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- EMDB-15291: Inactive levan utilisation machinery (utilisome) in the presence ... -

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Entry
Database: EMDB / ID: EMD-15291
TitleInactive levan utilisation machinery (utilisome) in the presence of levan fructo-oligosaccharides DP 15-25
Map dataSharpened map from post-processing with an ad-hoc low-pass filter of 3.2
Sample
  • Complex: Inactive levan utilisation machinery (utilisome) with levan fructo-oligosaccharides DP 15-25
    • 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 / : / CarboxypepD_reg-like domain / TonB-dependent outer membrane protein, SusC/RagA / TonB-dependent outer membrane protein SusC/RagA, conserved site / SusD-like, N-terminal / Starch-binding associating with outer membrane ...Domain of unknown function DUF4960 / Domain of unknown function (DUF4960) / GH32, BT1760-like, C-terminal domain / GH32, BT1760-like, C-terminal domain / : / CarboxypepD_reg-like domain / TonB-dependent outer membrane protein, SusC/RagA / TonB-dependent outer membrane protein SusC/RagA, conserved site / 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 (TBDR) proteins profile. / Vitamin B12 transporter BtuB-like / TonB-dependent receptor, plug domain superfamily / TonB-dependent receptor, plug domain / TonB-dependent Receptor Plug Domain / TonB-dependent receptor-like, beta-barrel domain superfamily / Glycosyl hydrolase, five-bladed beta-propellor domain superfamily / Tetratricopeptide-like helical domain superfamily / Prokaryotic membrane lipoprotein lipid attachment site profile. / 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: 3.1 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_15291.png

Downloads & links

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Map

FileDownload / File: emd_15291.map.gz / Format: CCP4 / Size: 103 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationSharpened map from post-processing with an ad-hoc low-pass filter of 3.2
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.07 Å/pix.
x 300 pix.
= 319.5 Å		1.07 Å/pix.
x 300 pix.
= 319.5 Å		1.07 Å/pix.
x 300 pix.
= 319.5 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.065 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.0425
Minimum - Maximum-0.061200738 - 0.18815258
Average (Standard dev.)0.0010507631 (±0.0067491736)
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_15291_msk_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_15291_half_map_1.map
Projections & Slices
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Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_15291_half_map_2.map
Projections & Slices
AxesZYX

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Slices (1/2)
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Histogram (log scale)

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Sample components

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Entire : Inactive levan utilisation machinery (utilisome) with levan fruct...

EntireName: Inactive levan utilisation machinery (utilisome) with levan fructo-oligosaccharides DP 15-25
Components
  • Complex: Inactive levan utilisation machinery (utilisome) with levan fructo-oligosaccharides DP 15-25
    • 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: Inactive levan utilisation machinery (utilisome) with levan fruct...

SupramoleculeName: Inactive levan utilisation machinery (utilisome) with levan fructo-oligosaccharides DP 15-25
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#4
Details: Levan utilisome composed of Bt1760-Bt1763. The levanase component, Bt1760, is inactivated (D42A mutation). The assembly includes bound levan fructo-oligosaccharides.
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: 59.25591 KDa
SequenceString: MMKNMILPIA FTALIASMTA CSDETDPILT QKNWDGTATY FQSSDEHGFS MYYKPQVGFV GAPMPFYDPV AKDFKVMYLQ DYRPNPEAT YHPIFGVATK DGATYESLGE LISCGGRDEQ DAAIGTGGTI YNPADKLYYT FYTGNKFKPS SDQNAQVVMV A TSPDFKTW ...String:
MMKNMILPIA FTALIASMTA CSDETDPILT QKNWDGTATY FQSSDEHGFS MYYKPQVGFV GAPMPFYDPV 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: 51.258098 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 INYLCKHHHH HH

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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: 65.029414 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 YTQNFGW

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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 / JCM 5827 / CCUG 10774 / NCTC 10582 / VPI-5482 / E50
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 #9: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 9 / 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 chlorideNaCl
0.03 % (w/v)DDM

Details: Supplemented with 0.5 mM levan fructo-oligosaccharides DP 15-25
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 current
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
Image recordingFilm or detector model: FEI FALCON III (4k x 4k) / Detector mode: COUNTING / Average electron dose: 37.8 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 3.0 µm / Nominal defocus min: 1.5 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: INSILICO MODEL
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 3.1 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.1) / Number images used: 27310
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
FSC plot (resolution estimation)

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