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- PDB-8z3a: Crystal structure of Sonic hedgehog in complex with antibody 5E1 ... -

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Basic information

Entry
Database: PDB / ID: 8z3a
TitleCrystal structure of Sonic hedgehog in complex with antibody 5E1 without metals
Components
  • Heavy chain of antibody 5E1
  • Light chain of antibody 5E1
  • Sonic hedgehog protein N-product
KeywordsIMMUNE 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
Hedgehog, N-terminal signalling domain / Hedgehog protein / Hedgehog protein, Hint domain / : / Hint module / Hedgehog amino-terminal signalling domain / Hedgehog signalling/DD-peptidase zinc-binding domain superfamily / Hint domain C-terminal / Hint (Hedgehog/Intein) domain C-terminal region / Intein N-terminal splicing region ...Hedgehog, N-terminal signalling domain / Hedgehog protein / Hedgehog protein, Hint domain / : / Hint module / Hedgehog amino-terminal signalling domain / Hedgehog signalling/DD-peptidase zinc-binding domain superfamily / Hint domain C-terminal / Hint (Hedgehog/Intein) domain C-terminal region / Intein N-terminal splicing region / Intein N-terminal splicing motif profile. / Hint domain N-terminal / Hint (Hedgehog/Intein) domain N-terminal region / Hint domain superfamily
Similarity search - Domain/homology
NITRATE ION / Sonic hedgehog protein
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 1.75 Å
AuthorsCaaveiro, J.M.M. / Kaneda, I. / Tsumoto, K.
Funding support Japan, 2items
OrganizationGrant numberCountry
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
CitationJournal: 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
DepositionApr 15, 2024Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Apr 16, 2025Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Sonic hedgehog protein N-product
H: Heavy chain of antibody 5E1
L: Light chain of antibody 5E1
hetero molecules


Theoretical massNumber of molelcules
Total (without water)71,6045
Polymers71,4503
Non-polymers1542
Water10,305572
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Unit cell
Length a, b, c (Å)69.370, 83.990, 126.850
Angle α, β, γ (deg.)90.00, 90.00, 90.00
Int Tables number19
Space group name H-MP212121

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Components

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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 of antibody 5E1


Mass: 26318.254 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)
#3: Antibody Light chain of 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 , 3 types, 574 molecules

#4: Chemical ChemComp-GOL / GLYCEROL / GLYCERIN / PROPANE-1,2,3-TRIOL


Mass: 92.094 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C3H8O3
#5: Chemical ChemComp-NO3 / NITRATE ION


Mass: 62.005 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: NO3
#6: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 572 / Source method: isolated from a natural source / Formula: H2O

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Details

Has ligand of interestN
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: 2.59 Å3/Da / Density % sol: 52.44 %
Crystal growTemperature: 293.15 K / Method: vapor diffusion, sitting drop / Details: 200mM KNO3, 20% PEG 3350

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: Photon Factory / Beamline: BL-5A / Wavelength: 1 Å
DetectorType: DECTRIS PILATUS3 S 6M / Detector: PIXEL / Date: Dec 18, 2018
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 1 Å / Relative weight: 1
ReflectionResolution: 1.75→53.5 Å / Num. obs: 75399 / % possible obs: 99.9 % / Redundancy: 9.3 % / CC1/2: 0.997 / Rmerge(I) obs: 0.117 / Rpim(I) all: 0.04 / Net I/σ(I): 11.8
Reflection shellResolution: 1.75→1.84 Å / Redundancy: 8.5 % / Rmerge(I) obs: 0.943 / Mean I/σ(I) obs: 2.3 / Num. unique obs: 10836 / CC1/2: 0.693 / Rpim(I) all: 0.339 / % possible all: 99.7

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Processing

Software
NameVersionClassification
REFMAC5.8.0238refinement
MOSFLM7.2.2data reduction
SCALA3.3.22data scaling
PHASER2.8.2phasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT / Resolution: 1.75→50.67 Å / Cor.coef. Fo:Fc: 0.964 / Cor.coef. Fo:Fc free: 0.951 / SU B: 4.827 / SU ML: 0.077 / Cross valid method: THROUGHOUT / ESU R: 0.098 / ESU R Free: 0.098 / Stereochemistry target values: MAXIMUM LIKELIHOOD / Details: HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS
RfactorNum. reflection% reflectionSelection details
Rfree0.20745 3093 4.1 %RANDOM
Rwork0.17546 ---
obs0.17677 72234 99.88 %-
Solvent computationIon probe radii: 0.8 Å / Shrinkage radii: 0.8 Å / VDW probe radii: 1.2 Å / Solvent model: MASK
Displacement parametersBiso mean: 25.3 Å2
Baniso -1Baniso -2Baniso -3
1--0.37 Å20 Å20 Å2
2--0.83 Å20 Å2
3----0.45 Å2
Refinement stepCycle: 1 / Resolution: 1.75→50.67 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms4467 0 10 572 5049
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
X-RAY DIFFRACTIONr_bond_refined_d0.0140.0134681
X-RAY DIFFRACTIONr_bond_other_d0.0010.0174218
X-RAY DIFFRACTIONr_angle_refined_deg1.8251.6476378
X-RAY DIFFRACTIONr_angle_other_deg1.4571.5779857
X-RAY DIFFRACTIONr_dihedral_angle_1_deg7.7645598
X-RAY DIFFRACTIONr_dihedral_angle_2_deg33.01923.04227
X-RAY DIFFRACTIONr_dihedral_angle_3_deg13.86715781
X-RAY DIFFRACTIONr_dihedral_angle_4_deg14.2281522
X-RAY DIFFRACTIONr_chiral_restr0.0850.2616
X-RAY DIFFRACTIONr_gen_planes_refined0.0110.025255
X-RAY DIFFRACTIONr_gen_planes_other0.0010.02967
X-RAY DIFFRACTIONr_nbd_refined
X-RAY DIFFRACTIONr_nbd_other
X-RAY DIFFRACTIONr_nbtor_refined
X-RAY DIFFRACTIONr_nbtor_other
X-RAY DIFFRACTIONr_xyhbond_nbd_refined
X-RAY DIFFRACTIONr_xyhbond_nbd_other
X-RAY DIFFRACTIONr_metal_ion_refined
X-RAY DIFFRACTIONr_metal_ion_other
X-RAY DIFFRACTIONr_symmetry_vdw_refined
X-RAY DIFFRACTIONr_symmetry_vdw_other
X-RAY DIFFRACTIONr_symmetry_hbond_refined
X-RAY DIFFRACTIONr_symmetry_hbond_other
X-RAY DIFFRACTIONr_symmetry_metal_ion_refined
X-RAY DIFFRACTIONr_symmetry_metal_ion_other
X-RAY DIFFRACTIONr_mcbond_it1.1621.4742327
X-RAY DIFFRACTIONr_mcbond_other1.1621.4732326
X-RAY DIFFRACTIONr_mcangle_it1.842.2032910
X-RAY DIFFRACTIONr_mcangle_other1.8392.2042911
X-RAY DIFFRACTIONr_scbond_it1.6991.6732354
X-RAY DIFFRACTIONr_scbond_other1.6971.6732352
X-RAY DIFFRACTIONr_scangle_it
X-RAY DIFFRACTIONr_scangle_other2.7212.423453
X-RAY DIFFRACTIONr_long_range_B_refined5.26918.3185273
X-RAY DIFFRACTIONr_long_range_B_other5.26918.3255274
X-RAY DIFFRACTIONr_rigid_bond_restr
X-RAY DIFFRACTIONr_sphericity_free
X-RAY DIFFRACTIONr_sphericity_bonded
LS refinement shellResolution: 1.75→1.795 Å / Total num. of bins used: 20
RfactorNum. reflection% reflection
Rfree0.343 229 -
Rwork0.282 5248 -
obs--99.58 %
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)
14.3753-0.56990.52721.8448-0.87972.31020.05770.1095-0.3212-0.0747-0.01340.15340.09640.0157-0.04430.01120.0154-0.00250.03470.00250.0351-1.5517-23.9202-0.0028
21.1241-0.1945-0.28550.95720.79473.222-0.0629-0.0196-0.1066-0.0345-0.05990.05670.0142-0.10840.12280.00740.0030.00180.01080.0090.0460.2695-14.1882-39.1737
30.4906-0.0185-0.2540.19490.70642.898-0.0995-0.0053-0.1261-0.0077-0.00540.00980.13190.12560.10490.08140.0550.02680.08030.02670.049314.091-26.9716-42.7316
Refinement TLS group
IDRefine-IDRefine TLS-IDAuth asym-IDAuth seq-ID
1X-RAY DIFFRACTION1A39 - 188
2X-RAY DIFFRACTION2H1 - 219
3X-RAY DIFFRACTION3L1 - 211

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