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

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Basic information

Entry
Database: PDB / ID: 8cyh
TitleNovel Anti-Mesothelin Antibodies Enable Crystallography of the Intact Mesothelin Ectodo- main and Engineering of Potent, T cell-engaging Bispecific Therapeutics
Components
  • A12 antibody heavy chain
  • A12 antibody light chain
  • Mesothelin, cleaved form
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: 3.38 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 23, 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
L: A12 antibody light chain
H: A12 antibody heavy chain
M: Mesothelin, cleaved form
hetero molecules


Theoretical massNumber of molelcules
Total (without water)84,0405
Polymers83,5973
Non-polymers4422
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)65.112, 124.862, 302.748
Angle α, β, γ (deg.)90.000, 90.000, 90.000
Int Tables number23
Space group name H-MI222

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Components

#1: Antibody A12 antibody light chain


Mass: 23325.744 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Production host: Homo sapiens (human)
#2: Antibody A12 antibody heavy chain


Mass: 23308.156 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / 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: Sugar ChemComp-NAG / 2-acetamido-2-deoxy-beta-D-glucopyranose / N-acetyl-beta-D-glucosamine / 2-acetamido-2-deoxy-beta-D-glucose / 2-acetamido-2-deoxy-D-glucose / 2-acetamido-2-deoxy-glucose / N-ACETYL-D-GLUCOSAMINE / N-Acetylglucosamine


Type: D-saccharide, beta linking / Mass: 221.208 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C8H15NO6
IdentifierTypeProgram
DGlcpNAcbCONDENSED IUPAC CARBOHYDRATE SYMBOLGMML 1.0
N-acetyl-b-D-glucopyranosamineCOMMON NAMEGMML 1.0
b-D-GlcpNAcIUPAC CARBOHYDRATE SYMBOLPDB-CARE 1.0
GlcNAcSNFG CARBOHYDRATE SYMBOLGMML 1.0
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: 3.68 Å3/Da / Density % sol: 66.58 %
Crystal growTemperature: 295 K / Method: vapor diffusion, hanging drop / pH: 8 / Details: Tris (8.0), sodium citrate, Peg3350

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: ALS / Beamline: 5.0.2 / Wavelength: 0.97926 Å
DetectorType: DECTRIS PILATUS 6M / Detector: PIXEL / Date: Aug 29, 2019
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.97926 Å / Relative weight: 1
ReflectionResolution: 3.35→50 Å / Num. obs: 17506 / % possible obs: 98.7 % / Redundancy: 5.8 % / Rmerge(I) obs: 0.149 / Rpim(I) all: 0.068 / Rrim(I) all: 0.164 / Χ2: 1.851 / Net I/σ(I): 8.4 / Num. measured all: 101701
Reflection shell

Diffraction-ID: 1

Resolution (Å)Redundancy (%)Rmerge(I) obsNum. unique obsCC1/2Rpim(I) allRrim(I) allΧ2% possible all
3.35-3.415.60.9348420.5980.4381.0350.87396.1
3.41-3.475.70.8248360.7170.380.910.92697.7
3.47-3.545.70.6918570.8430.3160.7630.96397.1
3.54-3.615.80.7548310.7540.3490.8331.1797.1
3.61-3.695.60.6518640.7820.3020.721.32897.7
3.69-3.775.40.4768420.8550.2250.5281.1795.6
3.77-3.875.70.4868560.8770.2220.5361.3799.2
3.87-3.975.60.3448560.9670.1590.381.21398.8
3.97-4.096.10.38790.970.1290.3271.27999.7
4.09-4.226.30.2558780.9830.110.2781.49299.8
4.22-4.376.20.218730.980.0910.231.91599.8
4.37-4.556.20.1798820.990.0780.1952.02599.9
4.55-4.756.10.1638800.9870.0720.1792.185100
4.75-55.60.1518840.9850.0690.1662.43100
5-5.3260.1388980.9910.0610.1512.278100
5.32-5.736.50.1348840.9920.0570.1452.30799.9
5.73-6.36.30.1288960.9910.0550.142.248100
6.3-7.215.70.0989040.9950.0450.1082.76899.9
7.21-9.085.40.0639110.9960.030.073.16399.2
9.08-504.70.0499530.9960.0250.0553.80896.9

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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: 4F3F

4f3f


Resolution: 3.38→44.38 Å / Cor.coef. Fo:Fc: 0.926 / Cor.coef. Fo:Fc free: 0.911 / SU B: 85.447 / SU ML: 0.542 / Cross valid method: THROUGHOUT / σ(F): 0 / ESU R Free: 0.519 / Stereochemistry target values: MAXIMUM LIKELIHOOD
Details: HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS U VALUES : WITH TLS ADDED
RfactorNum. reflection% reflectionSelection details
Rfree0.2676 851 4.9 %RANDOM
Rwork0.2296 ---
obs0.2315 16652 97.88 %-
Solvent computationIon probe radii: 0.8 Å / Shrinkage radii: 0.8 Å / VDW probe radii: 1.2 Å / Solvent model: MASK
Displacement parametersBiso max: 272.6 Å2 / Biso mean: 118.853 Å2 / Biso min: 78.63 Å2
Baniso -1Baniso -2Baniso -3
1-14.26 Å2-0 Å20 Å2
2---6.74 Å2-0 Å2
3----7.52 Å2
Refinement stepCycle: final / Resolution: 3.38→44.38 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms5216 0 28 0 5244
Biso mean--199.21 --
Num. residues----717
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
X-RAY DIFFRACTIONr_bond_refined_d0.0060.0135369
X-RAY DIFFRACTIONr_bond_other_d0.0010.0174805
X-RAY DIFFRACTIONr_angle_refined_deg1.141.6457356
X-RAY DIFFRACTIONr_angle_other_deg1.0931.57711060
X-RAY DIFFRACTIONr_dihedral_angle_1_deg8.6095713
X-RAY DIFFRACTIONr_dihedral_angle_2_deg37.67224.126206
X-RAY DIFFRACTIONr_dihedral_angle_3_deg19.06815752
X-RAY DIFFRACTIONr_dihedral_angle_4_deg26.631513
X-RAY DIFFRACTIONr_chiral_restr0.0320.2734
X-RAY DIFFRACTIONr_gen_planes_refined0.0020.026203
X-RAY DIFFRACTIONr_gen_planes_other00.021151
LS refinement shellResolution: 3.38→3.465 Å / Rfactor Rfree error: 0
RfactorNum. reflection% reflection
Rfree0.409 65 -
Rwork0.38 1072 -
obs--86.93 %
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)
11.8465-0.3226-0.54962.11360.73081.3104-0.06060.0959-0.0578-0.0894-0.1741-0.0768-0.28960.1360.23470.3199-0.0190.02950.32170.05470.5333-29.3227-29.679137.583
20.44790.4004-0.41983.6403-1.84386.0428-0.0287-0.43420.3199-0.29030.048-0.09710.0076-0.2801-0.01930.03680.03390.06490.5819-0.22720.6505-36.1595-21.352872.3703
31.8060.8463-0.87422.0256-1.33083.6913-0.5018-0.0556-0.3081-0.44730.0787-0.27680.2535-0.07310.42310.2661-0.04480.13430.2254-0.10010.6271-35.1653-50.380743.9882
42.4718-1.5012-0.94485.40620.29640.6204-0.0372-0.36950.122-0.3895-0.06210.08140.0908-0.14030.09920.0706-0.00870.00590.5167-0.06570.5772-48.9795-30.91467.6936
52.3913-1.0451-1.16367.64920.71454.1187-0.139-0.07590.16320.2692-0.1382-0.1270.2836-0.16660.27730.57770.0344-0.07610.19250.1480.2491-20.241510.7042-21.2692
60.0316-0.1173-0.36425.85950.10947.7735-0.084-0.0157-0.04110.0593-0.4595-0.0036-0.32890.33160.54360.57140.05710.03440.17170.19530.4184-16.3284-6.3566-14.1527
70.45560.45050.24764.4338-2.43862.04650.1446-0.09880.1783-0.1177-0.3071-0.14930.20580.1040.16250.50520.08870.18160.26380.12520.4516-17.8438-26.3658-1.384
80.1193-0.30440.16142.613-1.89195.060.03580.01370.1306-0.2489-0.33170.05490.4044-0.11770.29590.7178-0.08730.13950.2229-0.09870.2932-30.5628-46.555514.0654
Refinement TLS group
IDRefine-IDRefine TLS-IDAuth asym-IDAuth seq-ID
1X-RAY DIFFRACTION1L1 - 108
2X-RAY DIFFRACTION2L109 - 212
3X-RAY DIFFRACTION3H1 - 119
4X-RAY DIFFRACTION4H120 - 220
5X-RAY DIFFRACTION5M299 - 360
6X-RAY DIFFRACTION6M361 - 404
7X-RAY DIFFRACTION7M415 - 509
8X-RAY DIFFRACTION8M510 - 593

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