|Entry||Database: PDB / ID: 5a1a|
|Title||2.2 A resolution cryo-EM structure of beta-galactosidase in complex with a cell-permeant inhibitor|
|Keywords||HYDROLASE / NEAR-ATOMIC / 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 / ec:188.8.131.52: / 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
|Biological species||ESCHERICHIA COLI K-12 (bacteria)|
|Method||ELECTRON MICROSCOPY / 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.
SummaryFull reportAbout validation report
|Date||Deposition: Apr 29, 2015 / Release: May 6, 2015|
|Structure viewer||Molecule: |
Downloads & links
Mass: 116370.188 Da / Num. of mol.: 4 / Source method: isolated from a natural source / Source: (natural) ESCHERICHIA COLI K-12 (bacteria) / References: UniProt: P00722, EC: 184.108.40.206
Mass: 300.371 Da / Num. of mol.: 4
Source method: isolated from a genetically manipulated source
|#5: Water|| ChemComp-HOH / |
|Experiment||Method: ELECTRON MICROSCOPY|
|EM experiment||Aggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction|
|Component||Name: ESCHERICHIA COLI BETA- GALACTOSIDASE WITH PETG / Type: COMPLEX|
|Buffer solution||Name: 25 MM TRIS, PH 8.0, 50 MM NACL, 2 MM MGCL2, 0.5 MM TCEP|
Details: 25 MM TRIS, PH 8.0, 50 MM NACL, 2 MM MGCL2, 0.5 MM TCEP
|Specimen||Conc.: 2.3 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES|
|Specimen support||Details: HOLEY CARBON|
|Vitrification||Instrument: LEICA EM GP / Cryogen name: ETHANE|
Details: BLOT FOR 2 SECONDS BEFORE PLUNGING INTO LIQUID ETHANE (LEICA EM GP).
-Electron microscopy imaging
Model: Titan Krios / Image courtesy: FEI Company
|Microscopy||Model: FEI TITAN KRIOS / Date: Dec 15, 2014|
|Electron gun||Electron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM|
|Electron lens||Mode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 215000 X / Calibrated magnification: 215000 X / Nominal defocus max: 2000 nm / Nominal defocus min: 600 nm / Cs: 2.7 mm|
|Specimen holder||Temperature: 79.7 K|
|Image recording||Electron dose: 45 e/Å2 / Film or detector model: GATAN K2 (4k x 4k)|
|Image scans||Num. digital images: 1487|
|EM software||Name: FREALIGN / Category: 3D reconstruction|
|CTF correction||Details: EACH PARTICLE|
|Symmetry||Point symmetry: D2 (2x2 fold dihedral)|
|3D reconstruction||Resolution: 2.2 Å / Num. of particles: 41123 / Nominal pixel size: 0.637 Å / Actual pixel size: 0.637 Å|
Details: SUBMISSION BASED ON EXPERIMENTAL DATA FROM EMDB EMD-2984. (DEPOSITION ID: 13171).
Symmetry type: POINT
|Refinement||Highest resolution: 2.2 Å|
|Refinement step||Cycle: LAST / Highest resolution: 2.2 Å|
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