|Entry||Database: EMDB / ID: EMD-2984|
|Title||2.2 A resolution cryo-EM structure of beta-galactosidase in complex with a cell-permeant inhibitor|
|Sample||Escherichia coli beta-galactosidase bound to phenylethyl beta-D-thiogalactopyranoside (PETG):|
beta-galactosidase / ligand
|Keywords||near-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
Immunoglobulin-like fold / Galactose-binding-like domain superfamily / Glycosyl hydrolases family 2 / Beta-Galactosidase/glucuronidase domain superfamily / Beta-galactosidase, domain 4 / Glycoside hydrolase, family 2, beta-galactosidase / Glycoside hydrolase, family 2, active site / Glycoside hydrolase, family 2, conserved site / Glycoside hydrolase superfamily / Glycoside hydrolase-type carbohydrate-binding ...Immunoglobulin-like fold / Galactose-binding-like domain superfamily / Glycosyl hydrolases family 2 / Beta-Galactosidase/glucuronidase domain superfamily / Beta-galactosidase, domain 4 / Glycoside hydrolase, family 2, beta-galactosidase / Glycoside hydrolase, family 2, active site / Glycoside hydrolase, family 2, conserved site / Glycoside hydrolase superfamily / Glycoside hydrolase-type carbohydrate-binding / Beta galactosidase small chain / Galactose mutarotase-like domain superfamily / Glycosyl hydrolases family 2, sugar binding domain / Glycosyl hydrolases family 2, sugar binding domain / Glycoside hydrolase family 2, catalytic domain / Glycoside hydrolase, family 2, immunoglobulin-like beta-sandwich / Glycoside hydrolase, family 2 / Beta galactosidase small chain/ domain 5 / Domain of unknown function(DUF4981) / Glycosyl hydrolases family 2 acid/base catalyst. / Glycosyl hydrolases family 2 signature 1. / Glycosyl hydrolases family 2, TIM barrel domain
|Source||Escherichia coli K-12 (bacteria) / unidentified (others)|
|Method||single particle reconstruction / cryo EM / Resolution: 2.2 Å|
|Authors||Bartesaghi A / Merk A / Banerjee S / Matthies D / Wu X / Milne J / Subramaniam S|
|Citation||Journal: 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 ...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.
|Validation Report||PDB-ID: 5a1a|
SummaryFull reportAbout validation report
|Date||Deposition: Apr 26, 2015 / Header (metadata) release: May 6, 2015 / Map release: May 6, 2015 / Update: Oct 14, 2015|
|Structure viewer||EM map: |
Downloads & links
|File||Download / File: emd_2984.map.gz / Format: CCP4 / Size: 92.8 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)|
|Projections & slices|
Images are generated by Spider.
|Voxel size||X=Y=Z: 0.637 Å|
|Symmetry||Space group: 1|
CCP4 map header:
-Entire Escherichia coli beta-galactosidase bound to phenylethyl beta-D-t...
|Entire||Name: Escherichia coli beta-galactosidase bound to phenylethyl beta-D-thiogalactopyranoside (PETG)|
Details: The sample was monodisperse. / Number of components: 2 / Oligomeric State: tetramer
|Mass||Theoretical: 465 kDa|
Measured by: 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
-Component #1: protein, beta-galactosidase
|Protein||Name: beta-galactosidase / a.k.a: beta-gal, b-gal / Oligomeric Details: tetramer / Number of Copies: 1 / Recombinant expression: No|
|Mass||Theoretical: 465 kDa|
|Source||Species: Escherichia coli K-12 (bacteria)|
|Source (natural)||Location in cell: cytoplasm|
|External references||Gene Ontology: beta-galactosidase activity / UniProt: Beta-galactosidase|
-Component #2: ligand, phenylethyl beta-D-thiogalactopyranoside
|Ligand||Name: phenylethyl beta-D-thiogalactopyranoside / a.k.a: PETG / Oligomeric Details: monomer / Details: Molecular weight of PETG is 300.37 Da. / Recombinant expression: No / Number of Copies: 4|
|Source||Species: unidentified (others)|
|Specimen||Specimen state: Particle / Method: cryo EM|
|Sample solution||Specimen conc.: 2.3 mg/mL|
Buffer solution: 25 mM Tris, pH 8.0, 50 mM NaCl, 2 mM MgCl2, 1.0 mM TCEP
|Support film||200 mesh Quantifoil R2/2 grids, plasma cleaned|
|Vitrification||Instrument: LEICA EM GP / Cryogen name: ETHANE / Temperature: 90.15 K / Humidity: 90 % / Method: Blot for 2 seconds before plunging.|
-Electron microscopy imaging
Model: Titan Krios / Image courtesy: FEI Company
|Imaging||Microscope: FEI TITAN KRIOS / Date: Dec 15, 2014 / Details: Parallel beam illumination|
|Electron gun||Electron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Electron dose: 45 e/Å2 / Illumination mode: FLOOD BEAM|
|Lens||Magnification: 215000 X (nominal), 215000 X (calibrated)|
Astigmatism: Objective lens astigmatism was corrected at 215,000 times magnification.
Cs: 2.7 mm / Imaging mode: BRIGHT FIELD / Defocus: 600 - 2000 nm / Energy filter: Gatan, Inc. / Energy window: 0-20 eV
|Specimen Holder||Holder: Liquid nitrogen cooled / Model: FEI TITAN KRIOS AUTOGRID HOLDER / Temperature: 79.7 (79.6 - 79.8 K)|
|Camera||Detector: GATAN K2 (4k x 4k)|
|Image acquisition||Number of digital images: 1487 |
Details: Every image is the average of 38 frames recorded by the direct electron detector.
|Raw data||EMPIAR-10061 (Title: 2.2 A resolution cryo-EM structure of beta-galactosidase in complex with a cell-permeant inhibitor|
Data size: 12.4 TB
Data #1: Averages of aligned movie frames [micrographs - single frame]
Data #2: Raw movie frames [micrographs - multiframe])
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