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- PDB-8aa2: Inactive levan utilisation machinery (utilisome) in the presence ... -

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Basic information

Entry
Database: PDB / ID: 8aa2
TitleInactive levan utilisation machinery (utilisome) in the presence of levan fructo-oligosaccharides DP 15-25
Components
  • DUF4960 domain-containing protein
  • Glycoside hydrolase family 32
  • SusC homolog
  • SusD homolog
KeywordsTRANSPORT PROTEIN / Membrane protein transporter / glycan transporter / SusCD / utilisome / TonB dependent transporter / TBDT / levan
Function / homology
Function and homology information


beta-fructofuranosidase / hydrolase activity, hydrolyzing O-glycosyl compounds / cell outer membrane / carbohydrate metabolic process / membrane / metal ion binding
Similarity search - Function
Domain of unknown function DUF4960 / Domain of unknown function (DUF4960) / GH32, BT1760-like, C-terminal domain / GH32, BT1760-like, C-terminal domain / TonB-dependent outer membrane protein, SusC/RagA / TonB-dependent outer membrane protein SusC/RagA, conserved site / CarboxypepD_reg-like domain / SusD-like, N-terminal / Starch-binding associating with outer membrane / RagB/SusD domain ...Domain of unknown function DUF4960 / Domain of unknown function (DUF4960) / GH32, BT1760-like, C-terminal domain / GH32, BT1760-like, C-terminal domain / TonB-dependent outer membrane protein, SusC/RagA / TonB-dependent outer membrane protein SusC/RagA, conserved site / CarboxypepD_reg-like domain / SusD-like, N-terminal / Starch-binding associating with outer membrane / RagB/SusD domain / SusD family / Glycoside hydrolase, family 32 / Glycosyl hydrolase family 32, N-terminal / Glycosyl hydrolases family 32 N-terminal domain / Glycosyl hydrolases family 32 / Carboxypeptidase-like, regulatory domain superfamily / TonB-dependent receptor-like, beta-barrel / TonB dependent receptor-like, beta-barrel / TonB-dependent receptor, plug domain superfamily / TonB-dependent receptor, plug domain / TonB-dependent receptor-like, beta-barrel domain superfamily / TonB-dependent Receptor Plug Domain / Glycosyl hydrolase, five-bladed beta-propellor domain superfamily / Prokaryotic membrane lipoprotein lipid attachment site profile. / Tetratricopeptide-like helical domain superfamily / Concanavalin A-like lectin/glucanase domain superfamily
Similarity search - Domain/homology
SusC homolog / SusD homolog / DUF4960 domain-containing protein / beta-fructofuranosidase
Similarity search - Component
Biological speciesBacteroides thetaiotaomicron VPI-5482 (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.1 Å
AuthorsWhite, J.B.R. / Silale, A. / Ranson, N.A. / van den Berg, B.
Funding support United Kingdom, 3items
OrganizationGrant numberCountry
Wellcome Trust215064/Z/18/Z United Kingdom
Wellcome Trust214222/Z/18/Z United Kingdom
Wellcome Trust108466/Z/15/Z United Kingdom
CitationJournal: Nature / Year: 2023
Title: Outer membrane utilisomes mediate glycan uptake in gut Bacteroidetes.
Authors: Joshua B R White / Augustinas Silale / Matthew Feasey / Tiaan Heunis / Yiling Zhu / Hong Zheng / Akshada Gajbhiye / Susan Firbank / Arnaud Baslé / Matthias Trost / David N Bolam / Bert van ...Authors: Joshua B R White / Augustinas Silale / Matthew Feasey / Tiaan Heunis / Yiling Zhu / Hong Zheng / Akshada Gajbhiye / Susan Firbank / Arnaud Baslé / Matthias Trost / David N Bolam / Bert van den Berg / Neil A Ranson /
Abstract: Bacteroidetes are abundant members of the human microbiota, utilizing a myriad of diet- and host-derived glycans in the distal gut. Glycan uptake across the bacterial outer membrane of these bacteria ...Bacteroidetes are abundant members of the human microbiota, utilizing a myriad of diet- and host-derived glycans in the distal gut. Glycan uptake across the bacterial outer membrane of these bacteria is mediated by SusCD protein complexes, comprising a membrane-embedded barrel and a lipoprotein lid, which is thought to open and close to facilitate substrate binding and transport. However, surface-exposed glycan-binding proteins and glycoside hydrolases also play critical roles in the capture, processing and transport of large glycan chains. The interactions between these components in the outer membrane are poorly understood, despite being crucial for nutrient acquisition by our colonic microbiota. Here we show that for both the levan and dextran utilization systems of Bacteroides thetaiotaomicron, the additional outer membrane components assemble on the core SusCD transporter, forming stable glycan-utilizing machines that we term utilisomes. Single-particle cryogenic electron microscopy structures in the absence and presence of substrate reveal concerted conformational changes that demonstrate the mechanism of substrate capture, and rationalize the role of each component in the utilisome.
History
DepositionJun 29, 2022Deposition site: PDBE / Processing site: PDBE
Revision 1.0Jun 7, 2023Provider: repository / Type: Initial release
Revision 1.1Jun 21, 2023Group: Database references / Category: citation / citation_author
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_PubMed / _citation.title / _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
E: Glycoside hydrolase family 32
T: DUF4960 domain-containing protein
G: DUF4960 domain-containing protein
M: Glycoside hydrolase family 32
B: SusD homolog
A: SusC homolog
J: SusD homolog
I: SusC homolog
hetero molecules


Theoretical massNumber of molelcules
Total (without water)589,57419
Polymers582,0548
Non-polymers7,52011
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy, gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area47380 Å2
ΔGint26 kcal/mol
Surface area157910 Å2

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Components

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Protein , 4 types, 8 molecules EMTGBJAI

#1: Protein Glycoside hydrolase family 32 /


Mass: 59255.910 Da / Num. of mol.: 2 / Source method: isolated from a natural source
Source: (natural) Bacteroides thetaiotaomicron VPI-5482 (bacteria)
Strain: ATCC 29148 / DSM 2079 / JCM 5827 / CCUG 10774 / NCTC 10582 / VPI-5482 / E50
References: UniProt: Q8A6W6
#2: Protein DUF4960 domain-containing protein


Mass: 51258.098 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Bacteroides thetaiotaomicron VPI-5482 (bacteria)
Strain: ATCC 29148 / DSM 2079 / JCM 5827 / CCUG 10774 / NCTC 10582 / VPI-5482 / E50
Gene: BT_1761
Production host: Bacteroides thetaiotaomicron VPI-5482 (bacteria)
Strain (production host): ATCC 29148 / DSM 2079 / JCM 5827 / CCUG 10774 / NCTC 10582 / VPI-5482 / E50
References: UniProt: Q8A6W5
#3: Protein SusD homolog


Mass: 65029.414 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Bacteroides thetaiotaomicron VPI-5482 (bacteria)
Strain: ATCC 29148 / DSM 2079 / NCTC 10582 / E50 / VPI-5482 / Gene: BT_1762
Production host: Bacteroides thetaiotaomicron VPI-5482 (bacteria)
Strain (production host): ATCC 29148 / DSM 2079 / NCTC 10582 / E50 / VPI-5482
References: UniProt: Q8A6W4
#4: Protein SusC homolog


Mass: 115483.602 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Bacteroides thetaiotaomicron VPI-5482 (bacteria)
Strain: ATCC 29148 / DSM 2079 / JCM 5827 / CCUG 10774 / NCTC 10582 / VPI-5482 / E50
Gene: BT_1763
Production host: Bacteroides thetaiotaomicron VPI-5482 (bacteria)
Strain (production host): ATCC 29148 / DSM 2079 / JCM 5827 / CCUG 10774 / NCTC 10582 / VPI-5482 / E50
References: UniProt: Q8A6W3

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Sugars , 4 types, 7 molecules

#5: Polysaccharide beta-D-fructofuranose-(2-6)-beta-D-fructofuranose-(2-6)-beta-D-fructofuranose-(2-6)-beta-D- ...beta-D-fructofuranose-(2-6)-beta-D-fructofuranose-(2-6)-beta-D-fructofuranose-(2-6)-beta-D-fructofuranose-(2-6)-beta-D-fructofuranose-(2-6)-beta-D-fructofuranose-(2-6)-[beta-D-fructofuranose-(2-1)]beta-D-fructofuranose-(2-6)-beta-D-fructofuranose-(2-6)-beta-D-fructofuranose-(2-6)-beta-D-fructofuranose-(2-6)-beta-D-fructofuranose-(2-6)-beta-D-fructofuranose


Type: oligosaccharide / Mass: 2125.846 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
DescriptorTypeProgram
DFrufb2-6DFrufb2-6DFrufb2-6DFrufb2-6DFrufb2-6DFrufb2-6[DFrufb2-1]DFrufb2-6DFrufb2-6DFrufb2-6DFrufb2-6DFrufb2-6DFrufb2-Glycam Condensed SequenceGMML 1.0
WURCS=2.0/1,13,12/[ha122h-2b_2-5]/1-1-1-1-1-1-1-1-1-1-1-1-1/a6-b2_b6-c2_c6-d2_d6-e2_e6-f2_f1-g2_f6-h2_h6-i2_i6-j2_j6-k2_k6-l2_l6-m2WURCSPDB2Glycan 1.1.0
[][b-D-Fruf]{[(6+2)][b-D-Fruf]{[(6+2)][b-D-Fruf]{[(6+2)][b-D-Fruf]{[(6+2)][b-D-Fruf]{[(6+2)][b-D-Fruf]{[(6+2)][b-D-Fruf]{[(6+2)][b-D-Fruf]{[(6+2)][b-D-Fruf]{[(6+2)][b-D-Fruf]{[(6+2)][b-D-Fruf]{[(6+2)][b-D-Fruf]{}}}}}}}}}}}}LINUCSPDB-CARE
#6: Polysaccharide beta-D-fructofuranose-(2-6)-beta-D-fructofuranose-(2-6)-beta-D-fructofuranose-(2-6)-beta-D- ...beta-D-fructofuranose-(2-6)-beta-D-fructofuranose-(2-6)-beta-D-fructofuranose-(2-6)-beta-D-fructofuranose-(2-6)-beta-D-fructofuranose


Type: oligosaccharide / Mass: 828.719 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
DescriptorTypeProgram
DFrufb2-6DFrufb2-6DFrufb2-6DFrufb2-6DFrufb2-Glycam Condensed SequenceGMML 1.0
WURCS=2.0/1,5,4/[ha122h-2b_2-5]/1-1-1-1-1/a6-b2_b6-c2_c6-d2_d6-e2WURCSPDB2Glycan 1.1.0
[][b-D-Fruf]{[(6+2)][b-D-Fruf]{[(6+2)][b-D-Fruf]{[(6+2)][b-D-Fruf]{[(6+2)][b-D-Fruf]{}}}}}LINUCSPDB-CARE
#7: Polysaccharide beta-D-fructofuranose-(2-6)-beta-D-fructofuranose-(2-6)-beta-D-fructofuranose-(2-6)-beta-D- ...beta-D-fructofuranose-(2-6)-beta-D-fructofuranose-(2-6)-beta-D-fructofuranose-(2-6)-beta-D-fructofuranose-(2-6)-[beta-D-fructofuranose-(2-1)]beta-D-fructofuranose-(2-6)-beta-D-fructofuranose


Type: oligosaccharide / Mass: 1153.001 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
DescriptorTypeProgram
DFrufb2-6DFrufb2-6DFrufb2-6DFrufb2-6[DFrufb2-1]DFrufb2-6DFrufb2-Glycam Condensed SequenceGMML 1.0
WURCS=2.0/1,7,6/[ha122h-2b_2-5]/1-1-1-1-1-1-1/a6-b2_b1-c2_b6-d2_d6-e2_e6-f2_f6-g2WURCSPDB2Glycan 1.1.0
[][b-D-Fruf]{[(6+2)][b-D-Fruf]{[(6+2)][b-D-Fruf]{[(6+2)][b-D-Fruf]{[(6+2)][b-D-Fruf]{[(6+2)][b-D-Fruf]{}}}}}}LINUCSPDB-CARE
#8: Polysaccharide beta-D-fructofuranose-(2-6)-beta-D-fructofuranose-(2-6)-beta-D-fructofuranose-(2-6)-beta-D-fructofuranose


Type: oligosaccharide / Mass: 666.578 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
DescriptorTypeProgram
DFrufb2-6DFrufb2-6DFrufb2-6DFrufb2-Glycam Condensed SequenceGMML 1.0
WURCS=2.0/1,4,3/[ha122h-2b_2-5]/1-1-1-1/a6-b2_b6-c2_c6-d2WURCSPDB2Glycan 1.1.0
[][b-D-Fruf]{[(6+2)][b-D-Fruf]{[(6+2)][b-D-Fruf]{[(6+2)][b-D-Fruf]{}}}}LINUCSPDB-CARE

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Non-polymers , 1 types, 4 molecules

#9: Chemical
ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: Mg

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Details

Has ligand of interestY

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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: Inactive levan utilisation machinery (utilisome) with levan fructo-oligosaccharides DP 15-25
Type: COMPLEX
Details: Levan utilisome composed of Bt1760-Bt1763. The levanase component, Bt1760, is inactivated (D42A mutation). The assembly includes bound levan fructo-oligosaccharides.
Entity ID: #1-#4 / Source: NATURAL
Source (natural)Organism: Bacteroides thetaiotaomicron VPI-5482 (bacteria)
Buffer solutionpH: 7.5
Details: Supplemented with 0.5 mM levan fructo-oligosaccharides DP 15-25
Buffer component
IDConc.NameFormulaBuffer-ID
110 mMHEPES1
2100 mMSodium chlorideNaClSodium chloride1
30.03 % (w/v)DDM1
SpecimenConc.: 3 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: 30 mA current / Grid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277.15 K

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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 defocus max: 3000 nm / Nominal defocus min: 1500 nm
Image recordingElectron dose: 37.8 e/Å2 / Detector mode: COUNTING / Film or detector model: FEI FALCON III (4k x 4k)

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Processing

EM softwareName: RELION / Version: 3.1 / Category: 3D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.1 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 27310 / Symmetry type: POINT

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