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- PDB-6wkm: Fab Fragment of Anti-human LAG3 antibody (22D2) -

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Basic information

Entry
Database: PDB / ID: 6wkm
TitleFab Fragment of Anti-human LAG3 antibody (22D2)
Components
  • 22D2 Fab Heavy Chain
  • 22D2 Fab Light Chain
KeywordsIMMUNE SYSTEM / Anti Human LAG3
Function / homologyImmunoglobulins / Immunoglobulin-like / Sandwich / Mainly Beta
Function and homology information
Biological speciesMus musculus (house mouse)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 2.1 Å
AuthorsAgnihotri, P. / Mishra, A.K. / Mariuzza, R.A.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)1R01AI144422-01 United States
CitationJournal: Structure / Year: 2023
Title: CryoEM structure of a therapeutic antibody (favezelimab) bound to human LAG3 determined using a bivalent Fab as fiducial marker.
Authors: Arjun K Mishra / Salman Shahid / Sharanbasappa S Karade / Pragati Agnihotri / Alexander Kolesnikov / S Saif Hasan / Roy A Mariuzza /
Abstract: Lymphocyte activation gene 3 protein (LAG3) is an inhibitory receptor that is upregulated on exhausted T cells in tumors. LAG3 is a major target for cancer immunotherapy with many anti-LAG3 ...Lymphocyte activation gene 3 protein (LAG3) is an inhibitory receptor that is upregulated on exhausted T cells in tumors. LAG3 is a major target for cancer immunotherapy with many anti-LAG3 antibodies in clinical trials. However, there is no structural information on the epitopes recognized by these antibodies. We determined the single-particle cryoEM structure of a therapeutic antibody (favezelimab) bound to LAG3 to 3.5 Å resolution, revealing that favezelimab targets the LAG3-binding site for MHC class II, its canonical ligand. The small size of the complex between the conventional (monovalent) Fab of favezelimab and LAG3 (∼100 kDa) presented a challenge for cryoEM. Accordingly, we engineered a bivalent version of Fab favezelimab that doubled the size of the Fab-LAG3 complex and conferred a highly identifiable shape to the complex that facilitated particle selection and orientation for image processing. This study establishes bivalent Fabs as new fiducial markers for cryoEM analysis of small proteins.
History
DepositionApr 16, 2020Deposition site: RCSB / Processing site: RCSB
Revision 1.0Apr 21, 2021Provider: repository / Type: Initial release
Revision 1.1Sep 6, 2023Group: Data collection / Database references
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / citation / citation_author / database_2
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_ASTM / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year / _database_2.pdbx_DOI / _database_2.pdbx_database_accession
Revision 1.2Oct 18, 2023Group: Refinement description / Category: pdbx_initial_refinement_model
Revision 1.3Oct 23, 2024Group: Structure summary / Category: pdbx_entry_details / pdbx_modification_feature

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
H: 22D2 Fab Heavy Chain
L: 22D2 Fab Light Chain


Theoretical massNumber of molelcules
Total (without water)48,9772
Polymers48,9772
Non-polymers00
Water4,540252
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: gel filtration, Dimer
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area3480 Å2
ΔGint-28 kcal/mol
Surface area19860 Å2
MethodPISA
Unit cell
Length a, b, c (Å)97.800, 97.800, 103.120
Angle α, β, γ (deg.)90.000, 90.000, 90.000
Int Tables number92
Space group name H-MP41212
Space group name HallP4abw2nw
Symmetry operation#1: x,y,z
#2: -y+1/2,x+1/2,z+1/4
#3: y+1/2,-x+1/2,z+3/4
#4: x+1/2,-y+1/2,-z+3/4
#5: -x+1/2,y+1/2,-z+1/4
#6: -x,-y,z+1/2
#7: y,x,-z
#8: -y,-x,-z+1/2

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Components

#1: Antibody 22D2 Fab Heavy Chain


Mass: 25288.383 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 22D2 Fab Light Chain


Mass: 23688.984 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: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 252 / Source method: isolated from a natural source / Formula: H2O
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: 2.58 Å3/Da / Density % sol: 52.31 %
Crystal growTemperature: 296 K / Method: vapor diffusion, hanging drop / pH: 8
Details: PEG 8000, sodium phosphate dibasic, sodium chloride
PH range: 8-8.5

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: APS / Beamline: 23-ID-D / Wavelength: 0.9796 Å
DetectorType: DECTRIS PILATUS3 6M / Detector: PIXEL / Date: Nov 2, 2019
RadiationMonochromator: Double crystal cryo-cooled / Protocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.9796 Å / Relative weight: 1
ReflectionResolution: 2.1→45.65 Å / Num. obs: 29501 / % possible obs: 99.2 % / Redundancy: 6.5 % / Biso Wilson estimate: 39 Å2 / CC1/2: 0.98 / Rmerge(I) obs: 0.072 / Rpim(I) all: 0.04 / Rrim(I) all: 0.08 / Χ2: 1.23 / Net I/σ(I): 16.7
Reflection shellResolution: 2.1→2.16 Å / Redundancy: 6.4 % / Rmerge(I) obs: 0.3 / Mean I/σ(I) obs: 4.5 / Num. unique obs: 2378 / CC1/2: 0.95 / Rpim(I) all: 0.18 / Rrim(I) all: 0.34 / Χ2: 0.62 / % possible all: 98.8

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Processing

Software
NameVersionClassification
REFMACrefinement
PHENIX1.17.1_3660refinement
HKL-2000data reduction
Aimlessdata scaling
PHASERphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 1mim

1mim


Resolution: 2.1→45.61 Å / SU ML: 0.2451 / Cross valid method: FREE R-VALUE / σ(F): 1.37 / Phase error: 26.3459
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
RfactorNum. reflection% reflection
Rfree0.2347 1523 5.16 %
Rwork0.2027 27967 -
obs0.2044 29490 98.92 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL
Displacement parametersBiso mean: 42.71 Å2
Refinement stepCycle: LAST / Resolution: 2.1→45.61 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms3301 0 0 253 3554
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.00233377
X-RAY DIFFRACTIONf_angle_d0.52964594
X-RAY DIFFRACTIONf_chiral_restr0.0416515
X-RAY DIFFRACTIONf_plane_restr0.0042595
X-RAY DIFFRACTIONf_dihedral_angle_d12.73361211
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
2.1-2.170.31241500.26142449X-RAY DIFFRACTION98.34
2.17-2.250.27661240.2452487X-RAY DIFFRACTION98.19
2.25-2.340.31031400.24652498X-RAY DIFFRACTION98.73
2.34-2.440.2991270.24332510X-RAY DIFFRACTION98.8
2.44-2.570.30041390.24692519X-RAY DIFFRACTION98.66
2.57-2.730.25641200.23552517X-RAY DIFFRACTION98.8
2.73-2.940.29381270.24842530X-RAY DIFFRACTION98.96
2.94-3.240.25531440.22562545X-RAY DIFFRACTION99.19
3.24-3.710.27011400.20482567X-RAY DIFFRACTION99.34
3.71-4.670.19791670.16542588X-RAY DIFFRACTION99.57
4.67-45.610.17181450.16842757X-RAY DIFFRACTION99.52

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