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- EMDB-40646: CryoEM structure of a therapeutic antibody (favezelimab) bound to... -

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Basic information

Entry
Database: EMDB / ID: EMD-40646
TitleCryoEM structure of a therapeutic antibody (favezelimab) bound to human LAG3
Map data
Sample
  • Complex: therapeutic antibody (favezelimab) bound to human LAG3
    • Protein or peptide: Lymphocyte activation gene 3 protein
    • Protein or peptide: favezelimab Fab heavy chain
    • Protein or peptide: favezelimab Fab light chain
  • Ligand: 2-acetamido-2-deoxy-beta-D-glucopyranose
KeywordsLymphocyte activation gene 3 protein / favezelimab-LAG3 complex / cryoEM / Single particle reconstruction. / IMMUNOSUPPRESSANT
Function / homology
Function and homology information


plasmacytoid dendritic cell activation / negative regulation of regulatory T cell differentiation / positive regulation of natural killer cell mediated cytotoxicity / MHC class II protein binding / natural killer cell mediated cytotoxicity / negative regulation of interleukin-2 production / regulation of immune response / MHC class II antigen presentation / antigen binding / T cell activation ...plasmacytoid dendritic cell activation / negative regulation of regulatory T cell differentiation / positive regulation of natural killer cell mediated cytotoxicity / MHC class II protein binding / natural killer cell mediated cytotoxicity / negative regulation of interleukin-2 production / regulation of immune response / MHC class II antigen presentation / antigen binding / T cell activation / transmembrane signaling receptor activity / adaptive immune response / cell surface receptor signaling pathway / external side of plasma membrane / extracellular region / plasma membrane
Similarity search - Function
Interleukin-1 receptor family / Immunoglobulin subtype / Immunoglobulin / Ig-like domain profile. / Immunoglobulin-like domain / Immunoglobulin-like domain superfamily / Immunoglobulin-like fold
Similarity search - Domain/homology
Lymphocyte activation gene 3 protein
Similarity search - Component
Biological speciesHomo sapiens (human) / Mus musculus (house mouse)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.61 Å
AuthorsMishra AK / Shahid S / Karade SS / Mariuzza RA
Funding support United States, 1 items
OrganizationGrant numberCountry
National Institutes of Health/National Cancer Institute (NIH/NCI)AI144422 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 28, 2023-
Header (metadata) releaseSep 6, 2023-
Map releaseSep 6, 2023-
UpdateNov 13, 2024-
Current statusNov 13, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_40646.png

Downloads & links

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Map

FileDownload / File: emd_40646.map.gz / Format: CCP4 / Size: 125 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.12 Å/pix.
x 320 pix.
= 358.4 Å		1.12 Å/pix.
x 320 pix.
= 358.4 Å		1.12 Å/pix.
x 320 pix.
= 358.4 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.12 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.14
Minimum - Maximum-1.0148972 - 1.8230857
Average (Standard dev.)-0.00015731175 (±0.02433722)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions320320320
Spacing320320320
CellA=B=C: 358.4 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #2

Fileemd_40646_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_40646_half_map_2.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : therapeutic antibody (favezelimab) bound to human LAG3

EntireName: therapeutic antibody (favezelimab) bound to human LAG3
Components
  • Complex: therapeutic antibody (favezelimab) bound to human LAG3
    • Protein or peptide: Lymphocyte activation gene 3 protein
    • Protein or peptide: favezelimab Fab heavy chain
    • Protein or peptide: favezelimab Fab light chain
  • Ligand: 2-acetamido-2-deoxy-beta-D-glucopyranose

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Supramolecule #1: therapeutic antibody (favezelimab) bound to human LAG3

SupramoleculeName: therapeutic antibody (favezelimab) bound to human LAG3
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#3
Source (natural)Organism: Homo sapiens (human)

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Macromolecule #1: Lymphocyte activation gene 3 protein

MacromoleculeName: Lymphocyte activation gene 3 protein / type: protein_or_peptide / ID: 1 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 57.50827 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: MWEAQFLGLL FLQPLWVAPV KPLQPGAEVP VVWAQEGAPA QLPCSPTIPL QDLSLLRRAG VTWQHQPDSG PPAAAPGHPL APGPHPAAP SSWGPRPRRY TVLSVGPGGL RSGRLPLQPR VQLDERGRQR GDFSLWLRPA RRADAGEYRA AVHLRDRALS C RLRLRLGQ ...String:
MWEAQFLGLL FLQPLWVAPV KPLQPGAEVP VVWAQEGAPA QLPCSPTIPL QDLSLLRRAG VTWQHQPDSG PPAAAPGHPL APGPHPAAP SSWGPRPRRY TVLSVGPGGL RSGRLPLQPR VQLDERGRQR GDFSLWLRPA RRADAGEYRA AVHLRDRALS C RLRLRLGQ ASMTASPPGS LRASDWVILN CSFSRPDRPA SVHWFRNRGQ GRVPVRESPH HHLAESFLFL PQVSPMDSGP WG CILTYRD GFNVSIMYNL TVLGLEPPTP LTVYAGAGSR VGLPCRLPAG VGTRSFLTAK WTPPGGGPDL LVTGDNGDFT LRL EDVSQA QAGTYTCHIH LQEQQLNATV TLAIITVTPK SFGSPGSLGK LLCEVTPVSG QERFVWSSLD TPSQRSFSGP WLEA QEAQL LSQPWQCQLY QGERLLGAAV YFTELSSPGA QRSGRAPGAL PAGHLLLFLI LGVLSLLLLV TGAFGFHLWR RQWRP RRFS ALEQGIHPPQ AQSKIEELEQ EPEPEPEPEP EPEPEPEPEQ L

UniProtKB: Lymphocyte activation gene 3 protein

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Macromolecule #2: favezelimab Fab heavy chain

MacromoleculeName: favezelimab Fab heavy chain / type: protein_or_peptide / ID: 2 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Mus musculus (house mouse)
Molecular weightTheoretical: 27.245727 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: MGWTWIFLFF LSGTAGVLSE VLLLQSGPEL VKPGTSVKIP CKASGYTFTD YNVDWVKQRH GKGLEWIGDI NPNNGGTIYS QKFKGKATL TVDKSSSTAF MELRSLTSED TAVYFCARNY RWFGAMDHWG QGTSVTVSST KGPSVFPLAP SSKSTSGGTA A LGCLVKDY ...String:
MGWTWIFLFF LSGTAGVLSE VLLLQSGPEL VKPGTSVKIP CKASGYTFTD YNVDWVKQRH GKGLEWIGDI NPNNGGTIYS QKFKGKATL TVDKSSSTAF MELRSLTSED TAVYFCARNY RWFGAMDHWG QGTSVTVSST KGPSVFPLAP SSKSTSGGTA A LGCLVKDY FPEPVTVSWN SGALTSGVHT FPAVLQSSGL YSLSSVVTVP SSSLGTQTYI CNVNHKPSNT KVDKRVEPKS CD KTAGWSH PQFEK

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Macromolecule #3: favezelimab Fab light chain

MacromoleculeName: favezelimab Fab light chain / type: protein_or_peptide / ID: 3 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Mus musculus (house mouse)
Molecular weightTheoretical: 25.915627 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: METDTILLWV LLLWVPGSTG DIVLTQSPAS LAVSPGQRAT ISCKASQSLD YEGDSDMNWY QQKPGQPPRL LISGASNLES GIPARFSGS GSGTDFTVNI HPVEEEDAAT YYCQQSTEDP RTFGGGTKLE IKRTVAAPSV FIFPPSDEQL KSGTASVVCL L NNFYPREA ...String:
METDTILLWV LLLWVPGSTG DIVLTQSPAS LAVSPGQRAT ISCKASQSLD YEGDSDMNWY QQKPGQPPRL LISGASNLES GIPARFSGS GSGTDFTVNI HPVEEEDAAT YYCQQSTEDP RTFGGGTKLE IKRTVAAPSV FIFPPSDEQL KSGTASVVCL L NNFYPREA KYQWKVDNAL QSGNSQESVT EQDSKDSTYS LSSTLTLSKA DYEKHKVYAC EVTHQGLSSP VTKSFNRGEC

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Macromolecule #4: 2-acetamido-2-deoxy-beta-D-glucopyranose

MacromoleculeName: 2-acetamido-2-deoxy-beta-D-glucopyranose / type: ligand / ID: 4 / Number of copies: 4 / Formula: NAG
Molecular weightTheoretical: 221.208 Da
Chemical component information

ChemComp-NAG:
2-acetamido-2-deoxy-beta-D-glucopyranose

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

BufferpH: 8
GridMaterial: COPPER
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Number grids imaged: 1 / Number real images: 9508 / Average exposure time: 3.2 sec. / Average electron dose: 50.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: OTHER / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 2.5 µm / Nominal defocus min: 1.0 µm / Nominal magnification: 81000
Sample stageCooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: NONE
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.61 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 171144
Initial angle assignmentType: OTHER
Final angle assignmentType: OTHER

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Atomic model buiding 1

Initial modelPDB ID:

7tzg


Chain - Chain ID: C / Chain - Source name: PDB / Chain - Initial model type: experimental model
RefinementSpace: REAL / Protocol: RIGID BODY FIT
Output model

PDB-8so3:
CryoEM structure of a therapeutic antibody (favezelimab) bound to human LAG3

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