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- PDB-4ckd: Model of complex between the E.coli enzyme beta-galactosidase and... -

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Basic information

Entry
Database: PDB / ID: 4ckd
TitleModel of complex between the E.coli enzyme beta-galactosidase and four single chain Fv antibody domains scFv13R4.
Components
  • BETA-GALACTOSIDASE
  • SCFV13R4 ANTIBODY FV HEAVY CHAIN
  • SCFV13R4 ANTIBODY FV LIGHT CHAIN
KeywordsHYDROLASE/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 / magnesium ion binding / extracellular region / identical protein binding
Similarity search - Function
Glycoside hydrolase, family 2, beta-galactosidase / Beta galactosidase small chain/ domain 5 / Beta-galactosidase, domain 4 / Beta galactosidase small chain / Beta-galactosidase, domain 4 / Beta galactosidase small chain / : / Glycoside hydrolase, family 2, active site / Glycosyl hydrolases family 2 acid/base catalyst. / Glycoside hydrolase, family 2, conserved site ...Glycoside hydrolase, family 2, beta-galactosidase / Beta galactosidase small chain/ domain 5 / Beta-galactosidase, domain 4 / Beta galactosidase small chain / Beta-galactosidase, domain 4 / Beta galactosidase small chain / : / Glycoside hydrolase, family 2, active site / Glycosyl hydrolases family 2 acid/base catalyst. / Glycoside hydrolase, family 2, conserved site / Glycosyl hydrolases family 2 signature 1. / : / Glycoside hydrolase, family 2 / Glycosyl hydrolases family 2, sugar binding domain / Glycoside hydrolase family 2, catalytic domain / Glycosyl hydrolases family 2, sugar binding domain / Glycosyl hydrolases family 2, TIM barrel domain / Glycoside hydrolase, family 2, immunoglobulin-like beta-sandwich / Glycosyl hydrolases family 2 / Beta-Galactosidase/glucuronidase domain superfamily / Glycoside hydrolase-type carbohydrate-binding / : / Galactose mutarotase-like domain superfamily / Immunoglobulin subtype 2 / Immunoglobulin C-2 Type / Immunoglobulin V-Type / Galactose-binding-like domain superfamily / Immunoglobulin V-set domain / Immunoglobulin V-set domain / Immunoglobulin subtype / Immunoglobulin / Glycoside hydrolase superfamily / Ig-like domain profile. / Immunoglobulin-like domain / Immunoglobulin-like domain superfamily / Immunoglobulin-like fold
Similarity search - Domain/homology
Beta-galactosidase / Immunoglobulin kappa variable 5-48 / Ig heavy chain V region 36-60
Similarity search - Component
Biological speciesESCHERICHIA COLI K-12 (bacteria)
MUS MUSCULUS (house mouse)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 13 Å
AuthorsVinothkumar, K.R. / McMullan, G. / Henderson, R.
CitationJournal: 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
DepositionJan 3, 2014Deposition site: PDBE / Processing site: PDBE
Revision 1.0Jan 15, 2014Provider: repository / Type: Initial release
Revision 1.1Mar 26, 2014Group: Database references / Source and taxonomy / Structure summary
Revision 1.2Apr 23, 2014Group: Database references
Revision 1.3Aug 30, 2017Group: Data collection / Category: em_software
Item: _em_software.fitting_id / _em_software.image_processing_id / _em_software.name
Revision 1.4Oct 23, 2019Group: Data collection / Other / Category: cell / Item: _cell.Z_PDB
Revision 1.5Oct 16, 2024Group: Data collection / Database references ...Data collection / Database references / Refinement description / Structure summary
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / em_3d_fitting_list / pdbx_entry_details / pdbx_initial_refinement_model / pdbx_modification_feature
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id / _em_3d_fitting_list.source_name / _em_3d_fitting_list.type / _pdbx_entry_details.has_protein_modification

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Structure visualization

Movie
  • Deposited structure unit
  • Imaged by Jmol
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  • Superimposition on EM map
  • EMDB-2548
  • Imaged by UCSF Chimera
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Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: BETA-GALACTOSIDASE
B: BETA-GALACTOSIDASE
C: BETA-GALACTOSIDASE
D: BETA-GALACTOSIDASE
H: SCFV13R4 ANTIBODY FV HEAVY CHAIN
I: SCFV13R4 ANTIBODY FV HEAVY CHAIN
J: SCFV13R4 ANTIBODY FV HEAVY CHAIN
K: SCFV13R4 ANTIBODY FV HEAVY CHAIN
L: SCFV13R4 ANTIBODY FV LIGHT CHAIN
M: SCFV13R4 ANTIBODY FV LIGHT CHAIN
N: SCFV13R4 ANTIBODY FV LIGHT CHAIN
O: SCFV13R4 ANTIBODY FV LIGHT CHAIN


Theoretical massNumber of molelcules
Total (without water)564,09312
Polymers564,09312
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author&software
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
MethodPQS

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Components

#1: Protein
BETA-GALACTOSIDASE / BETA-GAL / LACTASE


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
SCFV13R4 ANTIBODY FV HEAVY CHAIN


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
SCFV13R4 ANTIBODY FV LIGHT CHAIN


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
Has protein modificationY
Sequence detailsTHE DEPOSITED BETA-GALACTOSIDASE SEQUENCE CONTAINS RESIDUES 4-1024 IN THE FULL GENE SEQUENCE ...THE DEPOSITED BETA-GALACTOSIDASE SEQUENCE CONTAINS RESIDUES 4-1024 IN THE FULL GENE SEQUENCE INCLUDING METHIONINE INITIATION CODON. THESE ARE NUMBERED FROM 3 TO 1023 IN THE DEPOSITION, FOLLOWING NORMAL PRACTICE FOR X- RAY DEPOSITIONS.

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: SINGLE CHAIN FV ANTIBODY DOMAIN BOUND TO THE ENZYME BETA-GALACTOSIDASE
Type: COMPLEX / Details: 49 BEST IMAGES SELECTED OUT OF 52 RECORDED
Buffer solutionName: 20% PHOSPHATE BUFFERED SALINE (PBS) / pH: 7.4 / Details: 20% PHOSPHATE BUFFERED SALINE (PBS)
SpecimenConc.: 0.9 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: HOLEY CARBON
VitrificationCryogen 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.

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Electron microscopy imaging

Experimental equipment
Model: Tecnai Polara / Image courtesy: FEI Company
MicroscopyModel: 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 gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 59000 X / Calibrated magnification: 81600 X / Nominal defocus max: 4027 nm / Nominal defocus min: 2678 nm / Cs: 2 mm
Specimen holderTemperature: 89 K
Image recordingElectron dose: 67 e/Å2 / Film or detector model: FEI FALCON II (4k x 4k)
Image scansNum. digital images: 49
Radiation wavelengthRelative weight: 1

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Processing

EM software
IDNameVersionCategory
1CTFFIND3CTF correction
2UCSF Chimeramodel fitting
3FREALIGN3D reconstruction
4MRC IMAGE PROCESSING PACKAGE3D reconstruction
CTF correctionDetails: DONE INSIDE FREALIGN
SymmetryPoint symmetry: D2 (2x2 fold dihedral)
3D reconstructionMethod: 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 buildingProtocol: RIGID BODY FIT / Space: REAL / Target criteria: FSC CURVE BETWEEN MAP AND MODEL / Details: METHOD--RIGID BODY REFINEMENT PROTOCOL--X-RAY
Atomic model buildingPDB-ID: 1F4A

1f4a


Accession code: 1F4A / Source name: PDB / Type: experimental model
RefinementHighest resolution: 13 Å
Refinement stepCycle: LAST / Highest resolution: 13 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms39672 0 0 0 39672

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