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- EMDB-7770: Atomic resolution cryo-EM structure of beta-galactosidase -

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Basic information

Entry
Database: EMDB / ID: EMD-7770
TitleAtomic resolution cryo-EM structure of beta-galactosidase
Map dataEscherichia coli beta-galactosidase
Sample
  • Complex: Escherichia coli beta-galactosidase bound to phenylethyl beta-D-thiogalactopyranoside (PETG)
    • Protein or peptide: Beta-galactosidase
  • Ligand: 2-phenylethyl 1-thio-beta-D-galactopyranoside
  • Ligand: MAGNESIUM ION
  • Ligand: SODIUM ION
  • Ligand: water
Keywordsdrift correction / radiation damage / drug discovery / precision medicine / computer-aided drug discovery / HYDROLASE
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 ...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 / Glycosyl hydrolases family 2, sugar binding domain / Glycoside hydrolase family 2, catalytic domain / Glycosyl hydrolases family 2, sugar binding domain / Glycosyl hydrolases family 2, TIM barrel domain / Glycoside hydrolase, family 2, immunoglobulin-like beta-sandwich / Glycosyl hydrolases family 2 / Beta-Galactosidase/glucuronidase domain superfamily / Glycoside hydrolase-type carbohydrate-binding / Galactose mutarotase-like domain superfamily / Galactose-binding-like domain superfamily / Glycoside hydrolase superfamily / Immunoglobulin-like fold
Similarity search - Domain/homology
Biological speciesEscherichia coli K-12 (bacteria) / Escherichia coli (strain K12) (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 1.9 Å
AuthorsSubramaniam S / Bartesaghi A / Banerjee S / Zhu X
CitationJournal: Structure / Year: 2018
Title: Atomic Resolution Cryo-EM Structure of β-Galactosidase.
Authors: Alberto Bartesaghi / Cecilia Aguerrebere / Veronica Falconieri / Soojay Banerjee / Lesley A Earl / Xing Zhu / Nikolaus Grigorieff / Jacqueline L S Milne / Guillermo Sapiro / Xiongwu Wu / Sriram Subramaniam /
Abstract: The advent of direct electron detectors has enabled the routine use of single-particle cryo-electron microscopy (EM) approaches to determine structures of a variety of protein complexes at near- ...The advent of direct electron detectors has enabled the routine use of single-particle cryo-electron microscopy (EM) approaches to determine structures of a variety of protein complexes at near-atomic resolution. Here, we report the development of methods to account for local variations in defocus and beam-induced drift, and the implementation of a data-driven dose compensation scheme that significantly improves the extraction of high-resolution information recorded during exposure of the specimen to the electron beam. These advances enable determination of a cryo-EM density map for β-galactosidase bound to the inhibitor phenylethyl β-D-thiogalactopyranoside where the ordered regions are resolved at a level of detail seen in X-ray maps at ∼ 1.5 Å resolution. Using this density map in conjunction with constrained molecular dynamics simulations provides a measure of the local flexibility of the non-covalently bound inhibitor and offers further opportunities for structure-guided inhibitor design.
History
DepositionMar 28, 2018-
Header (metadata) releaseMay 30, 2018-
Map releaseMay 30, 2018-
UpdateMar 13, 2024-
Current statusMar 13, 2024Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.52
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 0.52
  • Imaged by UCSF Chimera
  • Download
  • Surface view with fitted model
  • Atomic models: PDB-6cvm
  • Surface level: 0.52
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_7770.png

Downloads & links

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Map

FileDownload / File: emd_7770.map.gz / Format: CCP4 / Size: 147.3 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationEscherichia coli beta-galactosidase
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.64 Å/pix.
x 338 pix.
= 215.306 Å		0.64 Å/pix.
x 338 pix.
= 215.306 Å		0.64 Å/pix.
x 338 pix.
= 215.306 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

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.52 / Movie #1: 0.52
Minimum - Maximum-1.0223988 - 2.9114444
Average (Standard dev.)-0.007835173 (±0.15938693)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions338338338
Spacing338338338
CellA=B=C: 215.30602 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z0.6370.6370.637
M x/y/z338338338
origin x/y/z0.0000.0000.000
length x/y/z215.306215.306215.306
α/β/γ90.00090.00090.000
start NX/NY/NZ-199-199-199
NX/NY/NZ399399399
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS338338338
D min/max/mean-1.0222.911-0.008

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Supplemental data

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Mask #1

Fileemd_7770_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: Escherichia coli beta-galactosidase, sharpened map

Fileemd_7770_additional.map
AnnotationEscherichia coli beta-galactosidase, sharpened map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Escherichia coli beta-galactosidase, half map #1

Fileemd_7770_half_map_1.map
AnnotationEscherichia coli beta-galactosidase, half map #1
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Escherichia coli beta-galactosidase, half map #2

Fileemd_7770_half_map_2.map
AnnotationEscherichia coli beta-galactosidase, half map #2
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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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)
Components
  • Complex: Escherichia coli beta-galactosidase bound to phenylethyl beta-D-thiogalactopyranoside (PETG)
    • Protein or peptide: Beta-galactosidase
  • Ligand: 2-phenylethyl 1-thio-beta-D-galactopyranoside
  • Ligand: MAGNESIUM ION
  • Ligand: SODIUM ION
  • Ligand: water

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Supramolecule #1: Escherichia coli beta-galactosidase bound to phenylethyl beta-D-t...

SupramoleculeName: Escherichia coli beta-galactosidase bound to phenylethyl beta-D-thiogalactopyranoside (PETG)
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: Escherichia coli K-12 (bacteria)
Molecular weightTheoretical: 465 KDa

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Macromolecule #1: Beta-galactosidase

MacromoleculeName: Beta-galactosidase / type: protein_or_peptide / ID: 1 / Number of copies: 4 / Enantiomer: LEVO / EC number: beta-galactosidase
Source (natural)Organism: Escherichia coli (strain K12) (bacteria) / Strain: K12
Molecular weightTheoretical: 116.181031 KDa
SequenceString: MITDSLAVVL QRRDWENPGV TQLNRLAAHP PFASWRNSEE ARTDRPSQQL RSLNGEWRFA WFPAPEAVPE SWLECDLPEA DTVVVPSNW QMHGYDAPIY TNVTYPITVN PPFVPTENPT GCYSLTFNVD ESWLQEGQTR IIFDGVNSAF HLWCNGRWVG Y GQDSRLPS ...String:
MITDSLAVVL QRRDWENPGV TQLNRLAAHP PFASWRNSEE ARTDRPSQQL RSLNGEWRFA WFPAPEAVPE SWLECDLPEA DTVVVPSNW QMHGYDAPIY TNVTYPITVN PPFVPTENPT GCYSLTFNVD ESWLQEGQTR IIFDGVNSAF HLWCNGRWVG Y GQDSRLPS EFDLSAFLRA GENRLAVMVL RWSDGSYLED QDMWRMSGIF RDVSLLHKPT TQISDFHVAT RFNDDFSRAV LE AEVQMCG ELRDYLRVTV SLWQGETQVA SGTAPFGGEI IDERGGYADR VTLRLNVENP KLWSAEIPNL YRAVVELHTA DGT LIEAEA CDVGFRVVRI ENGLLLLNGK PLLIRGVNRH EHHPLHGQVM DEQTMVQDIL LMKQNNFNAV RCSHYPNHPL WYTL CDRYG LYVVDEANIE THGMVPMNRL TDDPRWLPAM SERVTRMVQR DRNHPSVIIW SLGNESGHGA NHDALYRWIK SVDPS RPVQ YEGGGADTTA TDIICPMYAR VDEDQPFPAV PKWSIKKWLS LPGETRPLIL CEYAHAMGNS LGGFAKYWQA FRQYPR LQG GFVWDWVDQS LIKYDENGNP WSAYGGDFGD TPNDRQFCMN GLVFADRTPH PALTEAKHQQ QFFQFRLSGQ TIEVTSE YL FRHSDNELLH WMVALDGKPL ASGEVPLDVA PQGKQLIELP ELPQPESAGQ LWLTVRVVQP NATAWSEAGH ISAWQQWR L AENLSVTLPA ASHAIPHLTT SEMDFCIELG NKRWQFNRQS GFLSQMWIGD KKQLLTPLRD QFTRAPLDND IGVSEATRI DPNAWVERWK AAGHYQAEAA LLQCTADTLA DAVLITTAHA WQHQGKTLFI SRKTYRIDGS GQMAITVDVV VASDTPHPAR IGLNCQLAQ VAERVNWLGL GPQENYPDRL TAACFDRWDL PLSDMYTPYV FPSENGLRCG TRELNYGPHQ WRGDFQFNIS R YSQQQLME TSHRHLLHAE EGTWLNIDGF HMGIGGDDSW SPSVSAEFQL SAGRYHYQLV WCQ

UniProtKB: Beta-galactosidase

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Macromolecule #2: 2-phenylethyl 1-thio-beta-D-galactopyranoside

MacromoleculeName: 2-phenylethyl 1-thio-beta-D-galactopyranoside / type: ligand / ID: 2 / Number of copies: 4 / Formula: PTQ
Molecular weightTheoretical: 300.371 Da
Chemical component information

ChemComp-PTQ:
2-phenylethyl 1-thio-beta-D-galactopyranoside

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Macromolecule #3: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 3 / Number of copies: 8 / Formula: MG
Molecular weightTheoretical: 24.305 Da

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Macromolecule #4: SODIUM ION

MacromoleculeName: SODIUM ION / type: ligand / ID: 4 / Number of copies: 8
Molecular weightTheoretical: 22.99 Da

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Macromolecule #5: water

MacromoleculeName: water / type: ligand / ID: 5 / Number of copies: 4194 / Formula: HOH
Molecular weightTheoretical: 18.015 Da
Chemical component information

ChemComp-HOH:
WATER

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration2.3 mg/mL
BufferpH: 8
Details: 25 mM Tris, pH 8.0, 50 mM NaCl, 2 mM MgCl2, 1.0 mM TCEP
GridModel: Quantifoil R2/2 / Material: COPPER / Mesh: 200 / Details: plasma cleaned
VitrificationCryogen name: ETHANE / Chamber humidity: 90 % / Chamber temperature: 90.15 K / Instrument: LEICA EM GP / Details: Blot for 2 seconds before plunging..

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Electron microscopy

MicroscopeFEI TITAN KRIOS
TemperatureMin: 79.6 K / Max: 79.8 K
Specialist opticsEnergy filter - Name: GIF Quantum LS / Energy filter - Lower energy threshold: 0 eV / Energy filter - Upper energy threshold: 20 eV
DetailsParallel beam illumination
Image recordingFilm or detector model: GATAN K2 QUANTUM (4k x 4k) / Detector mode: SUPER-RESOLUTION / Digitization - Frames/image: 1-38 / Number real images: 1539 / Average exposure time: 7.6 sec. / Average electron dose: 45.0 e/Å2 / Details: Raw micrographs are available from EMPIAR-10061.
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated defocus max: 2.0 µm / Calibrated magnification: 215000 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus min: 0.6 µm / Nominal magnification: 215000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 298715
Startup modelType of model: NONE
Final reconstructionApplied symmetry - Point group: D2 (2x2 fold dihedral) / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 1.9 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: FREALIGN (ver. 9.11) / Number images used: 150321
Initial angle assignmentType: PROJECTION MATCHING
Final angle assignmentType: PROJECTION MATCHING / Software - Name: FREALIGN (ver. 9.11)
FSC plot (resolution estimation)

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Atomic model buiding 1

RefinementSpace: REAL / Protocol: FLEXIBLE FIT
Output model

PDB-6cvm:
Atomic resolution cryo-EM structure of beta-galactosidase

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