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- EMDB-11273: Open-open state of the Bt1762-Bt1763 levan transport system -

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Basic information

Entry
Database: EMDB / ID: EMD-11273
TitleOpen-open state of the Bt1762-Bt1763 levan transport system
Map dataMap filtered by local resolution
Sample
  • Complex: Dimeric Bt1762-Bt1763 levan transporter complex
    • Complex: SusD homolog
      • Protein or peptide: SusD homolog
    • Complex: SusC homolog
      • Protein or peptide: SusC homolog
Function / homology
Function and homology information


cell outer membrane / membrane / metal ion binding
Similarity search - Function
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 / TonB-dependent receptor-like, beta-barrel / TonB dependent receptor ...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 / 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 / Prokaryotic membrane lipoprotein lipid attachment site profile. / Tetratricopeptide-like helical domain superfamily
Similarity search - Domain/homology
SusC homolog / SusD homolog
Similarity search - Component
Biological speciesBacteroides thetaiotaomicron (strain ATCC 29148 / DSM 2079 / NCTC 10582 / E50 / VPI-5482) (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.9 Å
AuthorsWhite JBR / van den Berg B / Ranson NA
Funding support United Kingdom, Switzerland, 5 items
OrganizationGrant numberCountry
Biotechnology and Biological Sciences Research Council (BBSRC)BB/P003192/1 United Kingdom
Wellcome Trust108466/Z/15/Z United Kingdom
Wellcome Trust215064/Z/18/Z United Kingdom
Medical Research Council (MRC, United Kingdom)MR/N020413/1 United Kingdom
Swiss National Science Foundation167125 Switzerland
CitationJournal: Nat Commun / Year: 2021
Title: Insights into SusCD-mediated glycan import by a prominent gut symbiont.
Authors: Declan A Gray / Joshua B R White / Abraham O Oluwole / Parthasarathi Rath / Amy J Glenwright / Adam Mazur / Michael Zahn / Arnaud Baslé / Carl Morland / Sasha L Evans / Alan Cartmell / ...Authors: Declan A Gray / Joshua B R White / Abraham O Oluwole / Parthasarathi Rath / Amy J Glenwright / Adam Mazur / Michael Zahn / Arnaud Baslé / Carl Morland / Sasha L Evans / Alan Cartmell / Carol V Robinson / Sebastian Hiller / Neil A Ranson / David N Bolam / Bert van den Berg /
Abstract: In Bacteroidetes, one of the dominant phyla of the mammalian gut, active uptake of large nutrients across the outer membrane is mediated by SusCD protein complexes via a "pedal bin" transport ...In Bacteroidetes, one of the dominant phyla of the mammalian gut, active uptake of large nutrients across the outer membrane is mediated by SusCD protein complexes via a "pedal bin" transport mechanism. However, many features of SusCD function in glycan uptake remain unclear, including ligand binding, the role of the SusD lid and the size limit for substrate transport. Here we characterise the β2,6 fructo-oligosaccharide (FOS) importing SusCD from Bacteroides thetaiotaomicron (Bt1762-Bt1763) to shed light on SusCD function. Co-crystal structures reveal residues involved in glycan recognition and suggest that the large binding cavity can accommodate several substrate molecules, each up to ~2.5 kDa in size, a finding supported by native mass spectrometry and isothermal titration calorimetry. Mutational studies in vivo provide functional insights into the key structural features of the SusCD apparatus and cryo-EM of the intact dimeric SusCD complex reveals several distinct states of the transporter, directly visualising the dynamics of the pedal bin transport mechanism.
History
DepositionJul 1, 2020-
Header (metadata) releaseNov 11, 2020-
Map releaseNov 11, 2020-
UpdateJan 20, 2021-
Current statusJan 20, 2021Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.038
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.038
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6zlt
  • Surface level: 0.038
  • Imaged by UCSF Chimera
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  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-6zlt
  • Imaged by Jmol
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_11273.png

Downloads & links

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Map

FileDownload / File: emd_11273.map.gz / Format: CCP4 / Size: 70.2 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationMap filtered by local resolution
Voxel sizeX=Y=Z: 1.07 Å
Density
Contour LevelBy AUTHOR: 0.038 / Movie #1: 0.038
Minimum - Maximum-0.21110451 - 0.32315978
Average (Standard dev.)0.00023944174 (±0.0069402237)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions264264264
Spacing264264264
CellA=B=C: 282.48 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.071.071.07
M x/y/z264264264
origin x/y/z0.0000.0000.000
length x/y/z282.480282.480282.480
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ400400400
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS264264264
D min/max/mean-0.2110.3230.000

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Supplemental data

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Mask #1

Fileemd_11273_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: Post-processed map output from RELION

Fileemd_11273_additional_1.map
AnnotationPost-processed map output from RELION
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: 3D refine map output from RELION

Fileemd_11273_additional_2.map
Annotation3D refine map output from RELION
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Dimeric Bt1762-Bt1763 levan transporter complex

EntireName: Dimeric Bt1762-Bt1763 levan transporter complex
Components
  • Complex: Dimeric Bt1762-Bt1763 levan transporter complex
    • Complex: SusD homolog
      • Protein or peptide: SusD homolog
    • Complex: SusC homolog
      • Protein or peptide: SusC homolog

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Supramolecule #1: Dimeric Bt1762-Bt1763 levan transporter complex

SupramoleculeName: Dimeric Bt1762-Bt1763 levan transporter complex / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Molecular weightTheoretical: 360 KDa

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Supramolecule #2: SusD homolog

SupramoleculeName: SusD homolog / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1
Source (natural)Organism: Bacteroides thetaiotaomicron (strain ATCC 29148 / DSM 2079 / NCTC 10582 / E50 / VPI-5482) (bacteria)
Recombinant expressionOrganism: Bacteroides thetaiotaomicron (bacteria)

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Supramolecule #3: SusC homolog

SupramoleculeName: SusC homolog / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #2
Source (natural)Organism: Bacteroides thetaiotaomicron (strain ATCC 29148 / DSM 2079 / NCTC 10582 / E50 / VPI-5482) (bacteria)

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Macromolecule #1: SusD homolog

MacromoleculeName: SusD homolog / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Bacteroides thetaiotaomicron (strain ATCC 29148 / DSM 2079 / NCTC 10582 / E50 / VPI-5482) (bacteria)
Molecular weightTheoretical: 64.17816 KDa
Recombinant expressionOrganism: Bacteroides thetaiotaomicron (bacteria)
SequenceString: CDDFLDRQVP QGIVTGDQIA SPEYVDNLVI SAYAIWATGD DINSSFSLWN YDVRSDDCYK GGSGTEDGGV FNALEISKGI NTTDWNIND IWKRLYQCIT RANTALQSLD QMDEKTYPLK NQRIAEMRFL RGHAHFMLKQ LFKKIVIVND ENMEPDAYNE L SNTTYTND ...String:
CDDFLDRQVP QGIVTGDQIA SPEYVDNLVI SAYAIWATGD DINSSFSLWN YDVRSDDCYK GGSGTEDGGV FNALEISKGI NTTDWNIND IWKRLYQCIT RANTALQSLD QMDEKTYPLK NQRIAEMRFL RGHAHFMLKQ LFKKIVIVND ENMEPDAYNE L SNTTYTND EQWQKIADDF QFAYDNLPEV QIEKGRPAQA AAAAYLAKTY LYKAYRQDGA DNALTGINEE DLKQVVKYTD PL IMAKGGY GLETDYSMNF LPQYENGAES VWAIQYSIND GTYNGNLNWG MGLTTPQILG CCDFHKPSQN LVNAFKTDSQ GKP LFSTYD NENYEVATDN VDPRLFHTVG MPGFPYKYNE GYIIQKNDDW SRSKGLYGYY VSLKENVDPD CDCLKKGSYW ASSL NHIVI RYADVLLMRA EALIQLNDGR ITDAISLINE VRSRAAGSTM LIFNYKEDYG VNFKVTPYDL KAYAQDEAMK MLKWE RRVE FGMESSRFFD LVRWGEAKDV INAYYVTEAS RCSIYKNAGF TENKNEYLPV PFEQISASNG NYTQNFGWAA AAHHHH HH

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Macromolecule #2: SusC homolog

MacromoleculeName: SusC homolog / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Bacteroides thetaiotaomicron (strain ATCC 29148 / DSM 2079 / NCTC 10582 / E50 / VPI-5482) (bacteria)
Molecular weightTheoretical: 103.915227 KDa
SequenceString: VVVTGYTTQR KADLTGAVSV VKVDEIQKQG ENNPVKALQG RVPGMNITAD GNPSGSATVR IRGIGTLNNN DPLYIIDGVP TKAGMHELN GNDIESIQVL KDAASASIYG SRAANGVIII TTKQGKKGQI KINFDASVSA SMYQSKMNVL NTEQYGRAMW Q AYVNDGEN ...String:
VVVTGYTTQR KADLTGAVSV VKVDEIQKQG ENNPVKALQG RVPGMNITAD GNPSGSATVR IRGIGTLNNN DPLYIIDGVP TKAGMHELN GNDIESIQVL KDAASASIYG SRAANGVIII TTKQGKKGQI KINFDASVSA SMYQSKMNVL NTEQYGRAMW Q AYVNDGEN PNGNALGYAY NWGYNADGNP VLYGMTLSKY LDSKNTMPVA DTDWFDEITR TGVIQQYNLS VSNGSEKGSS FF SLGYYKN LGVIKDTDFD RFSARMNSDY KLIDDILTIG QHFTLNRTSE VQAPGGIIET ALDIPSAIPV YASDGSWGGP VGG WPDRRN PRAVLEYNKD NRYTYWRMFG DAYVNLTPFK GFNLRSTFGL DYANKQARYF TYPYQEGTQT NNGKSAVEAK QEHW TKWMW NAIATYQLEV GKHRGDVMIG MELNREDDSH FSGYKEDFSI LTPDYMWPDA GSGTAQAYGA GEGYSLVSFF GKMNY SYAD RYLLSLTLRR DGSSRFGKNH RYATFPSVSL GWRITQENFM KELTWLDDLK LRASWGQTGN QEISNLARYT IYAPNY GTT DSFGGQSYGT AYDITGSNGG GVLPSGFKRN QIGNDNIKWE TTTQTNVGID FSLFKQSLYG SLEYYYKKAT DILTEMA GV GVLGEGGSRW INSGAMKNQG FEFNLGYRNK TAFGLTYDLN GNISTYRNEI LELPETVAAN GKFGGNGVKS VVGHTYGA Q VGYIADGIFK SQDEVDNHAT QEGAAVGRIR YRDIDHNGVI DERDQNWIYD PTPSFSYGLN IYLEYKNFDL TMFWQGVQG VDIISDVKKK SDFWSASNVG FLNKGTRLLN AWSPTNPNSD IPALTRSDTN NEQRVSTYFV ENGSFLKLRN IQLGYTVPAV ISKKMRMDR LRFYCSAQNL LTIKSKNFTG EDPENPNFSY PIPVNITFGL NIGF

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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

Concentration0.02 mg/mL
BufferpH: 7.5
Component:
ConcentrationNameFormula
10.0 mMHEPES
100.0 mMSodium chlorideNaClSodium chloride
0.05 % w/vLDAO
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
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Average electron dose: 63.84 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: INSILICO MODEL
In silico model: Starting model was generated de novo from the data by stochastic gradient descent in RELION
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
Final reconstructionApplied symmetry - Point group: C2 (2 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 3.9 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.0) / Number images used: 32190
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

6zaz

RefinementSpace: REAL / Protocol: FLEXIBLE FIT
Output model

PDB-6zlt:
Open-open state of the Bt1762-Bt1763 levan transport system

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