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- PDB-8cxc: Novel Anti-Mesothelin Antibodies Enable Crystallography of the In... -

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Basic information

Entry
Database: PDB / ID: 8cxc
TitleNovel Anti-Mesothelin Antibodies Enable Crystallography of the Intact Mesothelin Ectodo- main and Engineering of Potent, T cell-engaging Bispecific Therapeutics
Components
  • 3F2 Antibody heavy chain
  • 3F2 Antibody light chain
  • Mesothelin, cleaved form
  • scFv Amatuximab
KeywordsANTITUMOR PROTEIN / Antibody / complex / tumor associated antigen
Function / homology
Function and homology information


Post-translational modification: synthesis of GPI-anchored proteins / side of membrane / cell-matrix adhesion / Post-translational protein phosphorylation / Regulation of Insulin-like Growth Factor (IGF) transport and uptake by Insulin-like Growth Factor Binding Proteins (IGFBPs) / cell adhesion / endoplasmic reticulum lumen / Golgi apparatus / cell surface / extracellular region ...Post-translational modification: synthesis of GPI-anchored proteins / side of membrane / cell-matrix adhesion / Post-translational protein phosphorylation / Regulation of Insulin-like Growth Factor (IGF) transport and uptake by Insulin-like Growth Factor Binding Proteins (IGFBPs) / cell adhesion / endoplasmic reticulum lumen / Golgi apparatus / cell surface / extracellular region / membrane / plasma membrane
Similarity search - Function
Mesothelin / Stereocilin-related / Mesothelin
Similarity search - Domain/homology
Biological speciesMus musculus (house mouse)
Homo sapiens (human)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 4.31 Å
AuthorsBandaranayake, A.D. / Rupert, P.B. / Lin, I. / Pilat, K. / Ruff, R.O. / Friend, D.J. / Chan, M.K. / Clarke, M. / Carter, J. / Meshinchi, S. ...Bandaranayake, A.D. / Rupert, P.B. / Lin, I. / Pilat, K. / Ruff, R.O. / Friend, D.J. / Chan, M.K. / Clarke, M. / Carter, J. / Meshinchi, S. / Mehlin, C. / Olson, J.M. / Strong, R.K. / Correnti, C.E.
Funding support1items
OrganizationGrant numberCountry
Not funded
CitationJournal: Front. Drug Discov. / Year: 2023
Title: Novel mesothelin antibodies enable crystallography of the intact mesothelin ectodomain and engineering of potent, T cell-engaging bispecific therapeutics
Authors: Lin, I. / Rupert, P.B. / Pilat, K. / Ruff, R.O. / Friend, D.J. / Chan, M.K. / Clarke, M. / Hoffstrom, B.G. / Carter, J. / Meshinchi, S. / Bandaranayake, A.D. / Mehlin, C. / Olson, J.M. / ...Authors: Lin, I. / Rupert, P.B. / Pilat, K. / Ruff, R.O. / Friend, D.J. / Chan, M.K. / Clarke, M. / Hoffstrom, B.G. / Carter, J. / Meshinchi, S. / Bandaranayake, A.D. / Mehlin, C. / Olson, J.M. / Strong, R.K. / Correnti, C.E.
History
DepositionMay 20, 2022Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jun 7, 2023Provider: repository / Type: Initial release
Revision 1.1Oct 25, 2023Group: Data collection / Refinement description
Category: chem_comp_atom / chem_comp_bond / pdbx_initial_refinement_model
Revision 1.2Dec 20, 2023Group: Database references / Category: citation / citation_author
Item: _citation.journal_abbrev / _citation.journal_id_CSD ..._citation.journal_abbrev / _citation.journal_id_CSD / _citation.journal_volume / _citation.pdbx_database_id_DOI / _citation.title / _citation.year
Revision 2.0May 1, 2024Group: Advisory / Atomic model ...Advisory / Atomic model / Data collection / Database references / Derived calculations / Polymer sequence / Source and taxonomy / Structure summary
Category: atom_site / atom_site_anisotrop ...atom_site / atom_site_anisotrop / entity / entity_poly / entity_poly_seq / entity_src_gen / pdbx_poly_seq_scheme / pdbx_struct_assembly_gen / pdbx_struct_sheet_hbond / pdbx_unobs_or_zero_occ_atoms / pdbx_unobs_or_zero_occ_residues / pdbx_validate_torsion / struct_asym / struct_conn / struct_ref / struct_ref_seq / struct_sheet_range
Item: _atom_site.auth_asym_id / _atom_site.auth_seq_id ..._atom_site.auth_asym_id / _atom_site.auth_seq_id / _atom_site.label_asym_id / _atom_site.label_entity_id / _atom_site.label_seq_id / _atom_site_anisotrop.pdbx_auth_asym_id / _atom_site_anisotrop.pdbx_auth_seq_id / _atom_site_anisotrop.pdbx_label_asym_id / _atom_site_anisotrop.pdbx_label_seq_id / _entity_poly_seq.entity_id / _entity_poly_seq.num / _pdbx_poly_seq_scheme.asym_id / _pdbx_poly_seq_scheme.entity_id / _pdbx_poly_seq_scheme.ndb_seq_num / _pdbx_poly_seq_scheme.pdb_seq_num / _pdbx_poly_seq_scheme.pdb_strand_id / _pdbx_poly_seq_scheme.seq_id / _pdbx_struct_assembly_gen.asym_id_list / _pdbx_struct_sheet_hbond.range_1_auth_asym_id / _pdbx_struct_sheet_hbond.range_1_auth_seq_id / _pdbx_struct_sheet_hbond.range_1_label_asym_id / _pdbx_struct_sheet_hbond.range_1_label_seq_id / _pdbx_struct_sheet_hbond.range_2_auth_asym_id / _pdbx_struct_sheet_hbond.range_2_auth_seq_id / _pdbx_struct_sheet_hbond.range_2_label_asym_id / _pdbx_struct_sheet_hbond.range_2_label_seq_id / _pdbx_unobs_or_zero_occ_atoms.auth_asym_id / _pdbx_unobs_or_zero_occ_atoms.auth_seq_id / _pdbx_unobs_or_zero_occ_atoms.label_asym_id / _pdbx_unobs_or_zero_occ_atoms.label_seq_id / _pdbx_unobs_or_zero_occ_residues.auth_asym_id / _pdbx_unobs_or_zero_occ_residues.auth_seq_id / _pdbx_unobs_or_zero_occ_residues.label_asym_id / _pdbx_unobs_or_zero_occ_residues.label_seq_id / _pdbx_validate_torsion.auth_asym_id / _pdbx_validate_torsion.auth_seq_id / _struct_conn.ptnr1_auth_asym_id / _struct_conn.ptnr1_auth_seq_id / _struct_conn.ptnr1_label_asym_id / _struct_conn.ptnr1_label_seq_id / _struct_conn.ptnr2_auth_asym_id / _struct_conn.ptnr2_auth_seq_id / _struct_conn.ptnr2_label_asym_id / _struct_conn.ptnr2_label_seq_id / _struct_sheet_range.beg_auth_asym_id / _struct_sheet_range.beg_auth_seq_id / _struct_sheet_range.beg_label_asym_id / _struct_sheet_range.beg_label_seq_id / _struct_sheet_range.end_auth_asym_id / _struct_sheet_range.end_auth_seq_id / _struct_sheet_range.end_label_asym_id / _struct_sheet_range.end_label_seq_id

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
L: 3F2 Antibody light chain
H: 3F2 Antibody heavy chain
M: Mesothelin, cleaved form
A: scFv Amatuximab


Theoretical massNumber of molelcules
Total (without water)108,4664
Polymers108,4664
Non-polymers00
Water0
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)185.303, 204.175, 123.114
Angle α, β, γ (deg.)90.000, 90.000, 90.000
Int Tables number21
Space group name H-MC222

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Components

#1: Antibody 3F2 Antibody light chain


Mass: 23391.629 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mus musculus (house mouse) / Production host: Homo sapiens (human)
#2: Antibody 3F2 Antibody heavy chain


Mass: 23018.824 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mus musculus (house mouse) / Production host: Homo sapiens (human)
#3: Protein Mesothelin, cleaved form /


Mass: 36963.203 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: MSLN, MPF / Production host: Homo sapiens (human) / References: UniProt: Q13421
#4: Antibody scFv Amatuximab


Mass: 25092.773 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mus musculus (house mouse) / Production host: Homo sapiens (human)

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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: 5.37 Å3/Da / Density % sol: 77.08 %
Crystal growTemperature: 293 K / Method: vapor diffusion, hanging drop / pH: 8 / Details: tris 8.0, zinc chloride, ammonium sulfate

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: ALS / Beamline: 5.0.2 / Wavelength: 1 Å
DetectorType: DECTRIS PILATUS 6M / Detector: PIXEL / Date: Mar 28, 2019
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 1 Å / Relative weight: 1
ReflectionResolution: 4.25→50 Å / Num. obs: 13542 / % possible obs: 84.7 % / Redundancy: 2.6 % / Rmerge(I) obs: 0.121 / Rpim(I) all: 0.088 / Rrim(I) all: 0.151 / Χ2: 5.041 / Net I/σ(I): 12.6 / Num. measured all: 34767
Reflection shell

Diffraction-ID: 1

Resolution (Å)Redundancy (%)Rmerge(I) obsNum. unique obsCC1/2Rpim(I) allRrim(I) allΧ2% possible all
4.25-4.322.50.7846690.2630.5760.9793.4885.5
4.32-4.42.40.6546610.4410.4840.8192.95185.4
4.4-4.492.40.6776540.5770.4910.8433.49383.8
4.49-4.582.50.5416620.7580.3910.6723.73283.1
4.58-4.682.60.4026900.7550.2890.4993.58586.6
4.68-4.792.50.3576750.840.2540.4412.985.2
4.79-4.912.60.3166720.8990.220.3882.67386.9
4.91-5.042.70.2637180.9340.180.3212.67688.4
5.04-5.192.70.2166950.9220.1470.2623.26289.6
5.19-5.352.70.2227000.930.150.273.33488.5
5.35-5.542.70.1866950.9740.1270.2262.74788.8
5.54-5.772.70.2226930.9430.1540.2723.14586.8
5.77-6.032.70.1937010.9260.1350.2373.32386.9
6.03-6.352.60.1676880.9530.1140.2034.61186.4
6.35-6.742.50.1276810.9710.0890.1564.68584.1
6.74-7.262.60.116710.970.0790.1366.10283.9
7.26-7.992.80.086920.9840.0540.0976.38885.6
7.99-9.142.70.0696950.9880.0470.08410.46385.1
9.14-11.492.30.0546570.9930.0420.06912.46779.5
11.49-501.90.0685730.9820.0570.08928.48565.6

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Processing

Software
NameVersionClassification
SCALEPACKdata scaling
REFMAC5.8.0267refinement
PDB_EXTRACT3.27data extraction
HKL-2000data reduction
PHASERphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 4f33

4f33


Resolution: 4.31→45.78 Å / Cor.coef. Fo:Fc: 0.873 / Cor.coef. Fo:Fc free: 0.88 / SU B: 142.637 / SU ML: 0.788 / Cross valid method: THROUGHOUT / σ(F): 0 / ESU R Free: 1.029 / Stereochemistry target values: MAXIMUM LIKELIHOOD
Details: HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS U VALUES : WITH TLS ADDED
RfactorNum. reflection% reflectionSelection details
Rfree0.2997 667 4.9 %RANDOM
Rwork0.2435 ---
obs0.2463 12857 83.95 %-
Solvent computationIon probe radii: 0.8 Å / Shrinkage radii: 0.8 Å / VDW probe radii: 1.2 Å / Solvent model: MASK
Displacement parametersBiso max: 741.35 Å2 / Biso mean: 263.302 Å2 / Biso min: 100.06 Å2
Baniso -1Baniso -2Baniso -3
1--5.08 Å2-0 Å20 Å2
2---7.25 Å20 Å2
3---12.34 Å2
Refinement stepCycle: final / Resolution: 4.31→45.78 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms6984 0 0 0 6984
Num. residues----938
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
X-RAY DIFFRACTIONr_bond_refined_d0.0110.0137172
X-RAY DIFFRACTIONr_bond_other_d0.0010.0176458
X-RAY DIFFRACTIONr_angle_refined_deg1.9081.6419781
X-RAY DIFFRACTIONr_angle_other_deg1.1661.57514916
X-RAY DIFFRACTIONr_dihedral_angle_1_deg7.8635933
X-RAY DIFFRACTIONr_dihedral_angle_2_deg35.94223.241290
X-RAY DIFFRACTIONr_dihedral_angle_3_deg18.432151088
X-RAY DIFFRACTIONr_dihedral_angle_4_deg16.471524
X-RAY DIFFRACTIONr_chiral_restr0.060.2969
X-RAY DIFFRACTIONr_gen_planes_refined0.0070.028183
X-RAY DIFFRACTIONr_gen_planes_other0.0010.021573
LS refinement shellResolution: 4.314→4.426 Å / Rfactor Rfree error: 0 / Total num. of bins used: 20
RfactorNum. reflection% reflection
Rfree0.411 45 -
Rwork0.442 874 -
all-919 -
obs--79.16 %
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)
15.67343.49130.71917.06161.6277.44410.56580.05480.1191-0.8301-1.0865-0.28170.608-0.6530.52060.63990.3430.0780.7475-0.03730.11787.31274.00428.833
27.282-4.8945-0.309114.422710.52549.8294-0.03570.36120.7001-0.5618-0.0616-0.1164-0.20080.12020.09730.48460.0366-0.24470.73520.24690.2633-20.76196.31130.051
34.7269-1.7834-2.434612.35534.22158.81290.6982-0.67960.3370.075-0.2167-0.4030.9961-0.4208-0.48150.4177-0.1059-0.01490.4285-0.0390.0419.4978.37850.623
46.52562.66444.21293.84663.623812.54130.60520.0209-0.59270.7077-0.4619-0.01391.6019-0.7525-0.14330.2932-0.1803-0.08610.46560.14550.2133-24.16887.57344.025
511.2533-9.3701-0.60418.10061.43787.05280.34941.2314-1.6286-0.0485-0.5810.99821.69351.1320.23160.5920.3341-0.06850.7161-0.71511.068719.63342.151-27.783
66.2148.10660.592622.36079.09265.93490.9466-0.6043-0.5651-0.1045-0.7305-0.9557-0.79070.0532-0.21610.95950.6782-0.29861.11480.0140.504729.40845.426-11.853
720.58286.6502-10.36694.9044-8.111213.4709-0.0347-0.98151.1975-0.80350.05770.07041.4787-0.0011-0.0230.76610.57010.011.1727-0.12440.320926.81741.0770.876
83.5336-0.2074-0.79354.5421-0.819619.15180.41820.23030.00440.10030.1814-0.7251-0.0288-0.3466-0.59950.42490.5488-0.00130.7675-0.10080.742735.69755.24812.511
93.3601-1.67760.01251.7124-0.345920.8010.4483-0.0232-0.0988-0.12490.171-0.1130.66110.4406-0.61930.50040.5173-0.11350.6342-0.04640.456730.20958.67936.964
109.2746-0.2164.6680.4111-0.84083.88552.07832.2188-3.6048-0.431-0.06580.09652.20820.4568-2.01242.557-0.7641-0.95992.1028-1.16392.1996-9.51333.978-34.168
1113.6787-5.4356-1.326212.6346-0.51149.68970.3275-0.9866-1.0402-0.8373-0.49390.90062.0467-1.4310.16650.5446-0.3163-0.05780.48770.01130.1132-3.77747.994-16.712
Refinement TLS group
IDRefine-IDRefine TLS-IDAuth asym-IDAuth seq-ID
1X-RAY DIFFRACTION1L2 - 107
2X-RAY DIFFRACTION2L108 - 212
3X-RAY DIFFRACTION3H1 - 119
4X-RAY DIFFRACTION4H120 - 220
5X-RAY DIFFRACTION5M302 - 361
6X-RAY DIFFRACTION6M362 - 404
7X-RAY DIFFRACTION7M405 - 431
8X-RAY DIFFRACTION8M432 - 506
9X-RAY DIFFRACTION9M507 - 589
10X-RAY DIFFRACTION10A2 - 108
11X-RAY DIFFRACTION11B2 - 118

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