody VHH219 molecules.
95.6 kDa, 4 polymers
Search similar-shape structures of this assembly by Omokage search (details)

2

E: CFTR inhibitory factor

F: CFTR inhibitory factor

G: Nanobody VHH219

H: Nanobody VHH219

defined by author&software
Evidence: gel filtration, Cif is an obligate homodimer. The biological assembly contains one Cif dimer and two nanobody VHH219 molecules.
95.6 kDa, 4 polymers
Search similar-shape structures of this assembly by Omokage search (details)

3

I: CFTR inhibitory factor

J: CFTR inhibitory factor

K: Nanobody VHH219

L: Nanobody VHH219

defined by author&software
Evidence: gel filtration, Cif is an obligate homodimer. The biological assembly contains one Cif dimer and two nanobody VHH219 molecules.
95.6 kDa, 4 polymers
Search similar-shape structures of this assembly by Omokage search (details)

Unit cell

Length a, b, c (Å)	76.204, 162.599, 106.255
Angle α, β, γ (deg.)	90.000, 95.360, 90.000
Int Tables number	4
Space group name H-M	P12₁1
Space group name Hall	P2yb
Symmetry operation	#1: x,y,z #2: -x,y+1/2,-z

#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 VHH219 Mass: 13629.139 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 SUMO fusion / Production host: Escherichia coli BL21 (bacteria) / Variant (production host): DE3 RIL
#3: Water	ChemComp-HOH / water / Water Mass: 18.015 Da / Num. of mol.: 108 / Source method: isolated from a natural source / Formula: H₂O

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Experiment

Experiment	Method: X-RAY DIFFRACTION / Number of used crystals: 1

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Sample preparation

Crystal	Density Matthews: 2.18 Å³/Da / Density % sol: 43.71 %
Crystal grow	Temperature: 292.8 K / Method: vapor diffusion, hanging drop / pH: 4 Details: 14.5% (w/v) PEG3350, 375 mM sodium malonate pH 4, 5 mM cobalt (II) chloride

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Data collection

Diffraction	Mean temperature: 100 K / Serial crystal experiment: N
Diffraction source	Source: SYNCHROTRON / Site: NSLS-II / Beamline: 17-ID-2 / Wavelength: 0.979261 Å
Detector	Type: DECTRIS EIGER X 16M / Detector: PIXEL / Date: Dec 10, 2021
Radiation	Protocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelength	Wavelength: 0.979261 Å / Relative weight: 1
Reflection	Resolution: 2.4→43.75 Å / Num. obs: 99962 / % possible obs: 99.55 % / Redundancy: 3.5 % / B_iso Wilson estimate: 55.22 Å² / CC_1/2: 0.998 / CC star: 1 / R_merge(I) obs: 0.07373 / R_pim(I) all: 0.04621 / R_rim(I) all: 0.08721 / Net I/σ(I): 11.64
Reflection shell	Resolution: 2.4→2.486 Å / Redundancy: 3.5 % / R_merge(I) obs: 1.108 / Mean I/σ(I) obs: 1.25 / Num. unique obs: 9970 / CC_1/2: 0.501 / CC star: 0.817 / R_pim(I) all: 0.6915 / R_rim(I) all: 1.309 / % possible all: 99.89

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Processing

Software

Name	Version	Classification
XDS		data reduction
XSCALE		data scaling
PHASER		phasing
Coot		model building
PHENIX	1.20.1_4487	refinement

Refinement

Method to determine structure:

MOLECULAR REPLACEMENT
Starting model: 3KD2, 8E1C

3kd2

8e1c

Resolution: 2.4→43.75 Å / SU ML: 0.3735 / Cross valid method: FREE R-VALUE / σ(F): 1.36 / Phase error: 26.4988
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Details: Authors state that: The boundaries for stretches of missing residues were determined using a composite omit map with a one-sigma cutoff. Atoms modeled with zero occupancy could not be placed ...

	R_factor	Num. reflection	% reflection
R_free	0.2274	5000	5 %
R_work	0.1951	94942	-
obs	0.1967	99942	99.57 %

Solvent computation

Shrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL

Displacement parameters

B_iso mean: 69.39 Å²

Refinement step

Cycle: LAST / Resolution: 2.4→43.75 Å

	Protein	Nucleic acid	Ligand	Solvent	Total
Num. atoms	19052	0	0	108	19160

Refine LS restraints

Refine-ID	Type	Dev ideal	Number
X-RAY DIFFRACTION	f_bond_d	0.0086	19590
X-RAY DIFFRACTION	f_angle_d	1.0002	26579
X-RAY DIFFRACTION	f_chiral_restr	0.0585	2819
X-RAY DIFFRACTION	f_plane_restr	0.0097	3467
X-RAY DIFFRACTION	f_dihedral_angle_d	21.6788	7061

LS refinement shell

Resolution (Å)	R_factor R_free	Num. reflection R_free	R_factor R_work	Num. reflection R_work	Refine-ID	% reflection obs (%)
2.4-2.43	0.3595	250	0.3421	3076	X-RAY DIFFRACTION	99.88
2.43-2.46		0	0.3296	3364	X-RAY DIFFRACTION	99.88
2.46-2.49	0.3667	250	0.321	3030	X-RAY DIFFRACTION	99.94
2.49-2.52	0.3828	250	0.3129	3093	X-RAY DIFFRACTION	99.91
2.52-2.55		0	0.2951	3347	X-RAY DIFFRACTION	99.94
2.55-2.59	0.3267	250	0.2768	3077	X-RAY DIFFRACTION	99.88
2.59-2.62	0.3046	250	0.2704	3118	X-RAY DIFFRACTION	99.94
2.62-2.66		0	0.271	3299	X-RAY DIFFRACTION	99.91
2.66-2.7	0.3004	250	0.2559	3089	X-RAY DIFFRACTION	99.91
2.7-2.75	0.3057	250	0.2404	3038	X-RAY DIFFRACTION	99.97
2.75-2.79		0	0.2504	3348	X-RAY DIFFRACTION	99.76
2.79-2.85	0.2816	250	0.2542	3097	X-RAY DIFFRACTION	99.91
2.85-2.9	0.314	250	0.2566	3114	X-RAY DIFFRACTION	99.85
2.9-2.96		0	0.2603	3307	X-RAY DIFFRACTION	99.7
2.96-3.02	0.3176	250	0.2569	3067	X-RAY DIFFRACTION	99.73
3.02-3.09	0.3205	250	0.2502	3086	X-RAY DIFFRACTION	99.73
3.09-3.17		0	0.2201	3362	X-RAY DIFFRACTION	99.7
3.17-3.26	0.2353	250	0.216	3035	X-RAY DIFFRACTION	99.18
3.26-3.35	0.2531	250	0.2077	3064	X-RAY DIFFRACTION	99.28
3.35-3.46		0	0.1981	3364	X-RAY DIFFRACTION	99.26
3.46-3.58	0.2461	250	0.2064	3030	X-RAY DIFFRACTION	99.09
3.58-3.73	0.2221	250	0.1915	3078	X-RAY DIFFRACTION	99.46
3.73-3.9		0	0.1716	3314	X-RAY DIFFRACTION	99.19
3.9-4.1	0.185	250	0.1652	3066	X-RAY DIFFRACTION	99.13
4.1-4.36	0.1805	250	0.1601	3059	X-RAY DIFFRACTION	98.69
4.36-4.7		0	0.1465	3325	X-RAY DIFFRACTION	99.14
4.7-5.17	0.1688	250	0.148	3094	X-RAY DIFFRACTION	99.38
5.17-5.91	0.2062	250	0.1732	3128	X-RAY DIFFRACTION	99.7
5.91-7.45		0	0.1837	3343	X-RAY DIFFRACTION	99.43
7.45-43.75	0.1753	250	0.1514	3130	X-RAY DIFFRACTION	98.86

Refinement TLS params.

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

ID	L11 (°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)
1	2.66206065171	0.309002124181	0.500874220435	2.78821009773	-0.516581868903	2.70645711306	0.0963216750777	0.307288420477	-0.00967380919055	-0.981319017529	0.10246115504	0.851168869302	0.261405053584	-0.415746926569	-0.166540546301	0.73033740905	-0.0160906127228	-0.248252543468	0.38404361837	0.0467598569344	0.614114062153	1.20145771992	-21.8754467114	1.57903279344
2	4.02845203153	-0.951788089561	0.494985925578	3.43876731149	-0.106062922812	1.63564986306	0.159326076341	-0.0987941215142	0.0845069709795	-0.249967889563	-0.0414680740828	-0.842600807472	0.0853643311818	0.371797013729	-0.130582466966	0.401694218892	0.00897586922865	0.0354633660384	0.38172252355	0.0111397920564	0.543067145941	37.3182990858	-31.5576790575	20.9755427586
3	3.67999070348	0.298704532304	-1.1401632517	0.338245452529	-0.347560561017	2.84556104405	0.157450812007	0.436350981448	0.18330441134	-0.738626355923	-0.0464266412271	-0.513296326873	0.0243324340526	0.334795835063	-0.0707705897531	1.38235861971	0.158947374721	0.260544049033	0.538500917436	-0.0277585116132	0.719521039905	34.4541459319	-25.1365738463	-14.1431832074
4	8.45864116513	5.94372351549	-3.04243175256	6.10670182945	-2.80559058432	2.90678244109	0.0547299358323	-0.145053976049	0.268972258841	0.055087642328	0.0106375096464	0.401295682851	-0.162740246102	0.0180749521612	-0.0770604764183	0.412481192427	0.0204979456438	-0.0813959413643	0.272970319094	-0.0228915608809	0.363756337539	3.17563968566	-46.2657024515	27.7413707879
5	2.94002468689	0.652507854101	-0.32432922889	4.03420101694	0.734037922122	2.29397095662	0.0430738048067	-0.156761595394	-0.0353546191053	0.0886468475299	-0.0674886090128	0.319816494231	0.0719324036712	-0.338123136671	0.0216313385266	0.324751547867	0.0191018313319	-0.0423722262922	0.416350532159	-0.0371892408776	0.383581793141	-2.56089975951	-19.3765502984	47.1303286465
6	3.75668162656	0.312444762941	0.333929139459	2.63324887038	0.418107082487	1.94863465786	-0.029953037423	-0.280814934261	0.169878898779	0.180915491949	0.181539281639	-0.96372692014	-0.123326453659	0.777905196634	-0.124268928826	0.413863360651	-0.00887164145318	-0.0898218832653	0.72496540841	-0.187636403017	0.788014939301	34.1456624083	0.156259054535	52.7585431939
7	9.36324896441	-3.89466278112	5.25566971254	4.2000133055	-2.99809680594	5.2324901357	0.219704115975	0.374858788037	-0.285233361585	0.68053527399	-0.237798847463	-1.26005511051	0.834278356158	0.784534799908	-0.0752392132585	0.926563083421	0.0878849038114	-0.341842324296	0.66026277035	0.0418511517558	0.985593875395	26.1707859642	-34.4706975271	56.0717298771
8	8.71633526076	-6.05742057473	-5.56401900988	6.52909708295	4.00068982418	4.13834781889	-0.0572194227017	0.484859324597	-0.418683145886	-0.112780023263	-0.0733301190114	0.357584193325	0.120580586001	-0.186746454503	0.110940771854	0.451022277993	0.00381393684733	-0.121019643372	0.416168350723	-0.0544994335933	0.413886748001	0.0061913795182	12.9930045971	62.3902598282
9	4.47886987461	-0.331751682932	-0.0353708940682	2.22581098163	0.432665461313	2.73368419364	0.166098477775	-0.538701229285	0.1346047942	0.386443436922	-0.117976378018	0.482671876621	-0.257690340248	-0.407577322676	-0.0473409448786	0.584209037999	-0.0129650386311	0.0648170960414	0.412155355666	-0.0216184580484	0.480140314176	3.94087289025	16.8409648758	20.157575459
10	3.10493128346	-0.172019171564	0.00732053816638	2.76706099436	-0.183551372891	2.52207798617	0.117087807657	0.268926225605	-0.119463735904	-0.526540378158	-0.215079377237	-0.891136521461	0.00657011704235	0.564802620474	0.0920845617717	0.594307640589	0.0317281293866	0.122935805342	0.411570430356	0.076266882397	0.637829224362	39.7527228274	6.07045107299	0.979634562648
11	7.57373868061	0.953433964334	2.75019316105	3.39136832387	3.23801137022	4.73326768362	0.256383566288	-0.377025219533	0.0737590747139	0.343869090651	-0.230179160516	-0.642045005642	0.218919485151	0.315775009717	-0.0394920511033	0.701193305902	-0.12176762485	-0.0383431457477	0.478568524812	0.136205155442	0.624756261328	37.042126009	30.7073733718	26.0477086478
12	7.34566467725	-2.3115295414	3.96763523977	4.26517467684	-0.20117108105	6.91095205195	-0.02502213795	0.0609968755432	-0.741439219899	0.353860213024	0.129010167365	0.374692082641	0.857331653379	-0.544280192716	-0.129525273035	0.682741487539	-0.0090941533607	0.109625642045	0.440829455329	0.0600689300729	0.452724005638	5.13940595864	9.12943936857	-14.4250740032

Refinement TLS group

Refine-ID: X-RAY DIFFRACTION

ID	Refine TLS-ID	Selection details	Auth asym-ID	Label asym-ID	Auth seq-ID	Label seq-ID
1	1	chain 'A'	A	A	25 - 317	1 - 293
2	2	Chain 'B'	B	B	25 - 317	1 - 293
3	3	Chain 'C'	C	C	4 - 120	1 - 110
4	4	Chain 'D'	D	D	2 - 121	1 - 120
5	5	Chain 'E'	E	E	26 - 316	1 - 291
6	6	Chain 'F'	F	F	25 - 317	1 - 293
7	7	Chain 'G'	G	G	4 - 115	1 - 83
8	8	chain 'H'	H	H	3 - 121	1 - 119
9	9	Chain 'I'	I	I	25 - 317	1 - 293
10	10	Chain 'J'	J	J	25 - 316	1 - 292
11	11	Chain 'K'	K	K	3 - 119	1 - 108
12	12	Chain 'L'	L	L	3 - 121	1 - 119

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