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- EMDB-15289: Levan utilisation machinery (utilisome) with levan fructo-oligosa... -

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Basic information

Entry
Database: EMDB / ID: EMD-15289
TitleLevan utilisation machinery (utilisome) with levan fructo-oligosaccharides DP 8-12
Map dataSharpened map from post-processing using an ad-hoc low-pass filter of 3.3 A
Sample
  • Complex: Levan utilisation machinery (utilisome) with levan fructo-oligosaccharides DP8-12
    • Protein or peptide: Glycoside hydrolase family 32
    • Protein or peptide: DUF4960 domain-containing protein
    • Protein or peptide: SusD homolog
    • Protein or peptide: SusC homolog
  • Ligand: MAGNESIUM ION
KeywordsMembrane protein transporter / glycan transporter / SusCD / utilisome / TonB dependent transporter / TBDT / TRANSPORT PROTEIN / 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 / Carboxypeptidase-like, regulatory domain superfamily / Glycoside hydrolase, family 32 / Glycosyl hydrolase family 32, N-terminal / Glycosyl hydrolases family 32 N-terminal domain / Glycosyl hydrolases family 32 / TonB-dependent receptor-like, beta-barrel / TonB dependent receptor / 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)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.2 Å
AuthorsWhite JBR / Silale A / Ranson NA / van den Berg B
Funding support United Kingdom, 3 items
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, 2022-
Header (metadata) releaseJun 7, 2023-
Map releaseJun 7, 2023-
UpdateJun 21, 2023-
Current statusJun 21, 2023Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_15289.png

Downloads & links

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Map

FileDownload / File: emd_15289.map.gz / Format: CCP4 / Size: 103 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationSharpened map from post-processing using an ad-hoc low-pass filter of 3.3 A
Voxel sizeX=Y=Z: 1.065 Å
Density
Contour LevelBy AUTHOR: 0.0452
Minimum - Maximum-0.062261496 - 0.17972027
Average (Standard dev.)0.00096683117 (±0.0063738343)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions300300300
Spacing300300300
CellA=B=C: 319.50003 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_15289_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
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Histogram

Histogram (log scale)

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Additional map: Unsharpened map from 3D refinement

Fileemd_15289_additional_1.map
AnnotationUnsharpened map from 3D refinement
Projections & Slices
AxesZYX

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Histogram

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Half map: #2

Fileemd_15289_half_map_1.map
Projections & Slices
AxesZYX

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Histogram

Histogram (log scale)

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Half map: #1

Fileemd_15289_half_map_2.map
Projections & Slices
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Sample components

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Entire : Levan utilisation machinery (utilisome) with levan fructo-oligosa...

EntireName: Levan utilisation machinery (utilisome) with levan fructo-oligosaccharides DP8-12
Components
  • Complex: Levan utilisation machinery (utilisome) with levan fructo-oligosaccharides DP8-12
    • Protein or peptide: Glycoside hydrolase family 32
    • Protein or peptide: DUF4960 domain-containing protein
    • Protein or peptide: SusD homolog
    • Protein or peptide: SusC homolog
  • Ligand: MAGNESIUM ION

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Supramolecule #1: Levan utilisation machinery (utilisome) with levan fructo-oligosa...

SupramoleculeName: Levan utilisation machinery (utilisome) with levan fructo-oligosaccharides DP8-12
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#4 / Details: Levan utilisome comprised of Bt1760-Bt1763
Source (natural)Organism: Bacteroides thetaiotaomicron VPI-5482 (bacteria)

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Macromolecule #1: Glycoside hydrolase family 32

MacromoleculeName: Glycoside hydrolase family 32 / type: protein_or_peptide / ID: 1 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Bacteroides thetaiotaomicron VPI-5482 (bacteria)
Strain: ATCC 29148 / DSM 2079 / JCM 5827 / CCUG 10774 / NCTC 10582 / VPI-5482 / E50
Molecular weightTheoretical: 59.299926 KDa
Recombinant expressionOrganism: Bacteroides thetaiotaomicron VPI-5482 (bacteria)
SequenceString: MMKNMILPIA FTALIASMTA CSDETDPILT QKNWDGTATY FQSSDEHGFS MYYKPQVGFV GDPMPFYDPV AKDFKVMYLQ DYRPNPEAT YHPIFGVATK DGATYESLGE LISCGGRDEQ DAAIGTGGTI YNPADKLYYT FYTGNKFKPS SDQNAQVVMV A TSPDFKTW ...String:
MMKNMILPIA FTALIASMTA CSDETDPILT QKNWDGTATY FQSSDEHGFS MYYKPQVGFV GDPMPFYDPV AKDFKVMYLQ DYRPNPEAT YHPIFGVATK DGATYESLGE LISCGGRDEQ DAAIGTGGTI YNPADKLYYT FYTGNKFKPS SDQNAQVVMV A TSPDFKTW TKNRTFYLKG DTYGYDKNDF RDPFLFQTED GVYHMLIATR KNGKGHIAEF TSADLKEWES AGTFMTMMWD RF YECPDVF KMGDWWYLIY SEQASFMRKV QYFKGRTLED LKATTANDAG IWPDNREGML DSRAFYAGKT ASDGTNRYIW GWC PTRAGN DNGNVGDVEP EWAGNLVAQR LIQHEDGTLT LGVPDAIDRK YTSAQEVKVM AKDGNMIESG KTYTLGEGAS VIFN RLKVH NKISFTVKTA SNTDRFGISF VRGTDSASWY SIHVNADEGK ANFEKDGDDA KYLFDNKFNI PADNEYRVTI YSDQS VCVT YINDQLSFTN RIYQMQKNPW SLCCYKGEIT VSDVQVSTY

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Macromolecule #2: DUF4960 domain-containing protein

MacromoleculeName: DUF4960 domain-containing protein / type: protein_or_peptide / ID: 2 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Bacteroides thetaiotaomicron VPI-5482 (bacteria)
Strain: ATCC 29148 / DSM 2079 / JCM 5827 / CCUG 10774 / NCTC 10582 / VPI-5482 / E50
Molecular weightTheoretical: 50.429227 KDa
Recombinant expressionOrganism: Bacteroides thetaiotaomicron VPI-5482 (bacteria)
SequenceString: MKSIIKQLYT ILLVTVACLT VTGCSDDFKS GLRLDGDVWV NSIRLDEYAG TVDYQNKAIV VGVPYDYDIT RMVVTEMNLS EGAKASIAI GETIDFSLPV SLTVKNGDVQ MSYTITVKRD EAKILTFKLN DTYVGKVDQL SKTISVVVPL TVDITQLKGT F TVTDGATV ...String:
MKSIIKQLYT ILLVTVACLT VTGCSDDFKS GLRLDGDVWV NSIRLDEYAG TVDYQNKAIV VGVPYDYDIT RMVVTEMNLS EGAKASIAI GETIDFSLPV SLTVKNGDVQ MSYTITVKRD EAKILTFKLN DTYVGKVDQL SKTISVVVPL TVDITQLKGT F TVTDGATV TPASGSIQDF TNPVTYTATY RSAVTPYVVT VTQGNVIPTA FVGTASSVSL LTSPEEKAAA QWMMDNVSMS EY ISFKDVV DGKVDLGKYT AIWWHFHADN GDNPPLPDDA KAAAEKFKVY YQNGGNLLLT RYATFYIANL GIAKDERVPN NSW GGNEDS PEITSAPWSF LITGSESHPL FQDLRWKDGD KSTVYTCDAG YAITNSTAQW HIGTDWGGYD DLNAWRNLTG GIDL AHGGD GAVVIAEFEP RSNSGRTLCI GSGCYDWYGK GVDASADYYH YNVEQMTLNA INYLCK

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Macromolecule #3: SusD homolog

MacromoleculeName: SusD homolog / type: protein_or_peptide / ID: 3 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Bacteroides thetaiotaomicron VPI-5482 (bacteria) / Strain: ATCC 29148 / DSM 2079 / NCTC 10582 / E50 / VPI-5482
Molecular weightTheoretical: 66.142625 KDa
Recombinant expressionOrganism: Bacteroides thetaiotaomicron VPI-5482 (bacteria)
SequenceString: MKKIIYIATI GITLLTTSCD DFLDRQVPQG IVTGDQIASP EYVDNLVISA YAIWATGDDI NSSFSLWNYD VRSDDCYKGG SGTEDGGVF NALEISKGIN TTDWNINDIW KRLYQCITRA NTALQSLDQM DEKTYPLKNQ RIAEMRFLRG HAHFMLKQLF K KIVIVNDE ...String:
MKKIIYIATI GITLLTTSCD DFLDRQVPQG IVTGDQIASP EYVDNLVISA YAIWATGDDI NSSFSLWNYD VRSDDCYKGG SGTEDGGVF NALEISKGIN TTDWNINDIW KRLYQCITRA NTALQSLDQM DEKTYPLKNQ RIAEMRFLRG HAHFMLKQLF K KIVIVNDE NMEPDAYNEL SNTTYTNDEQ WQKIADDFQF AYDNLPEVQI EKGRPAQAAA AAYLAKTYLY KAYRQDGADN AL TGINEED LKQVVKYTDP LIMAKGGYGL ETDYSMNFLP QYENGAESVW AIQYSINDGT YNGNLNWGMG LTTPQILGCC DFH KPSQNL VNAFKTDSQG KPLFSTYDNE NYEVATDNVD PRLFHTVGMP GFPYKYNEGY IIQKNDDWSR SKGLYGYYVS LKEN VDPDC DCLKKGSYWA SSLNHIVIRY ADVLLMRAEA LIQLNDGRIT DAISLINEVR SRAAGSTMLI FNYKEDYGVN FKVTP YDLK AYAQDEAMKM LKWERRVEFG MESSRFFDLV RWGEAKDVIN AYYVTEASRC SIYKNAGFTE NKNEYLPVPF EQISAS NGN YTQNFGWAAA AHHHHHH

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Macromolecule #4: SusC homolog

MacromoleculeName: SusC homolog / type: protein_or_peptide / ID: 4 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Bacteroides thetaiotaomicron VPI-5482 (bacteria) / Strain: ATCC 29148 / DSM 2079 / NCTC 10582 / E50 / VPI-5482
Molecular weightTheoretical: 115.483602 KDa
Recombinant expressionOrganism: Bacteroides thetaiotaomicron VPI-5482 (bacteria)
SequenceString: MPGIMKNKKL LCSVCFLFAF MSALWGQNIT VKGNVTSKTD GQPIIGASVV ETTATTNGTI TDFDGNFTLS VPVNSTLKIT YIGYKPVTV KAAAIVNVLL EEDTQMVDEV VVTGYTTQRK ADLTGAVSVV KVDEIQKQGE NNPVKALQGR VPGMNITADG N PSGSATVR ...String:
MPGIMKNKKL LCSVCFLFAF MSALWGQNIT VKGNVTSKTD GQPIIGASVV ETTATTNGTI TDFDGNFTLS VPVNSTLKIT YIGYKPVTV KAAAIVNVLL EEDTQMVDEV VVTGYTTQRK ADLTGAVSVV KVDEIQKQGE NNPVKALQGR VPGMNITADG N PSGSATVR IRGIGTLNNN DPLYIIDGVP TKAGMHELNG NDIESIQVLK DAASASIYGS RAANGVIIIT TKQGKKGQIK IN FDASVSA SMYQSKMNVL NTEQYGRAMW QAYVNDGENP NGNALGYAYN WGYNADGNPV LYGMTLSKYL DSKNTMPVAD TDW FDEITR TGVIQQYNLS VSNGSEKGSS FFSLGYYKNL GVIKDTDFDR FSARMNSDYK LIDDILTIGQ HFTLNRTSEV QAPG GIIET ALDIPSAIPV YASDGSWGGP VGGWPDRRNP RAVLEYNKDN RYTYWRMFGD AYVNLTPFKG FNLRSTFGLD YANKQ ARYF TYPYQEGTQT NNGKSAVEAK QEHWTKWMWN AIATYQLEVG KHRGDVMIGM ELNREDDSHF SGYKEDFSIL TPDYMW PDA GSGTAQAYGA GEGYSLVSFF GKMNYSYADR YLLSLTLRRD GSSRFGKNHR YATFPSVSLG WRITQENFMK ELTWLDD LK LRASWGQTGN QEISNLARYT IYAPNYGTTD SFGGQSYGTA YDITGSNGGG VLPSGFKRNQ IGNDNIKWET TTQTNVGI D FSLFKQSLYG SLEYYYKKAT DILTEMAGVG VLGEGGSRWI NSGAMKNQGF EFNLGYRNKT AFGLTYDLNG NISTYRNEI LELPETVAAN GKFGGNGVKS VVGHTYGAQV GYIADGIFKS QDEVDNHATQ EGAAVGRIRY RDIDHNGVID ERDQNWIYDP TPSFSYGLN IYLEYKNFDL TMFWQGVQGV DIISDVKKKS DFWSASNVGF LNKGTRLLNA WSPTNPNSDI PALTRSDTNN E QRVSTYFV ENGSFLKLRN IQLGYTVPAV ISKKMRMDRL RFYCSAQNLL TIKSKNFTGE DPENPNFSYP IPVNITFGLN IG F

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Macromolecule #7: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 7 / Number of copies: 4 / Formula: MG
Molecular weightTheoretical: 24.305 Da

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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

Concentration3 mg/mL
BufferpH: 7.5
Component:
ConcentrationNameFormula
10.0 mMHEPES
100.0 mMSodium chlorideNaClSodium chloride
0.03 % (w/v)DDM

Details: Supplemented with 0.5 mM levan fructo-oligosaccharides DP 8-12
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 300 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 60 sec. / Pretreatment - Atmosphere: OTHER / Details: 30 mA
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277.15 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 3.0 µm / Nominal defocus min: 1.5 µm
Image recordingFilm or detector model: FEI FALCON III (4k x 4k) / Detector mode: COUNTING / Average electron dose: 38.5 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: INSILICO MODEL
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
Final reconstructionApplied symmetry - Point group: C2 (2 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 3.2 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 15012
FSC plot (resolution estimation)

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