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Yorodumi- PDB-4ckd: Model of complex between the E.coli enzyme beta-galactosidase and... -
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-Basic information
Entry | Database: PDB / ID: 4ckd | ||||||
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Title | Model of complex between the E.coli enzyme beta-galactosidase and four single chain Fv antibody domains scFv13R4. | ||||||
Components |
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Keywords | HYDROLASE/IMMUNE SYSTEM / HYDROLASE-IMMUNE SYSTEM COMPLEX | ||||||
Function / homology | Function and homology information alkali metal ion binding / lactose catabolic process / beta-galactosidase complex / beta-galactosidase / beta-galactosidase activity / immunoglobulin complex / immunoglobulin mediated immune response / antigen binding / carbohydrate binding / adaptive immune response ...alkali metal ion binding / lactose catabolic process / beta-galactosidase complex / beta-galactosidase / beta-galactosidase activity / immunoglobulin complex / immunoglobulin mediated immune response / antigen binding / carbohydrate binding / adaptive immune response / immune response / magnesium ion binding / extracellular space / identical protein binding Similarity search - Function | ||||||
Biological species | ESCHERICHIA COLI K-12 (bacteria) MUS MUSCULUS (house mouse) | ||||||
Method | ELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 13 Å | ||||||
Authors | Vinothkumar, K.R. / McMullan, G. / Henderson, R. | ||||||
Citation | Journal: Structure / Year: 2014 Title: Molecular mechanism of antibody-mediated activation of β-galactosidase. Authors: Kutti R Vinothkumar / Greg McMullan / Richard Henderson / Abstract: Binding of a single-chain Fv antibody to Escherichia coli β-galactosidase (β-gal) is known to stabilize the enzyme and activate several inactive point mutants, historically called antibody-mediated ...Binding of a single-chain Fv antibody to Escherichia coli β-galactosidase (β-gal) is known to stabilize the enzyme and activate several inactive point mutants, historically called antibody-mediated enzyme formation mutants. To understand the nature of this activation, we have determined by electron cryo-microscopy the structure of the complex between β-gal and the antibody scFv13R4. Our structure localizes the scFv13R4 binding site to the crevice between domains 1 and 3 in each β-gal subunit. The mutations that scFv13R4 counteracts are located between the antibody binding site and the active site of β-gal, at one end of the TIM-barrel that forms domain 3 where the substrate lactose is hydrolyzed. The mode of binding suggests how scFv stabilizes both the active site of β-gal and the tetrameric state. | ||||||
History |
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-Structure visualization
Movie |
Movie viewer |
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Structure viewer | Molecule: MolmilJmol/JSmol |
-Downloads & links
-Download
PDBx/mmCIF format | 4ckd.cif.gz | 965.1 KB | Display | PDBx/mmCIF format |
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PDB format | pdb4ckd.ent.gz | 803 KB | Display | PDB format |
PDBx/mmJSON format | 4ckd.json.gz | Tree view | PDBx/mmJSON format | |
Others | Other downloads |
-Validation report
Arichive directory | https://data.pdbj.org/pub/pdb/validation_reports/ck/4ckd ftp://data.pdbj.org/pub/pdb/validation_reports/ck/4ckd | HTTPS FTP |
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-Related structure data
Related structure data | 2548MC M: map data used to model this data C: citing same article (ref.) |
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Similar structure data |
-Links
-Assembly
Deposited unit |
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1 |
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-Components
#1: Protein | Mass: 116602.484 Da / Num. of mol.: 4 Source method: isolated from a genetically manipulated source Source: (gene. exp.) ESCHERICHIA COLI K-12 (bacteria) / Description: SIGMA CATALOGUE NUMBER G3153 / Production host: ESCHERICHIA COLI (E. coli) / References: UniProt: P00722, beta-galactosidase #2: Antibody | Mass: 12795.939 Da / Num. of mol.: 4 Source method: isolated from a genetically manipulated source Source: (gene. exp.) MUS MUSCULUS (house mouse) / Production host: ESCHERICHIA COLI (E. coli) / Strain (production host): BL21(DE3) / Variant (production host): PM12 PLYSS / References: UniProt: P01823*PLUS #3: Antibody | Mass: 11624.795 Da / Num. of mol.: 4 Source method: isolated from a genetically manipulated source Source: (gene. exp.) MUS MUSCULUS (house mouse) / Production host: ESCHERICHIA COLI (E. coli) / Strain (production host): BL21(DE3) / Variant (production host): PM12 PLYSS / References: UniProt: P01642*PLUS Sequence details | THE DEPOSITED BETA-GALACTOSIDASE SEQUENCE CONTAINS RESIDUES 4-1024 IN THE FULL GENE SEQUENCE ...THE DEPOSITED BETA-GALACTOSID | |
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-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: SINGLE CHAIN FV ANTIBODY DOMAIN BOUND TO THE ENZYME BETA-GALACTOSIDASE Type: COMPLEX / Details: 49 BEST IMAGES SELECTED OUT OF 52 RECORDED |
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Buffer solution | Name: 20% PHOSPHATE BUFFERED SALINE (PBS) / pH: 7.4 / Details: 20% PHOSPHATE BUFFERED SALINE (PBS) |
Specimen | Conc.: 0.9 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES |
Specimen support | Details: HOLEY CARBON |
Vitrification | Cryogen name: ETHANE Details: VITRIFICATION 1 -- CRYOGEN- ETHANE, HUMIDITY- 100, TEMPERATURE- 100, INSTRUMENT- OTHER, METHOD- BLOT FOR 10-20 SECONDS UNTIL DIAMETER OF BLOTTED MENISCUS CEASES TO EXPAND, BEFORE PLUNGING. |
-Electron microscopy imaging
Experimental equipment | Model: Tecnai Polara / Image courtesy: FEI Company |
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Microscopy | Model: FEI POLARA 300 / Date: Aug 2, 2012 Details: EXPOSURE INTENSITY SET TO GIVE 50 ELECTRONS PER PIXEL PER SECOND AT THE DETECTOR. THIS TRANSLATES INTO 16 ELECTRONS PER SQUARE ANGSTROM PER SECOND AT THE SPECIMEN. |
Electron gun | Electron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM |
Electron lens | Mode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 59000 X / Calibrated magnification: 81600 X / Nominal defocus max: 4027 nm / Nominal defocus min: 2678 nm / Cs: 2 mm |
Specimen holder | Temperature: 89 K |
Image recording | Electron dose: 67 e/Å2 / Film or detector model: FEI FALCON II (4k x 4k) |
Image scans | Num. digital images: 49 |
Radiation wavelength | Relative weight: 1 |
-Processing
EM software |
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CTF correction | Details: DONE INSIDE FREALIGN | ||||||||||||||||||||
Symmetry | Point symmetry: D2 (2x2 fold dihedral) | ||||||||||||||||||||
3D reconstruction | Method: PROJECTION MATCHING / Resolution: 13 Å / Num. of particles: 2965 / Nominal pixel size: 1.74 Å / Actual pixel size: 1.72 Å Magnification calibration: MAGNIFICATION REFINED BY MAXIMISING FSC AGAINST ATOMIC MODEL Details: FREALIGN MAP OBTAINED FROM 2965 PARTICLES USING D2 SYMMETRY THIS IS A MODEL MADE BY DOCKING TWO ATOMIC MODELS AS RIGID BODIES INTO A 13 ANGSTROM RESOLUTION DENSITY MAP. SUBMISSION BASED ON ...Details: FREALIGN MAP OBTAINED FROM 2965 PARTICLES USING D2 SYMMETRY THIS IS A MODEL MADE BY DOCKING TWO ATOMIC MODELS AS RIGID BODIES INTO A 13 ANGSTROM RESOLUTION DENSITY MAP. SUBMISSION BASED ON EXPERIMENTAL DATA FROM EMDB EMD-2548. (DEPOSITION ID: 12230). Symmetry type: POINT | ||||||||||||||||||||
Atomic model building | Protocol: RIGID BODY FIT / Space: REAL / Target criteria: FSC CURVE BETWEEN MAP AND MODEL / Details: METHOD--RIGID BODY REFINEMENT PROTOCOL--X-RAY | ||||||||||||||||||||
Atomic model building | PDB-ID: 1F4A | ||||||||||||||||||||
Refinement | Highest resolution: 13 Å | ||||||||||||||||||||
Refinement step | Cycle: LAST / Highest resolution: 13 Å
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