PDB-8t8i: Structure of VHH-Fab complex with engineered Elbow FNQIKG, Crysta...

ID or keywords:

Entry

Database: PDB / ID: 8t8i

Title

Structure of VHH-Fab complex with engineered Elbow FNQIKG, Crystal Kappa and SER substitutions

Components

Fab heavy chainFragment antigen-binding
Fab light chainFragment antigen-binding
VHH domain

Keywords

IMMUNE SYSTEM / NabFab /

Nanobody /

Antibody Fragment

Function / homology

DI(HYDROXYETHYL)ETHER /

PHOSPHATE ION /

SUCCINIC ACID

Function and homology information

Biological species

Homo sapiens (human)

Method

X-RAY DIFFRACTION /

SYNCHROTRON /

MOLECULAR REPLACEMENT / Resolution: 2.52 Å

Authors

Filippova, E.V. / Thompson, I. / Kossiakoff, A.A.

Funding support

United States, 1items

Citation

Journal: Protein Sci. / Year: 2024
Title: Engineered antigen-binding fragments for enhanced crystallization of antibody:antigen complexes.
Authors: Bruce, H.A. / Singer, A.U. / Filippova, E.V. / Blazer, L.L. / Adams, J.J. / Enderle, L. / Ben-David, M. / Radley, E.H. / Mao, D.Y.L. / Pau, V. / Orlicky, S. / Sicheri, F. / Kurinov, I. / ...

History

Deposition	Jun 22, 2023	Deposition site: RCSB / Processing site: RCSB
Revision 1.0	Nov 29, 2023	Provider: repository / Type: Initial release
Revision 1.1	Jan 3, 2024	Group: Database references / Category: citation / citation_author Item: _citation.journal_volume / _citation.title ..._citation.journal_volume / _citation.title / _citation.year / _citation_author.name

Structure viewer	Molecule: MolmilJmol/JSmol

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Download

PDBx/mmCIF format	8t8i.cif.gz	237.1 KB	Display	PDBx/mmCIF format
PDB format	pdb8t8i.ent.gz	190.8 KB	Display	PDB format
PDBx/mmJSON format	8t8i.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/t8/8t8i ftp://data.pdbj.org/pub/pdb/validation_reports/t8/8t8i	HTTPS FTP

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Related structure data

Related structure data	8t58C 8t6iC 8t7fC 8t7gC 8t7iC 8t9yC 8trsC 8trtC 8ts5C C: citing same article (ref.)
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	#136 - Apr 2011 Nanobodies similarity (1) #21 - Sep 2001 Antibodies similarity (1) #256 - Apr 2021 SARS-CoV-2 Spike and Antibodies similarity (1)

Deposited unit

A: VHH domain

L: Fab light chain

H: Fab heavy chain

hetero molecules

63.4 kDa, 3 polymers
Search similar-shape structures of this assembly by Omokage search (details)

1

Idetical with deposited unit
defined by author&software

Unit cell

Length a, b, c (Å)	52.221, 72.771, 242.398
Angle α, β, γ (deg.)	90.00, 90.00, 90.00
Int Tables number	19
Space group name H-M	P2₁2₁2₁

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Antibody , 3 types, 3 molecules ALH

#1: Antibody	VHH domain Mass: 14170.781 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Homo sapiens (human) / Plasmid: PET26B+ / Production host: Escherichia coli BL21(DE3) (bacteria)
#2: Antibody	Fab light chain / Fragment antigen-binding Mass: 23018.434 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Details: "QGTTS" - Crystal Kappa substitution "Q149S" and "K150Y" - SER substitution Source: (gene. exp.) Homo sapiens (human) / Plasmid: RH2.2 / Production host: Escherichia coli BL21(DE3) (bacteria) / Variant (production host): C43
#3: Antibody	Fab heavy chain / Fragment antigen-binding Mass: 25753.609 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Details: "FNQIKG" - elbow region substitution / Source: (gene. exp.) Homo sapiens (human) / Plasmid: RH2.2 / Production host: Escherichia coli BL21(DE3) (bacteria) / Variant (production host): C43

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Non-polymers , 5 types, 76 molecules

#4: Chemical	ChemComp-SIN / SUCCINIC ACID / Succinic acid Mass: 118.088 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C₄H₆O₄ / Feature type: SUBJECT OF INVESTIGATION
#5: Chemical	ChemComp-PEG / DI(HYDROXYETHYL)ETHER / Diethylene glycol Mass: 106.120 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C₄H₁₀O₃ / Feature type: SUBJECT OF INVESTIGATION
#6: Chemical	ChemComp-PO4 / PHOSPHATE ION / Phosphate Mass: 94.971 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: PO₄ / Feature type: SUBJECT OF INVESTIGATION
#7: Chemical	ChemComp-CL / CHLORIDE ION / Chloride Mass: 35.453 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Cl / Feature type: SUBJECT OF INVESTIGATION
#8: Water	ChemComp-HOH / water / Water Mass: 18.015 Da / Num. of mol.: 70 / Source method: isolated from a natural source / Formula: H₂O

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Details

Has ligand of interest	Y

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Experiment

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

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Sample preparation

Crystal	Density Matthews: 3.72 Å³/Da / Density % sol: 66.92 %
Crystal grow	Temperature: 293 K / Method: vapor diffusion, hanging drop / pH: 5.6 Details: 2% Tacsimate pH 5.0, 0.1 M Na citrate tribasic dihydrate, 16% PEG 3350

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Data collection

Diffraction	Mean temperature: 100 K / Serial crystal experiment: N
Diffraction source	Source: SYNCHROTRON / Site: APS / Beamline: 24-ID-E / Wavelength: 0.97918 Å
Detector	Type: DECTRIS EIGER2 X 16M / Detector: PIXEL / Date: Nov 1, 2022
Radiation	Protocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelength	Wavelength: 0.97918 Å / Relative weight: 1
Reflection	Resolution: 2.52→121.2 Å / Num. obs: 32276 / % possible obs: 100 % / Redundancy: 13.1 % / CC_1/2: 0.99 / R_pim(I) all: 0.04 / Net I/σ(I): 12.5
Reflection shell	Resolution: 2.52→2.62 Å / Redundancy: 13.6 % / Mean I/σ(I) obs: 0.8 / Num. unique obs: 3603 / CC_1/2: 0.37 / R_pim(I) all: 0.78 / % possible all: 100

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Processing

Software

Name	Version	Classification
REFMAC	5.8.0258	refinement
XDS		data reduction
Aimless		data scaling
PHASER		phasing
PDB_EXTRACT		data extraction

Refinement

Method to determine structure:

MOLECULAR REPLACEMENT / Resolution: 2.52→121.2 Å / Cor.coef. F_o:F_c: 0.968 / Cor.coef. F_o:F_c free: 0.954 / SU B: 26.907 / SU ML: 0.253 / Cross valid method: THROUGHOUT / ESU R: 0.287 / ESU R Free: 0.237 / Stereochemistry target values: MAXIMUM LIKELIHOOD / Details: HYDROGENS HAVE BEEN USED IF PRESENT IN THE INPUT

	R_factor	Num. reflection	% reflection	Selection details
R_free	0.24291	1595	5 %	RANDOM
R_work	0.19126	-	-	-
obs	0.1938	30590	99.91 %	-

Solvent computation

Ion probe radii: 0.8 Å / Shrinkage radii: 0.8 Å / VDW probe radii: 1.2 Å / Solvent model: MASK

Displacement parameters

B_iso mean: 83.249 Å²

	B_aniso -1	B_aniso -2	B_aniso -3
1-	1.81 Å²	0 Å²	-0 Å²
2-	-	-1.52 Å²	0 Å²
3-	-	-	-0.29 Å²

Refinement step

Cycle: 1 / Resolution: 2.52→121.2 Å

	Protein	Nucleic acid	Ligand	Solvent	Total
Num. atoms	4319	0	27	71	4417

Refine LS restraints

Refine-ID	Type	Dev ideal	Dev ideal target	Number
X-RAY DIFFRACTION	r_bond_refined_d	0.004	0.012	4449
X-RAY DIFFRACTION	r_bond_other_d
X-RAY DIFFRACTION	r_angle_refined_deg	1.118	1.634	6049
X-RAY DIFFRACTION	r_angle_other_deg
X-RAY DIFFRACTION	r_dihedral_angle_1_deg	7.437	5	562
X-RAY DIFFRACTION	r_dihedral_angle_2_deg	36.245	22.335	197
X-RAY DIFFRACTION	r_dihedral_angle_3_deg	18.942	15	694
X-RAY DIFFRACTION	r_dihedral_angle_4_deg	16.48	15	20
X-RAY DIFFRACTION	r_chiral_restr	0.09	0.2	578
X-RAY DIFFRACTION	r_gen_planes_refined	0.005	0.02	3388
X-RAY DIFFRACTION	r_gen_planes_other
X-RAY DIFFRACTION	r_nbd_refined
X-RAY DIFFRACTION	r_nbd_other
X-RAY DIFFRACTION	r_nbtor_refined
X-RAY DIFFRACTION	r_nbtor_other
X-RAY DIFFRACTION	r_xyhbond_nbd_refined
X-RAY DIFFRACTION	r_xyhbond_nbd_other
X-RAY DIFFRACTION	r_metal_ion_refined
X-RAY DIFFRACTION	r_metal_ion_other
X-RAY DIFFRACTION	r_symmetry_vdw_refined
X-RAY DIFFRACTION	r_symmetry_vdw_other
X-RAY DIFFRACTION	r_symmetry_hbond_refined
X-RAY DIFFRACTION	r_symmetry_hbond_other
X-RAY DIFFRACTION	r_symmetry_metal_ion_refined
X-RAY DIFFRACTION	r_symmetry_metal_ion_other
X-RAY DIFFRACTION	r_mcbond_it	2.149	5.616	2257
X-RAY DIFFRACTION	r_mcbond_other
X-RAY DIFFRACTION	r_mcangle_it	3.59	8.42	2816
X-RAY DIFFRACTION	r_mcangle_other
X-RAY DIFFRACTION	r_scbond_it	2.561	5.77	2192
X-RAY DIFFRACTION	r_scbond_other
X-RAY DIFFRACTION	r_scangle_it
X-RAY DIFFRACTION	r_scangle_other
X-RAY DIFFRACTION	r_long_range_B_refined	6.936	73.574	6184
X-RAY DIFFRACTION	r_long_range_B_other
X-RAY DIFFRACTION	r_rigid_bond_restr
X-RAY DIFFRACTION	r_sphericity_free
X-RAY DIFFRACTION	r_sphericity_bonded

LS refinement shell

Resolution: 2.52→2.586 Å / Total num. of bins used: 20

	R_factor	Num. reflection	% reflection
R_free	0.441	117	-
R_work	0.414	2219	-
obs	-	-	99.19 %

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	2.1357	-0.2241	2.6319	0.7147	-3.3102	16.6598	0.1124	-0.3654	0.1443	-0.1639	-0.0905	0.043	0.8193	-0.0258	-0.0219	0.1979	-0.0225	0.0132	0.3685	0.0118	0.2509	-21.7723	4.3825	0.9955
2	4.7387	3.7611	-0.8521	7.8558	-3.5353	2.3619	-0.0149	-0.2376	0.1014	0.1548	-0.0428	0.0722	-0.2481	-0.1618	0.0577	0.1346	0.0356	0.0586	0.346	-0.0515	0.0887	-19.6529	21.2295	1.691
3	1.5595	-1.2802	0.7249	3.0418	-0.0112	4.455	0.2189	-0.343	-0.0277	0.1955	-0.0602	0.1894	0.3662	-0.1953	-0.1587	0.1089	-0.1174	0.0189	0.5307	0.0191	0.0291	-11.9436	12.4673	7.6871
4	2.6379	-0.7433	1.877	6.4016	-3.2151	4.1613	0.3459	0.0976	-0.3937	-0.0864	0.0509	0.2195	0.5244	-0.2642	-0.3968	0.2185	-0.0487	-0.0232	0.3107	-0.0196	0.0774	-7.8635	6.9764	3.5147
5	1.4724	1.1492	-0.3703	12.0026	0.2961	0.398	-0.2323	-0.0745	0.093	0.1752	0.3221	0.01	0.053	-0.2379	-0.0898	0.1861	-0.0082	0.0225	0.3368	0.0159	0.0714	-16.6951	14.0467	-3.6538
6	3.4652	-2.9737	-0.2572	3.5288	-1.5799	4.3689	0.2027	0.1647	0.2672	0.2086	0.2933	0.2968	-0.6459	-0.5988	-0.4959	0.6248	0.1048	0.15	0.6245	0.0804	0.6245	-16.8418	30.6931	-29.596
7	7.1744	-1.2792	-3.7559	3.204	-0.4831	5.1581	-0.0555	0.0024	0.3098	-0.1811	0.2114	-0.0527	0.1389	-0.2359	-0.156	0.1746	0.0302	0.0508	0.312	0.0109	0.031	-14.1864	20.6014	-25.1865
8	1.6144	0.0119	1.8363	4.7066	-2.7029	3.9618	0.0414	-0.1511	0.1255	0.1669	-0.0589	-0.1419	-0.3257	-0.0486	0.0175	0.2937	-0.0166	0.1736	0.3375	-0.061	0.1681	-12.7543	27.2975	-18.0368
9	0.9016	-0.6636	-1.0206	2.7833	-1.474	4.6551	-0.0999	-0.1755	0.0381	-0.4205	0.1311	0.0012	-0.0168	-0.1081	-0.0312	0.4156	0.0885	0.0233	0.3504	-0.0264	0.125	-12.8047	24.5765	-27.8394
10	2.3144	-0.5792	-0.6772	7.6178	4.4148	6.3805	0.1942	0.0846	0.0318	-0.202	0.0087	-0.0066	-0.1603	-0.1585	-0.2028	0.2607	-0.0617	0.0963	0.0308	-0.0124	0.0607	-3.6732	27.0938	-57.823
11	1.3622	-0.5313	-0.7055	4.4953	2.1457	4.7282	0.1414	0.182	0.0892	-0.713	-0.1334	0.3995	-0.0761	-0.4297	-0.008	0.3466	-0.0465	0.0379	0.0641	-0.0071	0.1116	-8.4195	28.3365	-62.5542
12	1.6932	-0.6011	0.2545	0.2923	0.4616	6.5277	-0.1545	-0.0622	-0.2275	0.037	0.0221	0.0162	0.2927	0.1199	0.1324	0.2243	0.0184	0.0669	0.0348	0.046	0.2686	-8.7861	-0.0897	-32.4431
13	4.2332	2.0168	0.2863	1.8105	1.4915	2.97	-0.2158	-0.3832	-0.6027	-0.0158	-0.0486	-0.1323	0.5961	-0.189	0.2644	0.4387	-0.0815	0.1051	0.2993	0.0711	0.1286	-15.6725	2.4065	-24.8324
14	1.0018	-0.5431	-1.7555	0.62	1.1904	7.1637	0.0989	-0.2083	-0.1434	-0.2214	0.0012	-0.1034	0.4118	-0.2008	-0.1001	0.2316	-0.0361	0.0353	0.259	0.0692	0.1553	-14.8234	9.7949	-24.615
15	2.1877	0.4458	-1.3242	1.6703	-0.8064	4.7518	-0.3163	0.2684	-0.0317	-0.8936	-0.0092	-0.1056	0.6487	0.3949	0.3255	0.5667	0.0152	0.1046	0.3108	0.032	0.4314	2.9823	19.638	-61.9596
16	2.6713	1.2129	-0.7661	6.2267	-0.7546	1.0205	-0.1112	-0.1236	0.2023	0.0935	-0.0378	-0.2238	0.3655	0.0812	0.149	0.3621	-0.0176	0.1118	0.0239	-0.0127	0.0896	2.163	14.7693	-55.2541

Refinement TLS group

ID	Refine-ID	Refine TLS-ID	Auth asym-ID	Auth seq-ID
1	X-RAY DIFFRACTION	1	A	2 - 6
2	X-RAY DIFFRACTION	2	A	7 - 25
3	X-RAY DIFFRACTION	3	A	26 - 95
4	X-RAY DIFFRACTION	4	A	96 - 125
5	X-RAY DIFFRACTION	5	A	126 - 137
6	X-RAY DIFFRACTION	6	L	2 - 26
7	X-RAY DIFFRACTION	7	L	27 - 51
8	X-RAY DIFFRACTION	8	L	52 - 90
9	X-RAY DIFFRACTION	9	L	91 - 124
10	X-RAY DIFFRACTION	10	L	125 - 161
11	X-RAY DIFFRACTION	11	L	162 - 231
12	X-RAY DIFFRACTION	12	H	1 - 20
13	X-RAY DIFFRACTION	13	H	21 - 95
14	X-RAY DIFFRACTION	14	H	96 - 138
15	X-RAY DIFFRACTION	15	H	139 - 158
16	X-RAY DIFFRACTION	16	H	159 - 239

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