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- PDB-6zvf: Crystal structure of the recombinant Fab fragment derived from th... -

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Basic information

Entry
Database: PDB / ID: 6zvf
TitleCrystal structure of the recombinant Fab fragment derived from the hybridoma M3/38 in complex with a human Galectin-3 peptide
Components
  • (Chimeric Fab M3/38 (L,H)) x 2
  • Galectin-3
KeywordsIMMUNE SYSTEM / ANTIBODY / FAB FRAGMENT / PEPTIDE EPITOPE / PROTEIN ENGINEERING
Function / homology
Function and homology information


negative regulation of NK T cell activation / mononuclear cell migration / negative regulation of immunological synapse formation / disaccharide binding / negative regulation of T cell activation via T cell receptor contact with antigen bound to MHC molecule on antigen presenting cell / RUNX2 regulates genes involved in differentiation of myeloid cells / regulation of T cell apoptotic process / receptor ligand inhibitor activity / positive regulation of mononuclear cell migration / negative regulation of endocytosis ...negative regulation of NK T cell activation / mononuclear cell migration / negative regulation of immunological synapse formation / disaccharide binding / negative regulation of T cell activation via T cell receptor contact with antigen bound to MHC molecule on antigen presenting cell / RUNX2 regulates genes involved in differentiation of myeloid cells / regulation of T cell apoptotic process / receptor ligand inhibitor activity / positive regulation of mononuclear cell migration / negative regulation of endocytosis / IgE binding / eosinophil chemotaxis / regulation of extrinsic apoptotic signaling pathway via death domain receptors / signaling receptor inhibitor activity / RUNX1 regulates transcription of genes involved in differentiation of myeloid cells / negative regulation of T cell receptor signaling pathway / protein phosphatase inhibitor activity / positive chemotaxis / positive regulation of calcium ion import / chemoattractant activity / macrophage chemotaxis / monocyte chemotaxis / regulation of T cell proliferation / Advanced glycosylation endproduct receptor signaling / ficolin-1-rich granule membrane / immunological synapse / laminin binding / epithelial cell differentiation / neutrophil chemotaxis / RNA splicing / secretory granule membrane / negative regulation of extrinsic apoptotic signaling pathway / positive regulation of protein localization to plasma membrane / spliceosomal complex / positive regulation of protein-containing complex assembly / molecular condensate scaffold activity / mRNA processing / carbohydrate binding / protein phosphatase binding / : / mitochondrial inner membrane / innate immune response / Neutrophil degranulation / cell surface / extracellular space / RNA binding / extracellular exosome / extracellular region / nucleoplasm / nucleus / membrane / plasma membrane / cytosol / cytoplasm
Similarity search - Function
Galectin-like / Galactoside-binding lectin / Galectin / Galectin, carbohydrate recognition domain / Galactoside-binding lectin / Galactoside-binding lectin (galectin) domain profile. / Concanavalin A-like lectin/glucanase domain superfamily / Immunoglobulins / Immunoglobulin-like / Sandwich / Mainly Beta
Similarity search - Domain/homology
Biological speciesRattus norvegicus (Norway rat)
Homo sapiens (human)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / molecular replacement / Resolution: 1.9 Å
AuthorsSkerra, A. / Eichinger, A.
Funding support Germany, 1items
OrganizationGrant numberCountry
German Research Foundation (DFG)SK 33/11-1 Germany
CitationJournal: Sci Rep / Year: 2021
Title: Effective rational humanization of a PASylated anti-galectin-3 Fab for the sensitive PET imaging of thyroid cancer in vivo.
Authors: Peplau, E. / De Rose, F. / Eichinger, A. / Reder, S. / Mittelhauser, M. / Scafetta, G. / Schwaiger, M. / Weber, W.A. / Bartolazzi, A. / D'Alessandria, C. / Skerra, A.
History
DepositionJul 24, 2020Deposition site: PDBE / Processing site: PDBE
Revision 1.0Apr 14, 2021Provider: repository / Type: Initial release
Revision 1.1Jan 31, 2024Group: Data collection / Database references / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / pdbx_initial_refinement_model
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession
Revision 1.2Nov 20, 2024Group: Structure summary / Category: pdbx_entry_details / pdbx_modification_feature / Item: _pdbx_entry_details.has_protein_modification

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
L: Chimeric Fab M3/38 (L,H)
H: Chimeric Fab M3/38 (L,H)
P: Galectin-3


Theoretical massNumber of molelcules
Total (without water)49,2033
Polymers49,2033
Non-polymers00
Water3,621201
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: gel filtration, monomeric behavior
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area5130 Å2
ΔGint-41 kcal/mol
Surface area19700 Å2
MethodPISA
Unit cell
Length a, b, c (Å)94.134, 61.270, 80.909
Angle α, β, γ (deg.)90.000, 103.360, 90.000
Int Tables number5
Space group name H-MC121

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Components

#1: Antibody Chimeric Fab M3/38 (L,H)


Mass: 23927.803 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Details: Both immunoglobulin chains (L, H) are linked by a disulfide bond (CYS219L to CYS221H).
Source: (gene. exp.) Rattus norvegicus (Norway rat) / Production host: Escherichia coli (E. coli)
#2: Protein Chimeric Fab M3/38 (L,H)


Mass: 24392.615 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Details: Residues 222 - 227 represent an affinity tag (His6-tag). Both immunoglobulin chains (L, H) are linked by a disulfide bond (CYS219L to CYS221H).
Source: (gene. exp.) Rattus norvegicus (Norway rat) / Production host: Escherichia coli (E. coli)
#3: Protein/peptide Galectin-3 / Gal-3 / 35 kDa lectin / Carbohydrate-binding protein 35 / CBP 35 / Galactose-specific lectin 3 / ...Gal-3 / 35 kDa lectin / Carbohydrate-binding protein 35 / CBP 35 / Galactose-specific lectin 3 / Galactoside-binding protein / GALBP / IgE-binding protein / L-31 / Laminin-binding protein / Lectin L-29 / Mac-2 antigen


Mass: 882.959 Da / Num. of mol.: 1 / Source method: obtained synthetically
Details: The peptide QAPPGAYPG carries an acetyl (ACE) group at the N-terminus.
Source: (synth.) Homo sapiens (human) / References: UniProt: P17931
#4: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 201 / Source method: isolated from a natural source / Formula: H2O
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: 2.31 Å3/Da / Density % sol: 46.68 %
Crystal growTemperature: 293 K / Method: vapor diffusion, hanging drop / pH: 7 / Details: polyethylene glycol 4000

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: BESSY / Beamline: 14.1 / Wavelength: 0.9184 Å
DetectorType: DECTRIS PILATUS 6M / Detector: PIXEL / Date: Mar 22, 2018 / Details: Si mirror
RadiationMonochromator: Si 111 double-crystal / Protocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.9184 Å / Relative weight: 1
ReflectionResolution: 1.661→78.719 Å / Num. all: 34354 / Num. obs: 34354 / % possible obs: 97 % / Redundancy: 5.7 % / Rpim(I) all: 0.039 / Rrim(I) all: 0.096 / Rsym value: 0.088 / Net I/av σ(I): 5.7 / Net I/σ(I): 10.9 / Num. measured all: 197033
Reflection shell

Diffraction-ID: 1

Resolution (Å)Redundancy (%)Rmerge(I) obsMean I/σ(I) obsNum. measured allNum. unique obsRpim(I) allRrim(I) allRsym valueNet I/σ(I) obs% possible all
1.9-25.90.4111.82983650680.1820.4510.411497.9
2-2.125.70.32.42728947480.1340.3290.35.297.6
2.12-2.275.30.2173.22291843230.1010.2410.2176.694.9
2.27-2.455.60.1742316841210.0770.1870.178.296
2.45-2.695.80.1295.22227738310.0570.1420.12910.297.7
2.69-35.40.09371828633680.0420.1020.0931395.8
3-3.476.30.078.81956331230.030.0770.0718.398.5
3.47-4.255.80.0688.91508126190.030.0740.06821.697.8
4.25-6.015.90.05710.21198220160.0250.0620.05723.297.3
6.01-50.9255.80.04711.2663311370.020.0510.04722.196.5

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Phasing

PhasingMethod: molecular replacement
Phasing MRModel details: Phaser MODE: MR_AUTO
Highest resolutionLowest resolution
Rotation5.98 Å78.72 Å
Translation5.98 Å78.72 Å

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Processing

Software
NameVersionClassification
REFMAC5.8.0238refinement
MOSFLM7.2.1data reduction
SCALA3.3.22data scaling
PHASER2.7.17phasing
PDB_EXTRACT3.25data extraction
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 3O2V

3o2v


Resolution: 1.9→50.98 Å / Cor.coef. Fo:Fc: 0.943 / Cor.coef. Fo:Fc free: 0.921 / SU B: 8.385 / SU ML: 0.12 / Cross valid method: THROUGHOUT / σ(F): 0 / ESU R: 0.177 / ESU R Free: 0.159 / Stereochemistry target values: MAXIMUM LIKELIHOOD
Details: HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS U VALUES : WITH TLS ADDED
RfactorNum. reflection% reflectionSelection details
Rfree0.2434 1700 4.9 %RANDOM
Rwork0.2027 ---
obs0.2047 32653 96.75 %-
Solvent computationIon probe radii: 0.8 Å / Shrinkage radii: 0.8 Å / VDW probe radii: 1.2 Å / Solvent model: MASK
Displacement parametersBiso max: 88.63 Å2 / Biso mean: 25.385 Å2 / Biso min: 12.98 Å2
Baniso -1Baniso -2Baniso -3
1--0.95 Å2-0 Å2-1.06 Å2
2---2.05 Å20 Å2
3---3.15 Å2
Refinement stepCycle: final / Resolution: 1.9→50.98 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms3401 0 0 201 3602
Biso mean---32.31 -
Num. residues----450
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
X-RAY DIFFRACTIONr_bond_refined_d0.0070.0133522
X-RAY DIFFRACTIONr_bond_other_d0.0010.0173190
X-RAY DIFFRACTIONr_angle_refined_deg1.5191.6444802
X-RAY DIFFRACTIONr_angle_other_deg1.2571.5717483
X-RAY DIFFRACTIONr_dihedral_angle_1_deg7.7685458
X-RAY DIFFRACTIONr_dihedral_angle_2_deg34.09524.022138
X-RAY DIFFRACTIONr_dihedral_angle_3_deg15.05315581
X-RAY DIFFRACTIONr_dihedral_angle_4_deg22.307158
X-RAY DIFFRACTIONr_chiral_restr0.0610.2470
X-RAY DIFFRACTIONr_gen_planes_refined0.0070.023933
X-RAY DIFFRACTIONr_gen_planes_other0.0010.02675
LS refinement shellResolution: 1.9→1.949 Å / Rfactor Rfree error: 0 / Total num. of bins used: 20
RfactorNum. reflection% reflection
Rfree0.305 139 -
Rwork0.259 2442 -
all-2581 -
obs--98.25 %
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)
10.0394-0.02120.00410.4960.1450.2733-0.00640.02120.01050.0572-0.0087-0.02570.02820.02250.01510.11670.00480.01490.17490.01390.0298105.33344.08389.093
20.14090.0176-0.13890.58490.18890.24870.02420.0225-0.01370.0576-0.05060.14130.0125-0.01760.02640.1084-0.00120.02780.1511-0.00290.096187.59541.97890.978
32.4615-2.36352.99242.2916-2.88393.6493-0.3005-0.22560.09370.2280.1648-0.1384-0.3019-0.27870.13570.3284-0.00080.14280.24070.05940.121588.83147.138119.92
Refinement TLS group
IDRefine-IDRefine TLS-IDAuth asym-IDAuth seq-ID
1X-RAY DIFFRACTION1L1 - 214
2X-RAY DIFFRACTION2H1 - 221
3X-RAY DIFFRACTION3P0 - 9

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