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- EMDB-18244: ESIBD structure of beta-galactosidase -

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Basic information

Entry
Database: EMDB / ID: EMD-18244
TitleESIBD structure of beta-galactosidase
Map dataSharpened and local resolution-filtered
Sample
  • Complex: Beta-galactosidase from E. coli; tetrameric complex
    • Protein or peptide: Beta-galactosidase
KeywordsLactase / Beta-galactosidase / cryo-EM / native MS / 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 (E. coli) / Escherichia coli K-12 (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.6 Å
AuthorsEsser T / Boehning J / Bharat TAM / Rauschenbach S
Funding support United Kingdom, France, United States, European Union, 10 items
OrganizationGrant numberCountry
Biotechnology and Biological Sciences Research Council (BBSRC)EP/V051474/1 United Kingdom
Medical Research Council (MRC, United Kingdom)MC UP 1201/31 United Kingdom
Human Frontier Science Program (HFSP)RGY0074/2021 France
Engineering and Physical Sciences Research CouncilEP/V026623/1 United Kingdom
The Vallee Foundation Inc. United States
European Molecular Biology Organization (EMBO)European Union
Leverhulme Trust United Kingdom
The Lister Institute of Preventive Medicine United Kingdom
Wellcome Trust104633/Z/14/Z United Kingdom
Royal SocietyNIF/R1/192285 United Kingdom
CitationJournal: Sci Adv / Year: 2024
Title: Cryo-EM of soft-landed β-galactosidase: Gas-phase and native structures are remarkably similar.
Authors: Tim K Esser / Jan Böhning / Alpcan Önür / Dinesh K Chinthapalli / Lukas Eriksson / Marko Grabarics / Paul Fremdling / Albert Konijnenberg / Alexander Makarov / Aurelien Botman / Christine ...Authors: Tim K Esser / Jan Böhning / Alpcan Önür / Dinesh K Chinthapalli / Lukas Eriksson / Marko Grabarics / Paul Fremdling / Albert Konijnenberg / Alexander Makarov / Aurelien Botman / Christine Peter / Justin L P Benesch / Carol V Robinson / Joseph Gault / Lindsay Baker / Tanmay A M Bharat / Stephan Rauschenbach /
Abstract: Native mass spectrometry (MS) has become widely accepted in structural biology, providing information on stoichiometry, interactions, homogeneity, and shape of protein complexes. Yet, the fundamental ...Native mass spectrometry (MS) has become widely accepted in structural biology, providing information on stoichiometry, interactions, homogeneity, and shape of protein complexes. Yet, the fundamental assumption that proteins inside the mass spectrometer retain a structure faithful to native proteins in solution remains a matter of intense debate. Here, we reveal the gas-phase structure of β-galactosidase using single-particle cryo-electron microscopy (cryo-EM) down to 2.6-Å resolution, enabled by soft landing of mass-selected protein complexes onto cold transmission electron microscopy (TEM) grids followed by in situ ice coating. We find that large parts of the secondary and tertiary structure are retained from the solution. Dehydration-driven subunit reorientation leads to consistent compaction in the gas phase. By providing a direct link between high-resolution imaging and the capability to handle and select protein complexes that behave problematically in conventional sample preparation, the approach has the potential to expand the scope of both native mass spectrometry and cryo-EM.
History
DepositionAug 17, 2023-
Header (metadata) releaseJan 10, 2024-
Map releaseJan 10, 2024-
UpdateMar 6, 2024-
Current statusMar 6, 2024Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_18244.png

Downloads & links

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Map

FileDownload / File: emd_18244.map.gz / Format: CCP4 / Size: 274.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationSharpened and local resolution-filtered
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.83 Å/pix.
x 416 pix.
= 345.28 Å		0.83 Å/pix.
x 416 pix.
= 345.28 Å		0.83 Å/pix.
x 416 pix.
= 345.28 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.83 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.158
Minimum - Maximum-2.5955462 - 3.6415358
Average (Standard dev.)0.001471602 (±0.03986202)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions416416416
Spacing416416416
CellA=B=C: 345.28 Å
α=β=γ: 90.0 °

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Supplemental data

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Additional map: Sharpened

Fileemd_18244_additional_1.map
AnnotationSharpened
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_18244_half_map_1.map
Projections & Slices
AxesZYX

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Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #2

Fileemd_18244_half_map_2.map
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 from E. coli; tetrameric complex

EntireName: Beta-galactosidase from E. coli; tetrameric complex
Components
  • Complex: Beta-galactosidase from E. coli; tetrameric complex
    • Protein or peptide: Beta-galactosidase

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Supramolecule #1: Beta-galactosidase from E. coli; tetrameric complex

SupramoleculeName: Beta-galactosidase from E. coli; tetrameric complex / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all / Details: Sigma Aldrich Product Number G3153
Source (natural)Organism: Escherichia coli (E. coli)
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
Source (natural)Organism: Escherichia coli K-12 (bacteria)
Molecular weightTheoretical: 116.5725 KDa
Recombinant expressionOrganism: Escherichia coli K-12 (bacteria)
SequenceString: MTMITDSLAV VLQRRDWENP GVTQLNRLAA HPPFASWRNS EEARTDRPSQ QLRSLNGEWR FAWFPAPEAV PESWLECDLP EADTVVVPS NWQMHGYDAP IYTNVTYPIT VNPPFVPTEN PTGCYSLTFN VDESWLQEGQ TRIIFDGVNS AFHLWCNGRW V GYGQDSRL ...String:
MTMITDSLAV VLQRRDWENP GVTQLNRLAA HPPFASWRNS EEARTDRPSQ QLRSLNGEWR FAWFPAPEAV PESWLECDLP EADTVVVPS NWQMHGYDAP IYTNVTYPIT VNPPFVPTEN PTGCYSLTFN VDESWLQEGQ TRIIFDGVNS AFHLWCNGRW V GYGQDSRL PSEFDLSAFL RAGENRLAVM VLRWSDGSYL EDQDMWRMSG IFRDVSLLHK PTTQISDFHV ATRFNDDFSR AV LEAEVQM CGELRDYLRV TVSLWQGETQ VASGTAPFGG EIIDERGGYA DRVTLRLNVE NPKLWSAEIP NLYRAVVELH TAD GTLIEA EACDVGFRVV RIENGLLLLN GKPLLIRGVN RHEHHPLHGQ VMDEQTMVQD ILLMKQNNFN AVRCSHYPNH PLWY TLCDR YGLYVVDEAN IETHGMVPMN RLTDDPRWLP AMSERVTRMV QRDRNHPSVI IWSLGNESGH GANHDALYRW IKSVD PSRP VQYEGGGADT TATDIICPMY ARVDEDQPFP AVPKWSIKKW LSLPGETRPL ILCEYAHAMG NSLGGFAKYW QAFRQY PRL QGGFVWDWVD QSLIKYDENG NPWSAYGGDF GDTPNDRQFC MNGLVFADRT PHPALTEAKH QQQFFQFRLS GQTIEVT SE YLFRHSDNEL LHWMVALDGK PLASGEVPLD VAPQGKQLIE LPELPQPESA GQLWLTVRVV QPNATAWSEA GHISAWQQ W RLAENLSVTL PAASHAIPHL TTSEMDFCIE LGNKRWQFNR QSGFLSQMWI GDKKQLLTPL RDQFTRAPLD NDIGVSEAT RIDPNAWVER WKAAGHYQAE AALLQCTADT LADAVLITTA HAWQHQGKTL FISRKTYRID GSGQMAITVD VEVASDTPHP ARIGLNCQL AQVAERVNWL GLGPQENYPD RLTAACFDRW DLPLSDMYTP YVFPSENGLR CGTRELNYGP HQWRGDFQFN I SRYSQQQL METSHRHLLH AEEGTWLNID GFHMGIGGDD SWSPSVSAEF QLSAGRYHYQ LVWCQK

UniProtKB: Beta-galactosidase

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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

BufferpH: 6.9 / Details: Soft-landed sample
GridModel: Quantifoil R2/1 / Material: COPPER / Mesh: 400 / Support film - Material: CARBON / Support film - topology: HOLEY
VitrificationCryogen name: OTHER / Details: Soft-landed as described in manuscript.

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Specialist opticsEnergy filter - Name: GIF Bioquantum / Energy filter - Slit width: 20 eV
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Number real images: 4100 / Average electron dose: 34.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.5 µm / Nominal defocus min: 1.0 µm / Nominal magnification: 105000
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

Startup modelType of model: OTHER / Details: Ab-initio model generated in cryoSPARC
Final reconstructionApplied symmetry - Point group: D2 (2x2 fold dihedral) / Resolution.type: BY AUTHOR / Resolution: 2.6 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC / Software - details: Non-uniform refinement / Number images used: 463000
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC / Details: CryoSPARC
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

6drv


Chain - Source name: PDB / Chain - Initial model type: experimental model
DetailsInitial flexible fitting was performed using ISOLDE and Real-Space Refinement was performed using PHENIX.
RefinementSpace: REAL
Output model

PDB-8q7y:
ESIBD structure of beta-galactosidase

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