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- EMDB-18245: Plunge-frozen (control) map of beta-galactosidase -

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Basic information

Entry
Database: EMDB / ID: EMD-18245
TitlePlunge-frozen (control) map of beta-galactosidase
Map data
Sample
  • Complex: Beta-galactosidase from E. coli; tetrameric complex
    • Other: Beta-galactosidase
KeywordsLactase / Beta-galactosidase / cryo-EM / native MS / HYDROLASE
Biological speciesEscherichia coli (E. coli) / Escherichia coli K-12 (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.3 Å
AuthorsEsser TK / 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 31, 2024-
Map releaseJan 31, 2024-
UpdateMar 6, 2024-
Current statusMar 6, 2024Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_18245.png

Downloads & links

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Map

FileDownload / File: emd_18245.map.gz / Format: CCP4 / Size: 274.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 0.83 Å
Density
Contour LevelBy AUTHOR: 0.5
Minimum - Maximum-1.610418 - 2.5985234
Average (Standard dev.)0.00018434286 (±0.08306679)
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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Half map: #1

Fileemd_18245_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #2

Fileemd_18245_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
    • Other: 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: other / ID: 1 / Classification: other
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 AEACDVGFRV 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
Recombinant expressionOrganism: Escherichia coli K-12 (bacteria)

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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: 25 mM Tris, 50 mM NaCl, 10 mM EDTA, 2 mM MgCl2
GridModel: Quantifoil / Mesh: 200 / Support film - Material: CARBON / Support film - topology: HOLEY
VitrificationCryogen name: ETHANE / Details: Plunge-frozen.
Details2.5 uM protein concentration

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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: 2.3 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC / Software - details: Non-uniform refinement / Number images used: 273000
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:

6cvm


Chain - Source name: PDB / Chain - Initial model type: experimental model
DetailsThe PDB model 6CVM is an excellent fit for this map. No refinement was performed.
RefinementProtocol: RIGID BODY FIT

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