PDB-8yyz: Crystal structure of Sonic hedgehog in complex with antibody 5E1 ...

ID or keywords:

Entry

Database: PDB / ID: 8yyz

Title

Crystal structure of Sonic hedgehog in complex with antibody 5E1 mutant H-R102A in the absence of metals

Components

Heavy chain antibody 5E1 mutated
Light chain antibody 5E1
Sonic hedgehog protein N-product

Keywords

IMMUNE SYSTEM / Sonic hedgehog protein (SHH) / antibody / flexible/fluctuated epitope / affinity improvement

Function / homology

Function and homology information

regulation of nodal signaling pathway / positive regulation of skeletal muscle cell proliferation / right lung development / left lung development / primary prostatic bud elongation / regulation of mesenchymal cell proliferation involved in prostate gland development / mesenchymal smoothened signaling pathway involved in prostate gland development / positive regulation of sclerotome development / tracheoesophageal septum formation / negative regulation of ureter smooth muscle cell differentiation ...regulation of nodal signaling pathway / positive regulation of skeletal muscle cell proliferation / right lung development / left lung development / primary prostatic bud elongation / regulation of mesenchymal cell proliferation involved in prostate gland development / mesenchymal smoothened signaling pathway involved in prostate gland development / positive regulation of sclerotome development / tracheoesophageal septum formation / negative regulation of ureter smooth muscle cell differentiation / positive regulation of ureter smooth muscle cell differentiation / negative regulation of kidney smooth muscle cell differentiation / positive regulation of kidney smooth muscle cell differentiation / morphogen activity / regulation of odontogenesis / positive regulation of mesenchymal cell proliferation involved in ureter development / trunk neural crest cell migration / Formation of lateral plate mesoderm / hindgut morphogenesis / polarity specification of anterior/posterior axis / regulation of glial cell proliferation / negative regulation of alpha-beta T cell differentiation / regulation of prostatic bud formation / formation of anatomical boundary / positive regulation of striated muscle cell differentiation / metanephric mesenchymal cell proliferation involved in metanephros development / ventral midline development / trachea morphogenesis / cholesterol-protein transferase activity / HHAT G278V doesn't palmitoylate Hh-Np / telencephalon regionalization / bud outgrowth involved in lung branching / epithelial-mesenchymal cell signaling / Ligand-receptor interactions / laminin-1 binding / lung epithelium development / negative regulation of cholesterol efflux / salivary gland cavitation / spinal cord dorsal/ventral patterning / determination of left/right asymmetry in lateral mesoderm / negative regulation of mesenchymal cell apoptotic process / positive regulation of cerebellar granule cell precursor proliferation / negative regulation of T cell differentiation in thymus / cell development / spinal cord motor neuron differentiation / positive regulation of T cell differentiation in thymus / establishment of epithelial cell polarity / intermediate filament organization / prostate gland development / cerebellar granule cell precursor proliferation / limb bud formation / embryonic skeletal system development / skeletal muscle fiber differentiation / lung lobe morphogenesis / Activation of SMO / mesenchymal cell apoptotic process / patched binding / animal organ formation / embryonic digestive tract morphogenesis / embryonic foregut morphogenesis / hindbrain development / positive regulation of skeletal muscle tissue development / epithelial cell proliferation involved in salivary gland morphogenesis / somite development / ectoderm development / neuron fate commitment / negative regulation of dopaminergic neuron differentiation / mesenchymal cell proliferation involved in lung development / skeletal muscle cell proliferation / stem cell development / positive regulation of immature T cell proliferation in thymus / lymphoid progenitor cell differentiation / dorsal/ventral neural tube patterning / self proteolysis / smooth muscle tissue development / artery development / thalamus development / positive regulation of astrocyte differentiation / CD4-positive or CD8-positive, alpha-beta T cell lineage commitment / negative thymic T cell selection / pattern specification process / regulation of stem cell proliferation / oligodendrocyte development / positive regulation of epithelial cell proliferation involved in prostate gland development / male genitalia development / Release of Hh-Np from the secreting cell / branching involved in salivary gland morphogenesis / embryonic pattern specification / epithelial cell proliferation involved in prostate gland development / lung-associated mesenchyme development / dopaminergic neuron differentiation / intein-mediated protein splicing / glycosaminoglycan binding / Formation of axial mesoderm / Developmental Lineage of Pancreatic Acinar Cells / metanephros development / camera-type eye development / metanephric collecting duct development / positive thymic T cell selection / positive regulation of smoothened signaling pathway
Similarity search - Function

Biological species

Homo sapiens (human)

Method

X-RAY DIFFRACTION /

SYNCHROTRON /

MOLECULAR REPLACEMENT / Resolution: 1.9 Å

Authors

Caaveiro, J.M.M. / Senoo, A. / Kaneda, I. / Tsumoto, K.

Funding support

Japan, 2items

Organization	Grant number	Country
Japan Society for the Promotion of Science (JSPS)	JP18H02082, JP18H05425, 16H02420, JP19H05766, JP19H05760, JP20H02531	Japan
Japan Agency for Medical Research and Development (AMED)	JP23ama121033	Japan

Citation

Journal: To Be Published
Title: Biophysical insights to improve affinity of antibodies to fluctuated epitope of antigen: A case of anti-Shh antibody 5E1 against Shh antigen.
Authors: Kaneda, I. / Matsunaga, R. / Nagatoishi, S. / Senoo, A. / Caaveiro, J.M.M. / Kuroda, D. / Tsumoto, K.

History

Deposition	Apr 4, 2024	Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0	Apr 9, 2025	Provider: repository / Type: Initial release

Structure viewer	Molecule: MolmilJmol/JSmol

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PDBx/mmCIF format	8yyz.cif.gz	319.6 KB	Display	PDBx/mmCIF format
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Validation report

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

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Related structure data

Related structure data	8z2vC 8z39C 8z3aC C: citing same article (ref.)
Similar structure data	Similarity search - Function & homologyF&H Search Similarity search - FunctionF&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) #131 - Nov 2010 Inteins similarity (7) #219 - Mar 2018 Vacuolar ATPase similarity (7) #192 - Dec 2015 Vancomycin similarity (1)

Deposited unit

A: Sonic hedgehog protein N-product

H: Heavy chain antibody 5E1 mutated

L: Light chain antibody 5E1

hetero molecules

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

1

Idetical with deposited unit
defined by author
Evidence: surface plasmon resonance

Unit cell

Length a, b, c (Å)	69.510, 84.080, 126.040
Angle α, β, γ (deg.)	90.000, 90.000, 90.000
Int Tables number	19
Space group name H-M	P2₁2₁2₁

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Protein , 1 types, 1 molecules A

#1: Protein	Sonic hedgehog protein N-product / ShhN / Shh N-terminal processed signaling domains / ShhNp Mass: 19122.484 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Homo sapiens (human) / Gene: SHH / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: Q15465

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Antibody , 2 types, 2 molecules HL

#2: Antibody	Heavy chain antibody 5E1 mutated Mass: 26232.139 Da / Num. of mol.: 1 / Mutation: R102A Source method: isolated from a genetically manipulated source Source: (gene. exp.) Homo sapiens (human) / Gene: IgG / Cell line (production host): EXPI293 / Production host: Homo sapiens (human)
#3: Antibody	Light chain antibody 5E1 Mass: 26008.797 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Homo sapiens (human) / Gene: IgG / Cell line (production host): EXPI293 / Production host: Homo sapiens (human)

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Non-polymers , 4 types, 424 molecules

#4: Chemical	ChemComp-CL / CHLORIDE ION Mass: 35.453 Da / Num. of mol.: 5 / Source method: obtained synthetically / Formula: Cl
#5: Chemical	ChemComp-GOL / GLYCEROL / GLYCERIN / PROPANE-1,2,3-TRIOL Mass: 92.094 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C₃H₈O₃
#6: Chemical	ChemComp-IOD / IODIDE ION Mass: 126.904 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: I
#7: Water	ChemComp-HOH / water Mass: 18.015 Da / Num. of mol.: 416 / Source method: isolated from a natural source / Formula: H₂O

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Details

Has ligand of interest	N
Has protein modification	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: 2.58 Å³/Da / Density % sol: 52.34 %
Crystal grow	Temperature: 293.15 K / Method: vapor diffusion, sitting drop Details: 0.2M Ammonium iodide, 20% w/v Polyethylene glycol 3350

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Data collection

Diffraction	Mean temperature: 100 K / Serial crystal experiment: N
Diffraction source	Source: SYNCHROTRON / Site: Photon Factory / Beamline: AR-NW12A / Wavelength: 1 Å
Detector	Type: DECTRIS PILATUS3 S 2M / Detector: PIXEL / Date: Jun 22, 2019
Radiation	Protocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelength	Wavelength: 1 Å / Relative weight: 1
Reflection	Resolution: 1.9→42.04 Å / Num. obs: 58004 / % possible obs: 98.4 % / Redundancy: 7.3 % / CC_1/2: 0.99 / R_merge(I) obs: 0.116 / R_pim(I) all: 0.046 / Net I/σ(I): 9.4
Reflection shell	Resolution: 1.9→2 Å / Redundancy: 5.8 % / R_merge(I) obs: 0.53 / Mean I/σ(I) obs: 2.7 / Num. unique obs: 7699 / CC_1/2: 0.851 / R_pim(I) all: 0.23 / % possible all: 91.1

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Processing

Software

Name	Version	Classification
REFMAC	5.8.0419	refinement
MOSFLM	7.2.2	data reduction
SCALA	3.3.22	data scaling
PHASER	2.8.3	phasing

Refinement

Method to determine structure:

MOLECULAR REPLACEMENT / Resolution: 1.9→42.04 Å / Cor.coef. F_o:F_c: 0.955 / Cor.coef. F_o:F_c free: 0.933 / SU B: 6.196 / SU ML: 0.097 / Cross valid method: THROUGHOUT / ESU R: 0.133 / ESU R Free: 0.128
Details: Hydrogens have been added in their riding positions

	R_factor	Num. reflection	% reflection
R_free	0.2218	2422	4.182 %
R_work	0.1844	55499	-
all	0.186	-	-
obs	-	57921	98.229 %

Solvent computation

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

Displacement parameters

B_iso mean: 27.81 Å²

	B_aniso -1	B_aniso -2	B_aniso -3
1-	-1.202 Å²	0 Å²	-0 Å²
2-	-	2.156 Å²	-0 Å²
3-	-	-	-0.954 Å²

Refinement step

Cycle: LAST / Resolution: 1.9→42.04 Å

	Protein	Nucleic acid	Ligand	Solvent	Total
Num. atoms	4483	0	13	416	4912

Refine LS restraints

Refine-ID	Type	Dev ideal	Dev ideal target	Number
X-RAY DIFFRACTION	r_bond_refined_d	0.006	0.012	4630
X-RAY DIFFRACTION	r_bond_other_d	0.001	0.016	4240
X-RAY DIFFRACTION	r_angle_refined_deg	1.438	1.817	6297
X-RAY DIFFRACTION	r_angle_other_deg	0.51	1.757	9829
X-RAY DIFFRACTION	r_dihedral_angle_1_deg	7.417	5	585
X-RAY DIFFRACTION	r_dihedral_angle_2_deg	12.221	5	19
X-RAY DIFFRACTION	r_dihedral_angle_3_deg	12.229	10	765
X-RAY DIFFRACTION	r_dihedral_angle_6_deg	14.581	10	200
X-RAY DIFFRACTION	r_chiral_restr	0.07	0.2	698
X-RAY DIFFRACTION	r_gen_planes_refined	0.006	0.02	5420
X-RAY DIFFRACTION	r_gen_planes_other	0.001	0.02	1036
X-RAY DIFFRACTION	r_nbd_refined	0.203	0.2	744
X-RAY DIFFRACTION	r_symmetry_nbd_other	0.195	0.2	3780
X-RAY DIFFRACTION	r_nbtor_refined	0.177	0.2	2224
X-RAY DIFFRACTION	r_symmetry_nbtor_other	0.085	0.2	2362
X-RAY DIFFRACTION	r_xyhbond_nbd_refined	0.192	0.2	336
X-RAY DIFFRACTION	r_symmetry_xyhbond_nbd_other	0.113	0.2	1
X-RAY DIFFRACTION	r_symmetry_nbd_refined	0.116	0.2	4
X-RAY DIFFRACTION	r_nbd_other	0.155	0.2	38
X-RAY DIFFRACTION	r_symmetry_xyhbond_nbd_refined	0.152	0.2	18
X-RAY DIFFRACTION	r_mcbond_it	1.368	1.551	2319
X-RAY DIFFRACTION	r_mcbond_other	1.368	1.551	2319
X-RAY DIFFRACTION	r_mcangle_it	2.279	2.776	2896
X-RAY DIFFRACTION	r_mcangle_other	2.279	2.776	2897
X-RAY DIFFRACTION	r_scbond_it	1.786	1.758	2311
X-RAY DIFFRACTION	r_scbond_other	1.786	1.759	2312
X-RAY DIFFRACTION	r_scangle_it	2.946	3.116	3396
X-RAY DIFFRACTION	r_scangle_other	2.946	3.117	3397
X-RAY DIFFRACTION	r_lrange_it	5.487	15.994	5070
X-RAY DIFFRACTION	r_lrange_other	5.368	14.982	4972

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 (%)
1.9-1.949	0.313	152	0.258	3591	X-RAY DIFFRACTION	86.8647
1.949-2.003	0.238	140	0.232	3793	X-RAY DIFFRACTION	93.8439
2.003-2.06	0.255	157	0.214	3813	X-RAY DIFFRACTION	96.9475
2.06-2.124	0.253	175	0.192	3701	X-RAY DIFFRACTION	98.626
2.124-2.193	0.22	138	0.193	3717	X-RAY DIFFRACTION	99.7413
2.193-2.27	0.234	165	0.191	3533	X-RAY DIFFRACTION	99.9189
2.27-2.355	0.239	151	0.189	3444	X-RAY DIFFRACTION	99.9166
2.355-2.451	0.213	157	0.173	3294	X-RAY DIFFRACTION	99.9421
2.451-2.56	0.233	147	0.187	3195	X-RAY DIFFRACTION	100
2.56-2.684	0.239	133	0.182	3051	X-RAY DIFFRACTION	100
2.684-2.829	0.215	138	0.183	2905	X-RAY DIFFRACTION	99.9343
2.829-3	0.224	108	0.166	2778	X-RAY DIFFRACTION	99.9307
3-3.206	0.226	116	0.181	2606	X-RAY DIFFRACTION	99.9633
3.206-3.461	0.197	123	0.177	2395	X-RAY DIFFRACTION	99.9603
3.461-3.788	0.187	119	0.185	2242	X-RAY DIFFRACTION	99.9154
3.788-4.231	0.214	92	0.167	2043	X-RAY DIFFRACTION	100
4.231-4.878	0.188	58	0.148	1832	X-RAY DIFFRACTION	99.8943
4.878-5.954	0.249	76	0.18	1559	X-RAY DIFFRACTION	99.8778
5.954-8.337	0.226	46	0.224	1245	X-RAY DIFFRACTION	99.7682
8.337-42.04	0.227	31	0.198	762	X-RAY DIFFRACTION	99.8741

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	4.0919	-0.2191	0.0103	2.1337	-0.6757	2.0366	0.0283	0.0962	-0.3089	-0.1607	-0.0206	0.1088	0.1587	-0.0099	-0.0076	0.0292	0.0136	-0.0003	0.0176	0.0008	0.0328	-1.3108	-23.8712	0.0093
2	1.0661	-0.2325	-0.2769	0.9597	1.0614	3.7465	-0.0302	-0.015	-0.138	-0.0816	-0.1035	0.0739	-0.0756	-0.1544	0.1337	0.0811	0.013	-0.0091	0.0174	0.0146	0.1094	0.5194	-14.5314	-39.3569
3	0.353	0.0231	-0.2615	0.2422	0.816	3.1635	-0.0917	0.0074	-0.1244	-0.0199	0.0107	-0.0057	0.1225	0.0856	0.0811	0.151	0.0537	0.0148	0.0818	0.0134	0.1195	14.4167	-27.0231	-42.4046

Refinement TLS group

Selection: ALL

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Protein , 1 types, 1 molecules A

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