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- PDB-8q7y: ESIBD structure of beta-galactosidase -

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Basic information

Entry
Database: PDB / ID: 8q7y
TitleESIBD structure of beta-galactosidase
ComponentsBeta-galactosidase
KeywordsHYDROLASE / Lactase / Beta-galactosidase / cryo-EM / native MS
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 / 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
Biological speciesEscherichia coli K-12 (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.6 Å
AuthorsEsser, T. / Boehning, J. / Bharat, T.A.M. / Rauschenbach, S.
Funding support United Kingdom, France, United States, European Union, 10items
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, 2023Deposition site: PDBE / Processing site: PDBE
Revision 1.0Jan 10, 2024Provider: repository / Type: Initial release
Revision 1.1Mar 6, 2024Group: Database references / Category: citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year / _citation_author.identifier_ORCID / _citation_author.name

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Beta-galactosidase
B: Beta-galactosidase
C: Beta-galactosidase
D: Beta-galactosidase


Theoretical massNumber of molelcules
Total (without water)466,2904
Polymers466,2904
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area7040 Å2
ΔGint-31 kcal/mol
Surface area51880 Å2
MethodPISA

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Components

#1: Protein
Beta-galactosidase /


Mass: 116572.500 Da / Num. of mol.: 4
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli K-12 (bacteria) / Gene: lacZ / Production host: Escherichia coli K-12 (bacteria) / References: UniProt: P00722

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: Beta-galactosidase from E. coli; tetrameric complex / Type: COMPLEX / Details: Sigma Aldrich Product Number G3153 / Entity ID: all / Source: RECOMBINANT
Molecular weightValue: 0.465 MDa / Experimental value: NO
Source (natural)Organism: Escherichia coli (E. coli)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 6.9 / Details: Soft-landed sample
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 400 divisions/in. / Grid type: Quantifoil R2/1
VitrificationCryogen name: OTHER / Details: Soft-landed as described in manuscript

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 105000 X / Nominal defocus max: 2500 nm / Nominal defocus min: 1000 nm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 34 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) / Num. of real images: 4100
EM imaging opticsEnergyfilter name: GIF Bioquantum / Energyfilter slit width: 20 eV

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Processing

EM software
IDNameVersionCategoryDetails
2EPUimage acquisition
7ISOLDEmodel fitting
10cryoSPARCfinal Euler assignment
12cryoSPARC3D reconstructionNon-uniform refinement
13PHENIX1.2model refinement
CTF correctionDetails: As implemented in cryoSPARC / Type: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: D2 (2x2 fold dihedral)
3D reconstructionResolution: 2.6 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 463000 / Symmetry type: POINT
Atomic model buildingSpace: REAL
Details: Initial flexible fitting was performed using ISOLDE and Real-Space Refinement was performed using PHENIX.
Atomic model buildingPDB-ID: 6DRV

6drv


Accession code: 6DRV / Source name: PDB / Type: experimental model
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00310316
ELECTRON MICROSCOPYf_angle_d0.6313960
ELECTRON MICROSCOPYf_dihedral_angle_d4.1291316
ELECTRON MICROSCOPYf_chiral_restr0.0441412
ELECTRON MICROSCOPYf_plane_restr0.0051780

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