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- EMDB-2984: 2.2 A resolution cryo-EM structure of beta-galactosidase in compl... -

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Basic information

Entry
Database: EMDB / ID: EMD-2984
Title2.2 A resolution cryo-EM structure of beta-galactosidase in complex with a cell-permeant inhibitor
Map dataB-factor corrected reconstruction of PETG-bound beta-galactosidase.
Sample
  • Sample: Escherichia coli beta-galactosidase bound to phenylethyl beta-D-thiogalactopyranoside (PETG)
  • Protein or peptide: beta-galactosidase
  • Ligand: phenylethyl beta-D-thiogalactopyranoside
Keywordsnear-atomic resolution cryo-electron microscopy / single-particle cryo-EM / protein complexes / PETG
Function / homology
Function and homology information


alkali metal ion binding / lactose catabolic process / beta-galactosidase complex / beta-galactosidase / beta-galactosidase activity / carbohydrate binding / magnesium ion binding / 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 / Galactose-binding-like domain superfamily / Glycoside hydrolase superfamily / Immunoglobulin-like fold
Similarity search - Domain/homology
Biological speciesEscherichia coli K-12 (bacteria) / unidentified (others)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.2 Å
AuthorsBartesaghi A / Merk A / Banerjee S / Matthies D / Wu X / Milne J / Subramaniam S
CitationJournal: Science / Year: 2015
Title: 2.2 Å resolution cryo-EM structure of β-galactosidase in complex with a cell-permeant inhibitor.
Authors: Alberto Bartesaghi / Alan Merk / Soojay Banerjee / Doreen Matthies / Xiongwu Wu / Jacqueline L S Milne / Sriram Subramaniam /
Abstract: Cryo-electron microscopy (cryo-EM) is rapidly emerging as a powerful tool for protein structure determination at high resolution. Here we report the structure of a complex between Escherichia coli β- ...Cryo-electron microscopy (cryo-EM) is rapidly emerging as a powerful tool for protein structure determination at high resolution. Here we report the structure of a complex between Escherichia coli β-galactosidase and the cell-permeant inhibitor phenylethyl β-D-thiogalactopyranoside (PETG), determined by cryo-EM at an average resolution of ~2.2 angstroms (Å). Besides the PETG ligand, we identified densities in the map for ~800 water molecules and for magnesium and sodium ions. Although it is likely that continued advances in detector technology may further enhance resolution, our findings demonstrate that preparation of specimens of adequate quality and intrinsic protein flexibility, rather than imaging or image-processing technologies, now represent the major bottlenecks to routinely achieving resolutions close to 2 Å using single-particle cryo-EM.
History
DepositionApr 26, 2015-
Header (metadata) releaseMay 6, 2015-
Map releaseMay 6, 2015-
UpdateOct 14, 2015-
Current statusOct 14, 2015Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.05
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  • Surface view with fitted model
  • Atomic models: PDB-5a1a
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  • Surface view with fitted model
  • Atomic models: PDB-5a1a
  • Surface level: 0.05
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd-2984-bga...

Downloads & links

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Map

FileDownload / File: emd_2984.map.gz / Format: CCP4 / Size: 92.8 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationB-factor corrected reconstruction of PETG-bound beta-galactosidase.
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.64 Å/pix.
x 292 pix.
= 186.004 Å		0.64 Å/pix.
x 292 pix.
= 186.004 Å		0.64 Å/pix.
x 292 pix.
= 186.004 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.637 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.05 / Movie #1: 0.05
Minimum - Maximum-0.12121183 - 0.17074034
Average (Standard dev.)-0.0000077 (±0.01674482)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions292292292
Spacing292292292
CellA=B=C: 186.00401 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z0.6370.6370.637
M x/y/z292292292
origin x/y/z0.0000.0000.000
length x/y/z186.004186.004186.004
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS292292292
D min/max/mean-0.1210.171-0.000

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Supplemental data

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Supplemental map: emd 2984 additional 1.map

Fileemd_2984_additional_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Supplemental map: emd 2984 half map 1.map

Fileemd_2984_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Supplemental map: emd 2984 half map 2.map

Fileemd_2984_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 : Escherichia coli beta-galactosidase bound to phenylethyl beta-D-t...

EntireName: Escherichia coli beta-galactosidase bound to phenylethyl beta-D-thiogalactopyranoside (PETG)
Components
  • Sample: Escherichia coli beta-galactosidase bound to phenylethyl beta-D-thiogalactopyranoside (PETG)
  • Protein or peptide: beta-galactosidase
  • Ligand: phenylethyl beta-D-thiogalactopyranoside

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Supramolecule #1000: Escherichia coli beta-galactosidase bound to phenylethyl beta-D-t...

SupramoleculeName: Escherichia coli beta-galactosidase bound to phenylethyl beta-D-thiogalactopyranoside (PETG)
type: sample / ID: 1000 / Details: The sample was monodisperse. / Oligomeric state: tetramer / Number unique components: 2
Molecular weightTheoretical: 465 KDa
Method: ProtParam tool: Gasteiger E., Hoogland C., Gattiker A., Duvaud S., Wilkins M.R., Appel R.D., Bairoch A., Protein Identification and Analysis Tools on the ExPASy Server, (in) John M. Walker ...Method: ProtParam tool: Gasteiger E., Hoogland C., Gattiker A., Duvaud S., Wilkins M.R., Appel R.D., Bairoch A., Protein Identification and Analysis Tools on the ExPASy Server, (in) John M. Walker (ed): The Proteomics Protocols Handbook, Humana Press (2005), pp. 571-607

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Macromolecule #1: beta-galactosidase

MacromoleculeName: beta-galactosidase / type: protein_or_peptide / ID: 1 / Name.synonym: beta-gal, b-gal / Number of copies: 1 / Oligomeric state: tetramer / Recombinant expression: No
Source (natural)Organism: Escherichia coli K-12 (bacteria) / Location in cell: cytoplasm
Molecular weightTheoretical: 465 KDa
SequenceUniProtKB: Beta-galactosidase / GO: beta-galactosidase activity

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Macromolecule #2: phenylethyl beta-D-thiogalactopyranoside

MacromoleculeName: phenylethyl beta-D-thiogalactopyranoside / type: ligand / ID: 2 / Name.synonym: PETG / Details: Molecular weight of PETG is 300.37 Da. / Number of copies: 4 / Oligomeric state: monomer / Recombinant expression: No
Source (natural)Organism: unidentified (others)

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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

Concentration2.3 mg/mL
BufferpH: 8
Details: 25 mM Tris, pH 8.0, 50 mM NaCl, 2 mM MgCl2, 1.0 mM TCEP
GridDetails: 200 mesh Quantifoil R2/2 grids, plasma cleaned
VitrificationCryogen name: ETHANE / Chamber humidity: 90 % / Chamber temperature: 90.15 K / Instrument: LEICA EM GP / Method: Blot for 2 seconds before plunging.

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Electron microscopy

MicroscopeFEI TITAN KRIOS
TemperatureMin: 79.6 K / Max: 79.8 K / Average: 79.7 K
Alignment procedureLegacy - Astigmatism: Objective lens astigmatism was corrected at 215,000 times magnification.
Specialist opticsEnergy filter - Name: Gatan, Inc. / Energy filter - Lower energy threshold: 0.0 eV / Energy filter - Upper energy threshold: 20.0 eV
DetailsParallel beam illumination
DateDec 15, 2014
Image recordingCategory: CCD / Film or detector model: GATAN K2 (4k x 4k) / Number real images: 1487 / Average electron dose: 45 e/Å2
Details: Every image is the average of 38 frames recorded by the direct electron detector.
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 215000 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 2.0 µm / Nominal defocus min: 0.6 µm / Nominal magnification: 215000
Sample stageSpecimen holder: Liquid nitrogen cooled / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

CTF correctionDetails: each particle
Final reconstructionApplied symmetry - Point group: D2 (2x2 fold dihedral) / Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 2.2 Å / Resolution method: OTHER / Software - Name: FREALIGN
Details: Final map was obtained from 41,123 particles. D2 symmetry was applied throughout refinement. Map was corrected using a B-factor of -75 A^2.
Number images used: 41123
FSC plot (resolution estimation)

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