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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: 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.
SampleEscherichia coli beta-galactosidase bound to phenylethyl beta-D-thiogalactopyranoside (PETG):
beta-galactosidase / ligand
Keywordsnear-atomic resolution cryo-electron microscopy / single-particle cryo-EM / protein complexes / PETG
Function / homologyImmunoglobulin-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 / alkali metal ion binding / lactose catabolic process / beta-galactosidase complex / b-galactosidase / beta-galactosidase activity / carbohydrate binding / magnesium ion binding / identical protein binding / Beta-galactosidase
Function and homology information
SourceEscherichia coli K-12 (bacteria) / unidentified (others)
Methodsingle particle reconstruction / cryo EM / 2.2 Å resolution
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 ...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 ReportPDB-ID: 5a1a

SummaryFull reportAbout validation report
DateDeposition: Apr 26, 2015 / Header (metadata) release: May 6, 2015 / Map release: May 6, 2015 / Last update: Oct 14, 2015

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Structure visualization

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

Downloads & links

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Map

Fileemd_2984.map.gz (map file in CCP4 format, 97256 KB)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
292 pix
0.64 Å/pix.
= 186.004 Å
292 pix
0.64 Å/pix.
= 186.004 Å
292 pix
0.64 Å/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
Contour Level:0.05 (by author), 0.05 (movie #1):
Minimum - Maximum-0.12121183 - 0.17074034
Average (Standard dev.)-7.7E-6 (0.01674482)
Details

EMDB XML:

Space Group Number1
Map Geometry
Axis orderXYZ
Dimensions292292292
Origin000
Limit291291291
Spacing292292292
CellA=B=C: 186.00401 Å
α=β=γ: 90 deg.

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
start NX/NY/NZ
NX/NY/NZ
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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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)
Details: The sample was monodisperse. / Number of components: 2 / Oligomeric State: tetramer
MassTheoretical: 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

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Component #1: protein, beta-galactosidase

ProteinName: beta-galactosidase / a.k.a: beta-gal, b-gal / Oligomeric Details: tetramer / Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 465 kDa
SourceSpecies: Escherichia coli K-12 (bacteria)
Source (natural)Location in cell: cytoplasm
External referencesGene Ontology: beta-galactosidase activity / UniProt: Beta-galactosidase

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Component #2: ligand, phenylethyl beta-D-thiogalactopyranoside

LigandName: 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
SourceSpecies: unidentified (others)

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Experimental details

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Sample preparation

SpecimenSpecimen state: particle / Method: cryo EM
Sample solutionSpecimen conc.: 2.3 mg/ml
Buffer solution: 25 mM Tris, pH 8.0, 50 mM NaCl, 2 mM MgCl2, 1.0 mM TCEP
pH: 8
Support film200 mesh Quantifoil R2/2 grids, plasma cleaned
VitrificationInstrument: LEICA EM GP / Cryogen name: ETHANE / Temperature: 90.15 K / Humidity: 90 % / Method: Blot for 2 seconds before plunging.

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
ImagingMicroscope: FEI TITAN KRIOS / Date: Dec 15, 2014 / Details: Parallel beam illumination
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Electron dose: 45 e/Å2 / Illumination mode: FLOOD BEAM
LensMagnification: 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 HolderHolder: Liquid nitrogen cooled / Model: FEI TITAN KRIOS AUTOGRID HOLDER / Temperature: 79.7 K ( 79.6 - 79.8 K)
CameraDetector: GATAN K2 (4k x 4k)

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Image acquisition

Image acquisitionNumber of digital images: 1487
Details: Every image is the average of 38 frames recorded by the direct electron detector.
Raw dataEMPIAR-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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Image processing

ProcessingMethod: single particle reconstruction / Applied symmetry: D2 (2*2 fold dihedral) / Number of projections: 41123
3D reconstructionAlgorithm: phase residual minimization / Software: FREALIGN / CTF correction: each particle
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.
Resolution: 2.2 Å / Resolution method: FSC 0.143, semi-independent
FSC plot
(resolution estimation)

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Atomic model buiding

Output model

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