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Yorodumi- PDB-8a64: cryoEM structure of the catalytically inactive EndoS from S. pyog... -
+Open data
-Basic information
Entry | Database: PDB / ID: 8a64 | ||||||
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Title | cryoEM structure of the catalytically inactive EndoS from S. pyogenes in complex with the Fc region of immunoglobulin G1. | ||||||
Components |
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Keywords | HYDROLASE / Endoglycosidase S / EndoS / endo-b-N-acetylglucosaminidase / Fc region / antibody / immunoglobulin G1 / Streptococcus pyogenes / N-glycans | ||||||
Function / homology | : / Endo-beta-N-acetylglucosaminidase F2, Ig-like domain / Glycosyl hydrolases family 18 (GH18) active site / Glycosyl hydrolases family 18 (GH18) active site signature. / hydrolase activity, hydrolyzing O-glycosyl compounds / Leucine-rich repeat domain superfamily / Glycoside hydrolase superfamily / carbohydrate metabolic process / Endo-beta-N-acetylglucosaminidase F2 Function and homology information | ||||||
Biological species | Streptococcus pyogenes (bacteria) Homo sapiens (human) | ||||||
Method | ELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4.6 Å | ||||||
Authors | Trastoy, B. / Cifuente, J.O. / Du, J.J. / Sundberg, E.J. / Guerin, M.E. | ||||||
Funding support | United States, 1items
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Citation | Journal: Nat Commun / Year: 2023 Title: Mechanism of antibody-specific deglycosylation and immune evasion by Streptococcal IgG-specific endoglycosidases. Authors: Beatriz Trastoy / Jonathan J Du / Javier O Cifuente / Lorena Rudolph / Mikel García-Alija / Erik H Klontz / Daniel Deredge / Nazneen Sultana / Chau G Huynh / Maria W Flowers / Chao Li / ...Authors: Beatriz Trastoy / Jonathan J Du / Javier O Cifuente / Lorena Rudolph / Mikel García-Alija / Erik H Klontz / Daniel Deredge / Nazneen Sultana / Chau G Huynh / Maria W Flowers / Chao Li / Diego E Sastre / Lai-Xi Wang / Francisco Corzana / Alvaro Mallagaray / Eric J Sundberg / Marcelo E Guerin / Abstract: Bacterial pathogens have evolved intricate mechanisms to evade the human immune system, including the production of immunomodulatory enzymes. Streptococcus pyogenes serotypes secrete two multi- ...Bacterial pathogens have evolved intricate mechanisms to evade the human immune system, including the production of immunomodulatory enzymes. Streptococcus pyogenes serotypes secrete two multi-modular endo-β-N-acetylglucosaminidases, EndoS and EndoS2, that specifically deglycosylate the conserved N-glycan at Asn297 on IgG Fc, disabling antibody-mediated effector functions. Amongst thousands of known carbohydrate-active enzymes, EndoS and EndoS2 represent just a handful of enzymes that are specific to the protein portion of the glycoprotein substrate, not just the glycan component. Here, we present the cryoEM structure of EndoS in complex with the IgG1 Fc fragment. In combination with small-angle X-ray scattering, alanine scanning mutagenesis, hydrolytic activity measurements, enzyme kinetics, nuclear magnetic resonance and molecular dynamics analyses, we establish the mechanisms of recognition and specific deglycosylation of IgG antibodies by EndoS and EndoS2. Our results provide a rational basis from which to engineer novel enzymes with antibody and glycan selectivity for clinical and biotechnological applications. | ||||||
History |
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-Structure visualization
Structure viewer | Molecule: MolmilJmol/JSmol |
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-Downloads & links
-Download
PDBx/mmCIF format | 8a64.cif.gz | 210.2 KB | Display | PDBx/mmCIF format |
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PDB format | pdb8a64.ent.gz | Display | PDB format | |
PDBx/mmJSON format | 8a64.json.gz | Tree view | PDBx/mmJSON format | |
Others | Other downloads |
-Validation report
Summary document | 8a64_validation.pdf.gz | 1.1 MB | Display | wwPDB validaton report |
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Full document | 8a64_full_validation.pdf.gz | 1.1 MB | Display | |
Data in XML | 8a64_validation.xml.gz | 40.9 KB | Display | |
Data in CIF | 8a64_validation.cif.gz | 67.9 KB | Display | |
Arichive directory | https://data.pdbj.org/pub/pdb/validation_reports/a6/8a64 ftp://data.pdbj.org/pub/pdb/validation_reports/a6/8a64 | HTTPS FTP |
-Related structure data
Related structure data | 15205MC M: map data used to model this data C: citing same article (ref.) |
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Similar structure data | Similarity search - Function & homologyF&H Search |
-Links
-Assembly
Deposited unit |
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1 |
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-Components
#1: Protein | Mass: 108297.039 Da / Num. of mol.: 1 / Mutation: E235A Source method: isolated from a genetically manipulated source Source: (gene. exp.) Streptococcus pyogenes (bacteria) / Gene: endoS, M1GAS476_1618 / Production host: Escherichia coli (E. coli) / References: UniProt: J7M8R4 | ||||
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#2: Antibody | Mass: 49386.637 Da / Num. of mol.: 2 Source method: isolated from a genetically manipulated source Details: TCPPCPAPELLGGP SVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNS TYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDEL ...Details: TCPPCPAPELLGGP SVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNS TYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDEL TKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQ QGNVFSCSVMHEALHNHYTQKSLSLSPGK Source: (gene. exp.) Homo sapiens (human) / Production host: Cricetulus griseus (Chinese hamster) #3: Polysaccharide | beta-D-mannopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose-(1-4)-2-acetamido-2-deoxy-beta- ...beta-D-mannopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose | Source method: isolated from a genetically manipulated source Has ligand of interest | Y | |
-Experimental details
-Experiment
Experiment | Method: ELECTRON MICROSCOPY |
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EM experiment | Aggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction |
-Sample preparation
Component | Name: Complex of the EndoS from S. pyogenes and the Fc region of Immunoglobulin G Type: COMPLEX / Entity ID: #1-#2 / Source: RECOMBINANT |
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Molecular weight | Value: 0.150 MDa / Experimental value: YES |
Source (natural) | Organism: Streptococcus pyogenes (bacteria) |
Source (recombinant) | Organism: Escherichia coli (E. coli) |
Buffer solution | pH: 7.5 / Details: Standard PBS |
Specimen | Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES Details: Complex stabilized by glutaraldehyde crosslinking prepared by ultracentrifugation in a fixation glycerol gradient (Grafix) and size exclusion chromatography purified at approximately 0.2 mg/mL |
Specimen support | Grid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R1.2/1.3 |
Vitrification | Instrument: FEI VITROBOT MARK II / Cryogen name: ETHANE / Humidity: 80 % / Chamber temperature: 283 K / Details: single blot |
-Electron microscopy imaging
Experimental equipment | Model: Titan Krios / Image courtesy: FEI Company |
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Microscopy | Model: TFS KRIOS |
Electron gun | Electron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM |
Electron lens | Mode: BRIGHT FIELD / Nominal defocus max: 2500 nm / Nominal defocus min: 1300 nm / Alignment procedure: COMA FREE |
Specimen holder | Cryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER |
Image recording | Electron dose: 59.4 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 QUANTUM (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 5546 |
Image scans | Width: 3838 / Height: 3710 / Movie frames/image: 60 / Used frames/image: 1-60 |
-Processing
Software | Name: PHENIX / Version: 1.20.1_4487: / Classification: refinement | ||||||||||||||||||||||||||||||||||||||||||||
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EM software |
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CTF correction | Details: RELION CTFfind4 implementation / Type: PHASE FLIPPING AND AMPLITUDE CORRECTION | ||||||||||||||||||||||||||||||||||||||||||||
Particle selection | Num. of particles selected: 797351 | ||||||||||||||||||||||||||||||||||||||||||||
Symmetry | Point symmetry: C1 (asymmetric) | ||||||||||||||||||||||||||||||||||||||||||||
3D reconstruction | Resolution: 4.6 Å / Resolution method: FSC 3 SIGMA CUT-OFF / Num. of particles: 339309 / Symmetry type: POINT | ||||||||||||||||||||||||||||||||||||||||||||
Atomic model building | Protocol: OTHER Details: Initial PDB models were rigid-body refined for individual chains. Later, the model was real-space refined removing side chains that couldn't be assigned. | ||||||||||||||||||||||||||||||||||||||||||||
Atomic model building |
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Refine LS restraints |
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