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- EMDB-51072: AMo stained beta-galactosidase -

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Open data


ID or keywords:

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Basic information

Entry
Database: EMDB / ID: EMD-51072
TitleAMo stained beta-galactosidase
Map databeta-galactosidase, tetramer
Sample
  • Complex: tetrameric complex of beta-galactosidase
    • Protein or peptide: beta galactoside
Keywordshydrolysis / metabolism / CARBOHYDRATE
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 (E. coli) / Escherichia coli K-12 (bacteria)
Methodsingle particle reconstruction / negative staining / Resolution: 10.06 Å
AuthorsGunkel M / Behrmann E
Funding support Germany, 1 items
OrganizationGrant numberCountry
German Research Foundation (DFG)INST 216/512/1 FUGG Germany
CitationJournal: Acta Crystallogr F Struct Biol Commun / Year: 2024
Title: Revisiting sodium phosphotungstate and ammonium molybdate as nonradioactive negative-staining agents for single-particle analysis.
Authors: Monika Gunkel / Arthur Macha / Elmar Behrmann /
Abstract: This study reports the successful replacement of uranyl-based stains by either sodium phosphotungstate or ammonium molybdate in negative-staining electron microscopy. Using apoferritin as a test ...This study reports the successful replacement of uranyl-based stains by either sodium phosphotungstate or ammonium molybdate in negative-staining electron microscopy. Using apoferritin as a test specimen, it is demonstrated that in combination with a facile on-grid fixation step, both stains yield comparable images to uranyl formate. Subsequently, using β-galactosidase, it is shown that both stains can also successfully be employed for single-particle analysis, yielding virtually indistinguishable results from uranyl formate. As both replacement stains are nonradioactive, they are not subjected to the same handling restrictions as uranyl-based stains. Therefore they are not only cheaper to use, but also make decentralized sample-grid preparation, directly after purification, accessible to a broader range of scientists.
History
DepositionJul 16, 2024-
Header (metadata) releaseDec 11, 2024-
Map releaseDec 11, 2024-
UpdateDec 18, 2024-
Current statusDec 18, 2024Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_51072.png

Downloads & links

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Map

FileDownload / File: emd_51072.map.gz / Format: CCP4 / Size: 22.2 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationbeta-galactosidase, tetramer
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.86 Å/pix.
x 180 pix.
= 334.8 Å		1.86 Å/pix.
x 180 pix.
= 334.8 Å		1.86 Å/pix.
x 180 pix.
= 334.8 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.86 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.4
Minimum - Maximum-0.11901437 - 1.9702744
Average (Standard dev.)0.029796086 (±0.18789606)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions180180180
Spacing180180180
CellA=B=C: 334.8 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: beta-galactosidase, tetramer, halfmap odd

Fileemd_51072_half_map_1.map
Annotationbeta-galactosidase, tetramer, halfmap odd
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: beta-galactosidase, tetramer, halfmap even

Fileemd_51072_half_map_2.map
Annotationbeta-galactosidase, tetramer, halfmap even
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : tetrameric complex of beta-galactosidase

EntireName: tetrameric complex of beta-galactosidase
Components
  • Complex: tetrameric complex of beta-galactosidase
    • Protein or peptide: beta galactoside

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Supramolecule #1: tetrameric complex of beta-galactosidase

SupramoleculeName: tetrameric complex of beta-galactosidase / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Escherichia coli (E. coli) / Strain: K12
Molecular weightTheoretical: 466 KDa

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Macromolecule #1: beta galactoside

MacromoleculeName: beta galactoside / type: protein_or_peptide / ID: 1 / Enantiomer: LEVO / EC number: beta-galactosidase
Source (natural)Organism: Escherichia coli K-12 (bacteria)
SequenceString: MTMITDSLAV VLQRRDWENP GVTQLNRLAA HPPFASWRNS EEARTDRPSQ QLRSLNGEWR FAWFPAPEA VPESWLECDL PEADTVVVPS NWQMHGYDAP IYTNVTYPIT VNPPFVPTEN P TGCYSLTF NVDESWLQEG QTRIIFDGVN SAFHLWCNGR WVGYGQDSRL ...String:
MTMITDSLAV VLQRRDWENP GVTQLNRLAA HPPFASWRNS EEARTDRPSQ QLRSLNGEWR FAWFPAPEA VPESWLECDL PEADTVVVPS NWQMHGYDAP IYTNVTYPIT VNPPFVPTEN P TGCYSLTF NVDESWLQEG QTRIIFDGVN SAFHLWCNGR WVGYGQDSRL PSEFDLSAFL RA GENRLAV MVLRWSDGSY LEDQDMWRMS GIFRDVSLLH KPTTQISDFH VATRFNDDFS RAV LEAEVQ MCGELRDYLR VTVSLWQGET QVASGTAPFG GEIIDERGGY ADRVTLRLNV ENPK LWSAE IPNLYRAVVE LHTADGTLIE AEACDVGFRE VRIENGLLLL NGKPLLIRGV NRHEH HPLH GQVMDEQTMV QDILLMKQNN FNAVRCSHYP NHPLWYTLCD RYGLYVVDEA NIETHG MVP MNRLTDDPRW LPAMSERVTR MVQRDRNHPS VIIWSLGNES GHGANHDALY RWIKSVD PS RPVQYEGGGA DTTATDIICP MYARVDEDQP FPAVPKWSIK KWLSLPGETR PLILCEYA H AMGNSLGGFA KYWQAFRQYP RLQGGFVWDW VDQSLIKYDE NGNPWSAYGG DFGDTPNDR QFCMNGLVFA DRTPHPALTE AKHQQQFFQF RLSGQTIEVT SEYLFRHSDN ELLHWMVALD GKPLASGEV PLDVAPQGKQ LIELPELPQP ESAGQLWLTV RVVQPNATAW SEAGHISAWQ Q WRLAENLS VTLPAASHAI PHLTTSEMDF CIELGNKRWQ FNRQSGFLSQ MWIGDKKQLL TP LRDQFTR APLDNDIGVS EATRIDPNAW VERWKAAGHY QAEAALLQCT ADTLADAVLI TTA HAWQHQ GKTLFISRKT YRIDGSGQMA ITVDVEVASD TPHPARIGLN CQLAQVAERV NWLG LGPQE NYPDRLTAAC FDRWDLPLSD MYTPYVFPSE NGLRCGTREL NYGPHQWRGD FQFNI SRYS QQQLMETSHR HLLHAEEGTW LNIDGFHMGI GGDDSWSPSV SAEFQLSAGR YHYQLV WCQ K

UniProtKB: Beta-galactosidase

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Experimental details

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Structure determination

Methodnegative staining
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration2.1 mg/mL
BufferpH: 8
Component:
ConcentrationNameFormula
25.0 mMTris
50.0 mMSodiumclorideNaCl
2.0 mMMagnesiumchlorideMgCl2
0.5 mMTCEP

Details: 25 mM Tris at pH 8, 50 mM NaCl, 2 mM MgCl2, and 0.5 mM TCEP
StainingType: NEGATIVE / Material: Ammonium Molybdate
Details: Negative stained EM specimens were prepared using ammonium Molybdate negative staining.
GridModel: Quantifoil / Material: COPPER / Mesh: 200 / Support film - Material: CARBON / Support film - topology: CONTINUOUS / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 30 sec.
DetailsThis sample was monodisperse

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Electron microscopy

MicroscopeTFS TALOS L120C
DetailsPreliminary grid screening was performed manually.
Image recordingFilm or detector model: FEI CETA (4k x 4k) / Number grids imaged: 1 / Number real images: 246 / Average exposure time: 1.0 sec. / Average electron dose: 33.0 e/Å2 / Details: 163 were used
Electron beamAcceleration voltage: 120 kV / Electron source: LAB6
Electron opticsC2 aperture diameter: 100.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 2.0 µm / Nominal defocus min: 0.3 µm / Nominal magnification: 73000
Sample stageSpecimen holder model: SIDE ENTRY, EUCENTRIC / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Talos L120C / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 15955
Startup modelType of model: OTHER
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: D2 (2x2 fold dihedral) / Resolution.type: BY AUTHOR / Resolution: 10.06 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 4.4) / Number images used: 7919
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 4.4)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 4.4)
Final 3D classificationNumber classes: 2 / Software - Name: cryoSPARC (ver. 4.4)
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

6x1q


Chain - Source name: PDB / Chain - Initial model type: experimental model
RefinementProtocol: RIGID BODY FIT

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