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- PDB-8cz8: Novel Anti-Mesothelin Antibodies Enable Crystallography of the In... -

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Basic information

Entry
Database: PDB / ID: 8cz8
TitleNovel Anti-Mesothelin Antibodies Enable Crystallography of the Intact Mesothelin Ectodo- main and Engineering of Potent, T cell-engaging Bispecific Therapeutics
Components
  • Mesothelin, cleaved form
  • scFv Anetumab
  • scFv of A12 anti-mesothelin antibody
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 speciesHomo sapiens (human)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 2.6 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 24, 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

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
C: scFv Anetumab
M: Mesothelin, cleaved form
E: Mesothelin, cleaved form
D: scFv of A12 anti-mesothelin antibody
A: scFv Anetumab
B: scFv of A12 anti-mesothelin antibody
hetero molecules


Theoretical massNumber of molelcules
Total (without water)137,58612
Polymers137,0106
Non-polymers5766
Water1,56787
1
C: scFv Anetumab
E: Mesothelin, cleaved form
D: scFv of A12 anti-mesothelin antibody
hetero molecules


Theoretical massNumber of molelcules
Total (without water)68,8897
Polymers68,5053
Non-polymers3844
Water543
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area3620 Å2
ΔGint-60 kcal/mol
Surface area25540 Å2
MethodPISA
2
M: Mesothelin, cleaved form
A: scFv Anetumab
B: scFv of A12 anti-mesothelin antibody
hetero molecules


Theoretical massNumber of molelcules
Total (without water)68,6975
Polymers68,5053
Non-polymers1922
Water543
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area3120 Å2
ΔGint-37 kcal/mol
Surface area25550 Å2
MethodPISA
Unit cell
Length a, b, c (Å)83.795, 83.795, 230.450
Angle α, β, γ (deg.)90.000, 90.000, 120.000
Int Tables number145
Space group name H-MP32
Noncrystallographic symmetry (NCS)NCS domain:
IDEns-IDDetails
11C
21A
12C
22A
13M
23E
14D
24B
15D
25B

NCS domain segments:

Component-ID: 0 / Refine code: 0

Dom-IDEns-IDBeg auth comp-IDBeg label comp-IDEnd auth comp-IDEnd label comp-IDAuth asym-IDLabel asym-IDAuth seq-IDLabel seq-ID
11ILEILEVALVALCA2 - 1102 - 110
21ILEILEVALVALAE2 - 1102 - 110
12GLNGLNSERSERCA128 - 246128 - 246
22GLNGLNSERSERAE128 - 246128 - 246
13THRTHRGLYGLYMB442 - 58911 - 158
23THRTHRGLYGLYEC442 - 58911 - 158
14ASPASPILEILEDD1 - 1061 - 106
24ASPASPILEILEBF1 - 1061 - 106
15SERSERSERSERDD123 - 241123 - 241
25SERSERSERSERBF123 - 241123 - 241

NCS ensembles :
ID
1
2
3
4
5

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Components

#1: Antibody scFv Anetumab


Mass: 25534.037 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Production host: Homo sapiens (human)
#2: Protein Mesothelin, cleaved form /


Mass: 17676.039 Da / Num. of mol.: 2
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
#3: Antibody scFv of A12 anti-mesothelin antibody


Mass: 25294.922 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Production host: Homo sapiens (human)
#4: Chemical
ChemComp-SO4 / SULFATE ION / Sulfate


Mass: 96.063 Da / Num. of mol.: 6 / Source method: obtained synthetically / Formula: SO4
#5: Water ChemComp-HOH / water / Water


Mass: 18.015 Da / Num. of mol.: 87 / Source method: isolated from a natural source / Formula: H2O
Has ligand of interestN

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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: 3.41 Å3/Da / Density % sol: 63.92 %
Crystal growTemperature: 295 K / Method: vapor diffusion, hanging drop / pH: 9 / Details: bicine (9.0), glycerol, 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: 1.78652 Å
DetectorType: DECTRIS PILATUS 6M / Detector: PIXEL / Date: Jun 10, 2021
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 1.78652 Å / Relative weight: 1
ReflectionResolution: 2.6→50 Å / Num. obs: 55600 / % possible obs: 99.9 % / Redundancy: 4.7 % / CC1/2: 0.988 / Rmerge(I) obs: 0.093 / Net I/σ(I): 30.8
Reflection shellResolution: 2.6→2.64 Å / Rmerge(I) obs: 0.697 / Num. unique obs: 2751 / CC1/2: 0.9

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Processing

Software
NameVersionClassification
REFMAC5.8.0267refinement
PDB_EXTRACT3.27data extraction
HKL-2000data reduction
SCALEPACKdata scaling
PHASERphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 4F3F

4f3f


Resolution: 2.6→45.16 Å / Cor.coef. Fo:Fc: 0.957 / Cor.coef. Fo:Fc free: 0.939 / SU B: 21.137 / SU ML: 0.203 / Cross valid method: THROUGHOUT / σ(F): 0 / ESU R: 0.38 / ESU R Free: 0.247 / Stereochemistry target values: MAXIMUM LIKELIHOOD
Details: HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS U VALUES : WITH TLS ADDED
RfactorNum. reflection% reflectionSelection details
Rfree0.2241 2782 5 %RANDOM
Rwork0.19 ---
obs0.1918 52762 99.75 %-
Solvent computationIon probe radii: 0.8 Å / Shrinkage radii: 0.8 Å / VDW probe radii: 1.2 Å / Solvent model: MASK
Displacement parametersBiso max: 142.09 Å2 / Biso mean: 67.271 Å2 / Biso min: 30 Å2
Baniso -1Baniso -2Baniso -3
1--0.68 Å2-0.34 Å2-0 Å2
2---0.68 Å20 Å2
3---2.21 Å2
Refinement stepCycle: final / Resolution: 2.6→45.16 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms9056 0 2427 87 11570
Biso mean--68.76 57.58 -
Num. residues----878
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
X-RAY DIFFRACTIONr_bond_refined_d0.0030.0139290
X-RAY DIFFRACTIONr_bond_other_d0.0010.0188475
X-RAY DIFFRACTIONr_angle_refined_deg1.281.63912628
X-RAY DIFFRACTIONr_angle_other_deg1.1191.57719536
X-RAY DIFFRACTIONr_dihedral_angle_1_deg6.80151196
X-RAY DIFFRACTIONr_dihedral_angle_2_deg30.17522.3413
X-RAY DIFFRACTIONr_dihedral_angle_3_deg14.916151443
X-RAY DIFFRACTIONr_dihedral_angle_4_deg18.1781548
X-RAY DIFFRACTIONr_chiral_restr0.0430.21203
X-RAY DIFFRACTIONr_gen_planes_refined0.0030.0210646
X-RAY DIFFRACTIONr_gen_planes_other0.0010.022150
Refine LS restraints NCS

Refine-ID: X-RAY DIFFRACTION / Type: interatomic distance / Weight position: 0.05

Ens-IDDom-IDAuth asym-IDNumberRms dev position (Å)
11C31220.05
12A31220.05
21C33870.06
22A33870.06
31M42170.08
32E42170.08
41D31000.06
42B31000.06
51D34860.06
52B34860.06
LS refinement shellResolution: 2.6→2.667 Å / Rfactor Rfree error: 0 / Total num. of bins used: 20
RfactorNum. reflection% reflection
Rfree0.363 186 -
Rwork0.332 3861 -
all-4047 -
obs--98.13 %
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)
11.11941.373-0.18273.1740.19750.16650.2802-0.03620.22550.2607-0.21030.2933-0.0810.0097-0.06990.1321-0.05860.05690.1855-0.07480.221215.082942.9166-31.1577
22.86132.0351-0.08161.63670.02111.61970.2975-0.3627-0.21460.2275-0.3543-0.08560.08150.14390.05680.2129-0.0681-0.06520.16150.04910.099113.710722.7221-22.7951
30.96830.0886-0.27141.8169-2.08812.4623-0.04420.26320.1821-0.066-0.0981-0.15690.14490.04180.14230.1254-0.03740.00280.22940.05650.148414.01133.3018-59.8014
42.6961-0.4410.72940.7684-0.13661.01540.10930.18290.0996-0.0591-0.14130.00970.110.13010.0320.03460.073-0.00560.25390.06060.1152-11.252246.1351-76.5328
52.9651-1.37520.24461.1548-0.14250.2336-0.5281-0.46410.40350.13070.3463-0.29630.012-0.10090.18180.15190.0959-0.01380.3627-0.07670.1899-7.572651.3415-55.6994
61.2331-0.1810.3662.06770.02670.59590.00350.1519-0.05220.1208-0.04-0.01130.0977-0.11560.03650.14-0.10750.03460.1567-0.06020.105422.49766.5755-31.0379
70.80450.2826-0.49710.81160.03571.2050.01520.07450.00730.0831-0.0438-0.0118-0.1058-0.07890.02860.2218-0.0320.01310.1009-0.02490.108623.984786.5723-22.331
80.60040.20730.3462.06342.39842.89720.07610.1528-0.0369-0.0731-0.15920.0843-0.1743-0.16040.08310.13830.0191-0.01030.27690.02110.031722.620276.5108-59.6033
91.6181-0.21780.05940.67950.39760.7887-0.0442-0.0140.1025-0.05670.0242-0.2019-0.053-0.17910.020.07440.02310.01590.25490.03530.138247.700463.9187-76.1261
102.0011-1.0214-0.05120.98660.01011.1491-0.3205-0.41030.05570.05240.2514-0.0630.0333-0.09580.06910.10870.1023-0.00740.357-0.00920.013743.775758.5118-55.6122
Refinement TLS group
IDRefine-IDRefine TLS-IDAuth asym-IDAuth seq-ID
1X-RAY DIFFRACTION1B1 - 106
2X-RAY DIFFRACTION2B123 - 241
3X-RAY DIFFRACTION3M442 - 589
4X-RAY DIFFRACTION4A2 - 112
5X-RAY DIFFRACTION5A128 - 246
6X-RAY DIFFRACTION6D1 - 106
7X-RAY DIFFRACTION7D123 - 241
8X-RAY DIFFRACTION8E442 - 589
9X-RAY DIFFRACTION9C1 - 111
10X-RAY DIFFRACTION10C128 - 246

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