|Entry||Database: EMDB / ID: 2984|
|Title||2.2 A resolution cryo-EM structure of beta-galactosidase in complex with a cell-permeant inhibitor|
|Keywords||near-atomic resolution cryo-electron microscopy / single-particle cryo-EM / protein complexes / PETG|
|Sample||Escherichia coli beta-galactosidase bound to phenylethyl beta-D-thiogalactopyranoside (PETG)|
|Source||Escherichia coli k-12 / bacteria / image: Escherichia coli|
|Map data||B-factor corrected reconstruction of PETG-bound beta-galactosidase.|
|Method||single particle reconstruction, at 2.2 Å resolution|
|Authors||Bartesaghi A / Merk A / Banerjee S / Matthies D / Wu X / Milne J / Subramaniam S|
|Citation||Science, 2015, 348, 1147-1151|
|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 / Last update: Oct 14, 2015|
Downloads & links
|File||emd_2984.map.gz (map file in CCP4 format, 97256 KB)|
|Projections & slices|
Images are generated by Spider package.
|Voxel size||X=Y=Z: 0.637 Å|
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 / image: Escherichia coli|
|Source (natural)||Location in cell: cytoplasm|
|External references||Gene Ontology: GO: 0004565 / UniProt: UniProt: P00722|
-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|
|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 K ( 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])
-Atomic model buiding
-Oct 4, 2017. Three pioneers of this field were awarded Nobel Prize in Chemistry 2017
Three pioneers of this field were awarded Nobel Prize in Chemistry 2017
- Jacques Dubochet (University of Lausanne, Switzerland) is a pioneer of ice-embedding method of EM specimen (as known as cryo-EM), Most of 3DEM structures in EMDB and PDB are obtained using his method.
- Joachim Frank (Columbia University, New York, USA) is a pioneer of single particle reconstruction, which is the most used reconstruction method for 3DEM structures in EMDB and EM entries in PDB. And also, he is a develper of Spider, which is one of the most famous software in this field, and is used for some EM Navigor data (e.g. map projection/slice images).
- Richard Henderson (MRC Laboratory of Molecular Biology, Cambridge, UK) was determined the first biomolecule structure by EM. The first EM entry in PDB, PDB-1brd is determinedby him.
External links: The 2017 Nobel Prize in Chemistry - Press Release
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