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- EMDB-10563: Beta-galactosidase in complex with deoxygalacto-nojirimycin -

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Basic information

Entry
Database: EMDB / ID: EMD-10563
TitleBeta-galactosidase in complex with deoxygalacto-nojirimycin
Map dataNone
Sample
  • Complex: Beta-galactosidase
    • Protein or peptide: Beta-galactosidase
  • Ligand: MAGNESIUM ION
  • Ligand: (2R,3S,4R,5S)-2-(hydroxymethyl)piperidine-3,4,5-triol
  • Ligand: water
Function / homology
Function and homology information


alkali metal ion binding / organic substance catabolic process / lactose catabolic process / beta-galactosidase complex / beta-galactosidase / beta-galactosidase activity / carbohydrate binding / carbohydrate metabolic process / 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. / 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 / Galactose mutarotase-like domain superfamily / Galactose-binding-like domain superfamily / Glycoside hydrolase superfamily / Immunoglobulin-like fold
Similarity search - Domain/homology
Beta-galactosidase / Beta-galactosidase
Similarity search - Component
Biological speciesEscherichia coli (E. coli)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.3 Å
AuthorsSaur M / Hartshorn MJ / Dong J / Reeks J / Bunkoczi G / Jhoti H / Williams PA
CitationJournal: Drug Discov Today / Year: 2020
Title: Fragment-based drug discovery using cryo-EM.
Authors: Michael Saur / Michael J Hartshorn / Jing Dong / Judith Reeks / Gabor Bunkoczi / Harren Jhoti / Pamela A Williams /
Abstract: Recent advances in electron cryo-microscopy (cryo-EM) structure determination have pushed the resolutions obtainable by the method into the range widely considered to be of utility for drug discovery. ...Recent advances in electron cryo-microscopy (cryo-EM) structure determination have pushed the resolutions obtainable by the method into the range widely considered to be of utility for drug discovery. Here, we review the use of cryo-EM in fragment-based drug discovery (FBDD) based on in-house method development. We demonstrate not only that cryo-EM can reveal details of the molecular interactions between fragments and a protein, but also that the current reproducibility, quality, and throughput are compatible with FBDD. We exemplify this using the test system β-galactosidase (Bgal) and the oncology target pyruvate kinase 2 (PKM2).
History
DepositionDec 20, 2019-
Header (metadata) releaseJan 8, 2020-
Map releaseJan 8, 2020-
UpdateJan 27, 2021-
Current statusJan 27, 2021Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.05
  • Imaged by UCSF Chimera
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  • 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-6tsh
  • Surface level: 0.05
  • Imaged by UCSF Chimera
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  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-6tsh
  • Imaged by Jmol
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_10563.png

Downloads & links

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Map

FileDownload / File: emd_10563.map.gz / Format: CCP4 / Size: 329.4 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationNone
Voxel sizeX=Y=Z: 0.68 Å
Density
Contour LevelBy AUTHOR: 0.05 / Movie #1: 0.05
Minimum - Maximum-0.15401648 - 0.265189
Average (Standard dev.)0.0024545165 (±0.023397842)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions220141295
Spacing141220295
CellA: 95.88 Å / B: 149.6 Å / C: 200.6 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z0.680.680.68
M x/y/z141220295
origin x/y/z0.0000.0000.000
length x/y/z95.880149.600200.600
α/β/γ90.00090.00090.000
start NX/NY/NZ1081340
NX/NY/NZ13584349
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS141220295
D min/max/mean-0.1540.2650.002

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Supplemental data

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Additional map: Relion post-process unmasked map.

Fileemd_10563_additional.map
AnnotationRelion post-process unmasked map.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: Relion post-process unmasked map.

Fileemd_10563_additional_1.map
AnnotationRelion post-process unmasked map.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Relion auto-refine halfmap 2

Fileemd_10563_half_map_1.map
AnnotationRelion auto-refine halfmap 2
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Relion auto-refine halfmap 1

Fileemd_10563_half_map_2.map
AnnotationRelion auto-refine halfmap 1
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Beta-galactosidase

EntireName: Beta-galactosidase
Components
  • Complex: Beta-galactosidase
    • Protein or peptide: Beta-galactosidase
  • Ligand: MAGNESIUM ION
  • Ligand: (2R,3S,4R,5S)-2-(hydroxymethyl)piperidine-3,4,5-triol
  • Ligand: water

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Supramolecule #1: Beta-galactosidase

SupramoleculeName: Beta-galactosidase / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: Escherichia coli (E. coli)
Recombinant expressionOrganism: Escherichia coli (E. coli) / Recombinant plasmid: pET-15b
Molecular weightTheoretical: 464 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 (E. coli)
Molecular weightTheoretical: 118.395336 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MGSSHHHHHH SSGLVPRGSH MLEDPVVLQR RDWENPGVTQ LNRLAAHPPF ASWRNSEEAR TDRPSQQLRS LNGEWRFAWF PAPEAVPES WLECDLPEAD TVVVPSNWQM HGYDAPIYTN VTYPITVNPP FVPTENPTGC YSLTFNVDES WLQEGQTRII F DGVNSAFH ...String:
MGSSHHHHHH SSGLVPRGSH MLEDPVVLQR RDWENPGVTQ LNRLAAHPPF ASWRNSEEAR TDRPSQQLRS LNGEWRFAWF PAPEAVPES WLECDLPEAD TVVVPSNWQM HGYDAPIYTN VTYPITVNPP FVPTENPTGC YSLTFNVDES WLQEGQTRII F DGVNSAFH LWCNGRWVGY GQDSRLPSEF DLSAFLRAGE NRLAVMVLRW SDGSYLEDQD MWRMSGIFRD VSLLHKPTTQ IS DFHVATR FNDDFSRAVL EAEVQMCGEL RDYLRVTVSL WQGETQVASG TAPFGGEIID ERGGYADRVT LRLNVENPKL WSA EIPNLY RAVVELHTAD GTLIEAEACD VGFREVRIEN GLLLLNGKPL LIRGVNRHEH HPLHGQVMDE QTMVQDILLM KQNN FNAVR CSHYPNHPLW YTLCDRYGLY VVDEANIETH GMVPMNRLTD DPRWLPAMSE RVTRMVQRDR NHPSVIIWSL GNESG HGAN HDALYRWIKS VDPSRPVQYE GGGADTTATD IICPMYARVD EDQPFPAVPK WSIKKWLSLP GETRPLILCE YAHAMG NSL GGFAKYWQAF RQYPRLQGGF VWDWVDQSLI KYDENGNPWS AYGGDFGDTP NDRQFCMNGL VFADRTPHPA LTEAKHQ QQ FFQFRLSGQT IEVTSEYLFR HSDNELLHWM VALDGKPLAS GEVPLDVAPQ GKQLIELPEL PQPESAGQLW LTVRVVQP N ATAWSEAGHI SAWQQWRLAE NLSVTLPAAS HAIPHLTTSE MDFCIELGNK RWQFNRQSGF LSQMWIGDKK QLLTPLRDQ FTRAPLDNDI GVSEATRIDP NAWVERWKAA GHYQAEAALL QCTADTLADA VLITTAHAWQ HQGKTLFISR KTYRIDGSGQ MAITVDVEV ASDTPHPARI GLNCQLAQVA ERVNWLGLGP QENYPDRLTA ACFDRWDLPL SDMYTPYVFP SENGLRCGTR E LNYGPHQW RGDFQFNISR YSQQQLMETS HRHLLHAEEG TWLNIDGFHM GIGGDDSWSP SVSAEFQLSA GRYHYQLVWC QK

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Macromolecule #2: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 2 / Number of copies: 4 / Formula: MG
Molecular weightTheoretical: 24.305 Da

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Macromolecule #3: (2R,3S,4R,5S)-2-(hydroxymethyl)piperidine-3,4,5-triol

MacromoleculeName: (2R,3S,4R,5S)-2-(hydroxymethyl)piperidine-3,4,5-triol / type: ligand / ID: 3 / Number of copies: 4 / Formula: DGJ
Molecular weightTheoretical: 163.172 Da
Chemical component information

ChemComp-DGJ:
(2R,3S,4R,5S)-2-(hydroxymethyl)piperidine-3,4,5-triol / medication, Galafold*YM / Migalastat

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Macromolecule #4: water

MacromoleculeName: water / type: ligand / ID: 4 / Number of copies: 864 / Formula: HOH
Molecular weightTheoretical: 18.015 Da
Chemical component information

ChemComp-HOH:
WATER / 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

Concentration0.17 mg/mL
BufferpH: 6.8
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 300 / Support film - Material: GRAPHENE OXIDE / Support film - topology: CONTINUOUS / Pretreatment - Type: GLOW DISCHARGE
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy
Image recordingFilm or detector model: FEI FALCON III (4k x 4k) / Detector mode: COUNTING / Number grids imaged: 476 / Number real images: 476 / Average exposure time: 59.98 sec. / Average electron dose: 59.69 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 146239
Startup modelType of model: EMDB MAP
EMDB ID:

4116

Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
Final reconstructionApplied symmetry - Point group: D2 (2x2 fold dihedral) / Resolution.type: BY AUTHOR / Resolution: 2.3 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.04) / Number images used: 73037

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