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- EMDB-2548: Single particle electron cryomicroscopy of the complex between th... -

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Basic information

Entry
Database: EMDB / ID: EMD-2548
TitleSingle particle electron cryomicroscopy of the complex between the E.coli enzyme beta-galactosidase and the single chain Fv antibody scFv13R4.
Map dataMap of the structure of the complex between beta-galactosidase and the scFv13R4 single chain antibody domain.
Sample
  • Sample: Single chain Fv antibody domain bound to the enzyme beta-galactosidase
  • Protein or peptide: beta-galactosidase
  • Protein or peptide: SCFV13R4 ANTIBODY FV HEAVY CHAIN
  • Protein or peptide: SCFV13R4 ANTIBODY FV LIGHT CHAIN
Keywordsbeta-galactosidase / antibody / scFv13R4 / single particle cryoEM
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
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, 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 / Glycoside hydrolase family 2, catalytic domain / Glycosyl hydrolases family 2, TIM barrel domain / Glycoside hydrolase, family 2, immunoglobulin-like beta-sandwich / Glycosyl hydrolases family 2, sugar binding domain / Glycosyl hydrolases family 2 / Glycosyl hydrolases family 2, sugar binding domain / Beta-Galactosidase/glucuronidase domain superfamily / Glycoside hydrolase-type carbohydrate-binding / Glycoside hydrolase family 1 / 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 / Ig kappa chain V-V region L7 / Ig heavy chain V region 36-60
Similarity search - Component
Biological speciesEscherichia coli (E. coli) / Mus musculus (house mouse)
Methodsingle particle reconstruction / cryo EM / negative staining / Resolution: 13.0 Å
AuthorsVinothkumar KR / 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 2, 2014-
Header (metadata) releaseJan 15, 2014-
Map releaseJan 15, 2014-
UpdateApr 23, 2014-
Current statusApr 23, 2014Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.13
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 0.13
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-4ckd
  • Surface level: 0.13
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_2548.tif

Downloads & links

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Map

FileDownload / File: emd_2548.map.gz / Format: CCP4 / Size: 3.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationMap of the structure of the complex between beta-galactosidase and the scFv13R4 single chain antibody domain.
Voxel sizeX=Y=Z: 3 Å
Density
Contour LevelBy AUTHOR: 0.13 / Movie #1: 0.13
Minimum - Maximum-0.05654818 - 0.32245788
Average (Standard dev.)-0.00343177 (±0.04594183)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions100100100
Spacing100100100
CellA=B=C: 300.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z333
M x/y/z100100100
origin x/y/z0.0000.0000.000
length x/y/z300.000300.000300.000
α/β/γ90.00090.00090.000
start NX/NY/NZ-207-207-206
NX/NY/NZ414414414
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS100100100
D min/max/mean-0.0570.322-0.003

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Supplemental data

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Sample components

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Entire : Single chain Fv antibody domain bound to the enzyme beta-galactosidase

EntireName: Single chain Fv antibody domain bound to the enzyme beta-galactosidase
Components
  • Sample: Single chain Fv antibody domain bound to the enzyme beta-galactosidase
  • Protein or peptide: beta-galactosidase
  • Protein or peptide: SCFV13R4 ANTIBODY FV HEAVY CHAIN
  • Protein or peptide: SCFV13R4 ANTIBODY FV LIGHT CHAIN

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Supramolecule #1000: Single chain Fv antibody domain bound to the enzyme beta-galactosidase

SupramoleculeName: Single chain Fv antibody domain bound to the enzyme beta-galactosidase
type: sample / ID: 1000
Details: The occupancy of the antibody domains was estimated to be 90%
Oligomeric state: one D2 symmetry tetramer, with 4 Fv antibody domains (heavy & light chains)
Number unique components: 3
Molecular weightTheoretical: 560 KDa
Method: Known stoichiometry and known molecular weights of the components, which are 450 kiloDaltons for the D2 beta-galactosidase and 28 kiloDaltons for each Fv domain.

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Macromolecule #1: beta-galactosidase

MacromoleculeName: beta-galactosidase / type: protein_or_peptide / ID: 1 / Details: Central tetrameric enzyme in complex / Number of copies: 4 / Oligomeric state: tetramer / Recombinant expression: Yes
Source (natural)Organism: Escherichia coli (E. coli) / Strain: K-12
Molecular weightTheoretical: 465 KDa
SequenceUniProtKB: Beta-galactosidase / GO: beta-galactosidase complex / InterPro: Glycoside hydrolase family 1

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Macromolecule #2: SCFV13R4 ANTIBODY FV HEAVY CHAIN

MacromoleculeName: SCFV13R4 ANTIBODY FV HEAVY CHAIN / type: protein_or_peptide / ID: 2 / Number of copies: 4 / Oligomeric state: Tetrameric / Recombinant expression: Yes
Source (natural)Organism: Mus musculus (house mouse) / synonym: HOUSE MOUSE
Recombinant expressionOrganism: Escherichia coli (E. coli) / Recombinant strain: BL21(DE3) / Recombinant plasmid: pet16B

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Macromolecule #3: SCFV13R4 ANTIBODY FV LIGHT CHAIN

MacromoleculeName: SCFV13R4 ANTIBODY FV LIGHT CHAIN / type: protein_or_peptide / ID: 3 / Number of copies: 4 / Oligomeric state: Tetrameric / Recombinant expression: Yes
Source (natural)Organism: Mus musculus (house mouse) / synonym: HOUSE MOUSE
Recombinant expressionOrganism: Escherichia coli (E. coli) / Recombinant strain: BL21(DE3) / Recombinant plasmid: pet16b

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Experimental details

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Structure determination

Methodnegative staining, cryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration0.9 mg/mL
BufferpH: 7.4 / Details: 20% phosphate buffered saline (PBS)
StainingType: NEGATIVE
Details: Sample applied to glow-discharged (30 seconds in residual air) grids, blotted and frozen rapidly in an environmental plunge-freeze apparatus (Bellare et al, J. Electr. Micros. Tech., 1988, 10, 87-111).
GridDetails: Qunatifoil grids with 1.2 micron holes
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 100 K / Instrument: OTHER
Method: Blot for 10-20 seconds until diameter of blotted meniscus ceases to expand, before plunging.

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Electron microscopy

MicroscopeFEI POLARA 300
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 81600 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.0 mm / Nominal defocus max: 4.027 µm / Nominal defocus min: 2.678 µm / Nominal magnification: 59000
Sample stageSpecimen holder: Polara cartridges / Specimen holder model: OTHER
TemperatureMin: 88 K / Max: 90 K / Average: 89 K
Alignment procedureLegacy - Astigmatism: Corrected at 200,000x once at start of session
Legacy - Electron beam tilt params: 0
DetailsExposure intensity set to give 50 electrons/pixel/second at the detector. This translates into 16 electron/square_Angstrom/second at the specimen.
DateAug 2, 2012
Image recordingCategory: CCD / Film or detector model: FEI FALCON II (4k x 4k) / Digitization - Sampling interval: 14 µm / Number real images: 49 / Average electron dose: 67 e/Å2
Details: Every image is the average of all frames recorded by the Falcon II direct electron detector
Bits/pixel: 16
Experimental equipment
Model: Tecnai Polara / Image courtesy: FEI Company

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Image processing

CTF correctionDetails: done inside FREALIGN
Final angle assignmentDetails: as determined by FREALIGN. Orientations were determined using the data out to a resolution limited to 14 Angstroms
Final reconstructionApplied symmetry - Point group: D2 (2x2 fold dihedral) / Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 13.0 Å / Resolution method: OTHER / Software - Name: MRC, CTFFIND3, FREALIGN
Details: Map was calculated from a single dataset of 2965 particles.
Number images used: 2965
DetailsThe map was obtained using Frealign, with a starting model consisting of a similar map from 43,000 single particle images of beta-galactosidase without antibody.

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Atomic model buiding 1

Initial modelPDB ID:

1f4a


Chain - #0 - Chain ID: A / Chain - #1 - Chain ID: B / Chain - #2 - Chain ID: C / Chain - #3 - Chain ID: D
SoftwareName: Chimera
DetailsSince there are three orthogonal two-fold axes, there are actually no parameters required for the fitting, except to make sure the three axes are correctly assigned (which they were at an earlier stage) and relative magnification, which was tested from 0.95 to 1.05, determined. In this case, at this resolution, a relative magnification of 1.00 was adequate. For the higher resolution map of beta-galactosidase without antibody, a 2.5% correction was necessary. In addition to 1F4A, we also fitted the coordinates of Fv antibody HYHEL-10 (anti-hen egg lysozyme) with PDB coordinates 3A6B to all four antibody domains in the map.
RefinementSpace: REAL / Protocol: RIGID BODY FIT / Overall B value: 0 / Target criteria: FSC curve between map and model
Output model

PDB-4ckd:
Model of complex between the E.coli enzyme beta-galactosidase and four single chain Fv antibody domains scFv13R4.

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