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- PDB-5a1a: 2.2 A resolution cryo-EM structure of beta-galactosidase in compl... -

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Basic information

Entry
Database: PDB / ID: 5a1a
Title2.2 A resolution cryo-EM structure of beta-galactosidase in complex with a cell-permeant inhibitor
ComponentsBETA-GALACTOSIDASE
KeywordsHYDROLASE / 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 / 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 / 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. / Beta-galactosidase; Chain A, domain 5 - #10 / 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 / Beta-galactosidase; Chain A, domain 5 / Galactose mutarotase-like domain superfamily / Galactose-binding domain-like / Galactose-binding-like domain superfamily / Distorted Sandwich / Glycosidases / Glycoside hydrolase superfamily / Jelly Rolls / Immunoglobulins / TIM Barrel / Alpha-Beta Barrel / Immunoglobulin-like fold / Immunoglobulin-like / Sandwich / Mainly Beta / Alpha Beta
Similarity search - Domain/homology
2-phenylethyl 1-thio-beta-D-galactopyranoside / Beta-galactosidase
Similarity search - Component
Biological speciesESCHERICHIA COLI K-12 (bacteria)
MethodELECTRON MICROSCOPY / single 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 29, 2015Deposition site: PDBE / Processing site: PDBE
Revision 1.0May 6, 2015Provider: repository / Type: Initial release
Revision 1.1May 13, 2015Group: Database references
Revision 1.2May 20, 2015Group: Database references
Revision 1.3Jun 24, 2015Group: Database references
Revision 1.4Oct 3, 2018Group: Data collection / Category: em_software / Item: _em_software.image_processing_id / _em_software.name

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Structure visualization

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  • Deposited structure unit
  • Imaged by Jmol
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  • Superimposition on EM map
  • EMDB-2984
  • Imaged by UCSF Chimera
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  • Superimposition on EM map
  • EMDB-2984
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Structure viewerMolecule:
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Assembly

Deposited unit
A: BETA-GALACTOSIDASE
B: BETA-GALACTOSIDASE
C: BETA-GALACTOSIDASE
D: BETA-GALACTOSIDASE
hetero molecules


Theoretical massNumber of molelcules
Total (without water)467,06124
Polymers465,4814
Non-polymers1,58020
Water13,980776
1


  • Idetical with deposited unit
  • defined by software
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
MethodPISA

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Components

#1: Protein
BETA-GALACTOSIDASE / / BETA-GAL / LACTASE


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, beta-galactosidase
#2: Sugar
ChemComp-PTQ / 2-phenylethyl 1-thio-beta-D-galactopyranoside / 2-Phenylethyl beta-D-thiogalactoside, PETG


Type: D-saccharide / Mass: 300.371 Da / Num. of mol.: 4
Source method: isolated from a genetically manipulated source
Formula: C14H20O5S
#3: Chemical
ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 8 / Source method: obtained synthetically / Formula: Mg
#4: Chemical
ChemComp-NA / SODIUM ION


Mass: 22.990 Da / Num. of mol.: 8 / Source method: obtained synthetically / Formula: Na
#5: Water ChemComp-HOH / water / Water


Mass: 18.015 Da / Num. of mol.: 776 / Source method: isolated from a natural source / Formula: H2O

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: ESCHERICHIA COLI BETA- GALACTOSIDASE WITH PETG / Type: COMPLEX
Buffer solutionName: 25 MM TRIS, PH 8.0, 50 MM NACL, 2 MM MGCL2, 0.5 MM TCEP
pH: 8
Details: 25 MM TRIS, PH 8.0, 50 MM NACL, 2 MM MGCL2, 0.5 MM TCEP
SpecimenConc.: 2.3 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: HOLEY CARBON
VitrificationInstrument: LEICA EM GP / Cryogen name: ETHANE
Details: BLOT FOR 2 SECONDS BEFORE PLUNGING INTO LIQUID ETHANE (LEICA EM GP).

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS / Date: Dec 15, 2014
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: 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 holderTemperature: 79.7 K
Image recordingElectron dose: 45 e/Å2 / Film or detector model: GATAN K2 (4k x 4k)
Image scansNum. digital images: 1487

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Processing

EM softwareName: FREALIGN / Category: 3D reconstruction
CTF correctionDetails: EACH PARTICLE
SymmetryPoint symmetry: D2 (2x2 fold dihedral)
3D reconstructionResolution: 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
RefinementHighest resolution: 2.2 Å
Refinement stepCycle: LAST / Highest resolution: 2.2 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms32824 0 96 776 33696

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