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- PDB-8z2v: Crystal structure of Sonic hedgehog in complex with antibody 5E1 ... -

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Basic information

Entry
Database: PDB / ID: 8z2v
TitleCrystal structure of Sonic hedgehog in complex with antibody 5E1 mutant H-R102A with 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 / : / 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 / : / 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 / negative regulation of alpha-beta T cell differentiation / regulation of prostatic bud formation / formation of anatomical boundary / positive regulation of striated muscle cell differentiation / regulation of glial cell proliferation / 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 / negative regulation of mesenchymal cell apoptotic process / determination of left/right asymmetry in lateral mesoderm / spinal cord motor neuron differentiation / negative regulation of T cell differentiation in thymus / cell development / establishment of epithelial cell polarity / positive regulation of T cell differentiation in thymus / skeletal muscle cell proliferation / prostate gland development / intermediate filament organization / limb bud formation / embryonic skeletal system development / stem cell development / skeletal muscle fiber differentiation / positive regulation of cerebellar granule cell precursor proliferation / animal organ formation / mesenchymal cell apoptotic process / patched binding / embryonic digestive tract morphogenesis / somite development / hindbrain development / positive regulation of skeletal muscle tissue development / ectoderm development / embryonic foregut morphogenesis / epithelial cell proliferation involved in salivary gland morphogenesis / cerebellar granule cell precursor proliferation / mesenchymal cell proliferation involved in lung development / neuron fate commitment / negative regulation of dopaminergic neuron differentiation / Activation of SMO / positive regulation of immature T cell proliferation in thymus / self proteolysis / smooth muscle tissue development / dorsal/ventral neural tube patterning / lung lobe morphogenesis / artery development / lymphoid progenitor cell differentiation / Release of Hh-Np from the secreting cell / positive regulation of astrocyte differentiation / CD4-positive or CD8-positive, alpha-beta T cell lineage commitment / regulation of stem cell proliferation / positive regulation of epithelial cell proliferation involved in prostate gland development / negative thymic T cell selection / pattern specification process / male genitalia development / branching involved in salivary gland morphogenesis / embryonic pattern specification / epithelial cell proliferation involved in prostate gland development / lung-associated mesenchyme development / intein-mediated protein splicing / glycosaminoglycan binding / Formation of axial mesoderm / dopaminergic neuron differentiation / metanephros development / thalamus development / Developmental Lineage of Pancreatic Acinar Cells / metanephric collecting duct development / camera-type eye development / positive thymic T cell selection / positive regulation of smoothened signaling pathway / oligodendrocyte development
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
Sonic hedgehog protein
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 1.89 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 13, 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)71,6378
Polymers71,3633
Non-polymers2735
Water7,368409
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)96.641, 69.752, 119.270
Angle α, β, γ (deg.)90.00, 108.73, 90.00
Int Tables number5
Space group name H-MC121
Components on special symmetry positions
IDModelComponents
11H-533-

HOH

21H-557-

HOH

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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 (E. coli) / Strain (production host): BL-21(DE3) / References: UniProt: Q15465

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Antibody , 2 types, 2 molecules HL

#2: Antibody Heavy chain of antibody 5E1


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 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 , 5 types, 414 molecules

#4: Chemical ChemComp-CL / CHLORIDE ION


Mass: 35.453 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Cl
#5: Chemical ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Zn / Feature type: SUBJECT OF INVESTIGATION
#6: Chemical ChemComp-CA / CALCIUM ION


Mass: 40.078 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Ca / Feature type: SUBJECT OF INVESTIGATION
#7: Chemical ChemComp-GOL / GLYCEROL / GLYCERIN / PROPANE-1,2,3-TRIOL


Mass: 92.094 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C3H8O3
#8: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 409 / Source method: isolated from a natural source / Formula: H2O

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Details

Has ligand of interestY
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: 2.67 Å3/Da / Density % sol: 53.89 %
Crystal growTemperature: 293.15 K / Method: vapor diffusion, sitting drop / Details: 200mM MgCl2, 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-1A / Wavelength: 1 Å
DetectorType: DECTRIS EIGER X 4M / Detector: PIXEL / Date: Mar 19, 2019
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 1 Å / Relative weight: 1
ReflectionResolution: 1.89→56.48 Å / Num. obs: 58458 / % possible obs: 97.3 % / Redundancy: 4.9 % / CC1/2: 0.999 / Rmerge(I) obs: 0.064 / Rpim(I) all: 0.032 / Net I/σ(I): 11.8
Reflection shellResolution: 1.89→1.99 Å / Rmerge(I) obs: 0.436 / Mean I/σ(I) obs: 3.2 / Num. unique obs: 8161 / CC1/2: 0.854 / Rpim(I) all: 0.226 / % possible all: 93.1

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Processing

Software
NameVersionClassification
REFMAC5.8.0238refinement
MOSFLM7.2.2data reduction
SCALA3.3.22data scaling
PHASERphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT / Resolution: 1.89→56.48 Å / Cor.coef. Fo:Fc: 0.965 / Cor.coef. Fo:Fc free: 0.946 / SU B: 9.165 / SU ML: 0.13 / Cross valid method: THROUGHOUT / ESU R: 0.132 / ESU R Free: 0.129 / Stereochemistry target values: MAXIMUM LIKELIHOOD / Details: HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS
RfactorNum. reflection% reflectionSelection details
Rfree0.22514 2384 4.1 %RANDOM
Rwork0.18587 ---
obs0.18747 56074 96.98 %-
Solvent computationIon probe radii: 0.8 Å / Shrinkage radii: 0.8 Å / VDW probe radii: 1.2 Å / Solvent model: MASK
Displacement parametersBiso mean: 29.98 Å2
Baniso -1Baniso -2Baniso -3
1-3.71 Å20 Å2-3.37 Å2
2---1.03 Å20 Å2
3----0.33 Å2
Refinement stepCycle: 1 / Resolution: 1.89→56.48 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms4463 0 10 409 4882
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
X-RAY DIFFRACTIONr_bond_refined_d0.0180.0134609
X-RAY DIFFRACTIONr_bond_other_d0.0030.0174139
X-RAY DIFFRACTIONr_angle_refined_deg2.141.6466270
X-RAY DIFFRACTIONr_angle_other_deg1.5111.5769663
X-RAY DIFFRACTIONr_dihedral_angle_1_deg18.2865.342599
X-RAY DIFFRACTIONr_dihedral_angle_2_deg33.38623.178214
X-RAY DIFFRACTIONr_dihedral_angle_3_deg15.4315749
X-RAY DIFFRACTIONr_dihedral_angle_4_deg18.8721520
X-RAY DIFFRACTIONr_chiral_restr0.0930.2607
X-RAY DIFFRACTIONr_gen_planes_refined0.0120.025745
X-RAY DIFFRACTIONr_gen_planes_other0.0020.02947
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_it0.9471.1182304
X-RAY DIFFRACTIONr_mcbond_other0.9461.1172303
X-RAY DIFFRACTIONr_mcangle_it1.5861.672876
X-RAY DIFFRACTIONr_mcangle_other1.5861.6712877
X-RAY DIFFRACTIONr_scbond_it1.3191.2692305
X-RAY DIFFRACTIONr_scbond_other1.3191.272306
X-RAY DIFFRACTIONr_scangle_it
X-RAY DIFFRACTIONr_scangle_other2.1481.8333389
X-RAY DIFFRACTIONr_long_range_B_refined5.61914.2955199
X-RAY DIFFRACTIONr_long_range_B_other5.52213.645103
X-RAY DIFFRACTIONr_rigid_bond_restr
X-RAY DIFFRACTIONr_sphericity_free
X-RAY DIFFRACTIONr_sphericity_bonded
LS refinement shellResolution: 1.89→1.939 Å / Total num. of bins used: 20
RfactorNum. reflection% reflection
Rfree0.331 150 -
Rwork0.324 3838 -
obs--89.9 %
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)
11.72530.21430.783.17660.00963.42840.1722-0.1606-0.04160.5148-0.09410.07060.292-0.2016-0.07810.244-0.0465-0.03090.1306-0.02020.1482.907316.199757.3239
21.6893-0.09751.47481.4178-0.06432.926-0.05820.03950.10180.1773-0.0411-0.0016-0.03620.11360.09940.0503-0.02050.060.11120.01090.1726-7.356413.534718.2777
31.00010.231.39450.68570.60032.08750.1522-0.074-0.01980.1746-0.13530.11070.2842-0.2067-0.01690.0901-0.08950.06880.20290.01980.2013-20.1895-0.181521.6284
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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