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- EMDB-17575: BtuB3G3 bound to cyanocobalamin with ordered EL8 -

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Basic information

Entry
Database: EMDB / ID: EMD-17575
TitleBtuB3G3 bound to cyanocobalamin with ordered EL8
Map dataMain sharpened map
Sample
  • Complex: Complex of TonB-dependent transporter BtuB3 with surface lipoprotein BtuG3 bound to cyanocobalamin (state 1)
    • Protein or peptide: Vitamin B12 transporter BtuB
    • Protein or peptide: Putative surface layer protein
  • Ligand: CYANOCOBALAMIN
KeywordsCyanocobalamin / transporter / lipoprotein / complex / MEMBRANE PROTEIN
Function / homology
Function and homology information


Protein of unknown function DUF5074 / Domain of unknown function (DUF5074) / Cytochrome cd1-nitrite reductase-like, haem d1 domain superfamily / Vitamin B12 transporter BtuB-like / 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. / WD40/YVTN repeat-like-containing domain superfamily
Similarity search - Domain/homology
Vitamin B12 transporter BtuB / Surface layer protein
Similarity search - Component
Biological speciesBacteroides thetaiotaomicron VPI-5482 (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.97 Å
AuthorsSilale A / Abellon-Ruiz J / van den Berg B
Funding support United Kingdom, 1 items
OrganizationGrant numberCountry
Wellcome Trust214222/Z/18/Z United Kingdom
CitationJournal: Nat Commun / Year: 2023
Title: BtuB TonB-dependent transporters and BtuG surface lipoproteins form stable complexes for vitamin B uptake in gut Bacteroides.
Authors: Javier Abellon-Ruiz / Kalyanashis Jana / Augustinas Silale / Andrew M Frey / Arnaud Baslé / Matthias Trost / Ulrich Kleinekathöfer / Bert van den Berg /
Abstract: Vitamin B (cobalamin) is required for most human gut microbes, many of which are dependent on scavenging to obtain this vitamin. Since bacterial densities in the gut are extremely high, competition ...Vitamin B (cobalamin) is required for most human gut microbes, many of which are dependent on scavenging to obtain this vitamin. Since bacterial densities in the gut are extremely high, competition for this keystone micronutrient is severe. Contrasting with Enterobacteria, members of the dominant genus Bacteroides often encode several BtuB vitamin B outer membrane transporters together with a conserved array of surface-exposed B-binding lipoproteins. Here we show that the BtuB transporters from Bacteroides thetaiotaomicron form stable, pedal bin-like complexes with surface-exposed BtuG lipoprotein lids, which bind B with high affinities. Closing of the BtuG lid following B capture causes destabilisation of the bound B by a conserved BtuB extracellular loop, causing translocation of the vitamin to BtuB and subsequent transport. We propose that TonB-dependent, lipoprotein-assisted small molecule uptake is a general feature of Bacteroides spp. that is important for the success of this genus in colonising the human gut.
History
DepositionJun 5, 2023-
Header (metadata) releaseAug 16, 2023-
Map releaseAug 16, 2023-
UpdateSep 20, 2023-
Current statusSep 20, 2023Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_17575.png

Downloads & links

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Map

FileDownload / File: emd_17575.map.gz / Format: CCP4 / Size: 512 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationMain sharpened map
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.74 Å/pix.
x 512 pix.
= 378.88 Å		0.74 Å/pix.
x 512 pix.
= 378.88 Å		0.74 Å/pix.
x 512 pix.
= 378.88 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.74 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.113
Minimum - Maximum-0.43137366 - 0.6863303
Average (Standard dev.)0.00009287989 (±0.014491084)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions512512512
Spacing512512512
CellA=B=C: 378.88 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_17575_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map B

Fileemd_17575_half_map_1.map
AnnotationHalf map B
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map A

Fileemd_17575_half_map_2.map
AnnotationHalf map A
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Complex of TonB-dependent transporter BtuB3 with surface lipoprot...

EntireName: Complex of TonB-dependent transporter BtuB3 with surface lipoprotein BtuG3 bound to cyanocobalamin (state 1)
Components
  • Complex: Complex of TonB-dependent transporter BtuB3 with surface lipoprotein BtuG3 bound to cyanocobalamin (state 1)
    • Protein or peptide: Vitamin B12 transporter BtuB
    • Protein or peptide: Putative surface layer protein
  • Ligand: CYANOCOBALAMIN

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Supramolecule #1: Complex of TonB-dependent transporter BtuB3 with surface lipoprot...

SupramoleculeName: Complex of TonB-dependent transporter BtuB3 with surface lipoprotein BtuG3 bound to cyanocobalamin (state 1)
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#2
Source (natural)Organism: Bacteroides thetaiotaomicron VPI-5482 (bacteria)
Molecular weightTheoretical: 122 KDa

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Macromolecule #1: Vitamin B12 transporter BtuB

MacromoleculeName: Vitamin B12 transporter BtuB / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Bacteroides thetaiotaomicron VPI-5482 (bacteria)
Molecular weightTheoretical: 81.169516 KDa
Recombinant expressionOrganism: Bacteroides thetaiotaomicron VPI-5482 (bacteria)
SequenceString: MRRNILLVQF VGVLLLLPSI MSGQQHSSDN KNWAEKGIVI REVEILGKRP MKDIGVQQTR FDSLVLKENI ALSMADILTF NSSIFVKSY GRATLSTVSF RGTSASHTQV TWNGMRINNP MLGMTDFSMI PSYFIDDASL LHGTSSVNMA GGGLGGLVKL S TVPAHQEG ...String:
MRRNILLVQF VGVLLLLPSI MSGQQHSSDN KNWAEKGIVI REVEILGKRP MKDIGVQQTR FDSLVLKENI ALSMADILTF NSSIFVKSY GRATLSTVSF RGTSASHTQV TWNGMRINNP MLGMTDFSMI PSYFIDDASL LHGTSSVNMA GGGLGGLVKL S TVPAHQEG FGMQYVQGIG SFSTFDEFLQ LKYGDKHWQI STRAVYQSSP NDYKYRNHDK KENIYDDKYN IIEQYYPIER NR SGAYKDL HILQEVYYNT GKGDRFGLNA WYTDSNRELA LLTTDQGDLM DFENRQREHT LRSVLSWDHT RENWKVSARG GYV HTWLAY DYKRDLGNGI MATMTRSRSK VNTFYGQLDG EYFFSDKLLL TAGVSAHQHL VNSLDKDLNK DDNKNDKYGQ GRKN DSIVY FDKGRIELSG NVSLKWQPVN RLGMSLVLRG EMFGTKWAPV IPAFFVDYVL SKRGNIMAKA SITRNYRFPT LNDLY FLPG GNPALNNESG FTYETGLSFS VDKDNVYTLS GSASWFDQHI NDWIIWLPTS KGFYSPVNLK KVHAYGVEVQ ADYAVA IDK AWKLGLNGTF AWTPSINEGE PTSKADQSVG KQLPYIPEYS ATLSGRLTYR SWGLLYKWCY YSERYTMTSN AVSYTGH LP PYLMSNVTLE KGFSLRWADL SLKGTVNNLF DEEYLSVLSR PMPGINFEFF IGITPKWGKK KKGGGHHHHH H

UniProtKB: Vitamin B12 transporter BtuB

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Macromolecule #2: Putative surface layer protein

MacromoleculeName: Putative surface layer protein / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Bacteroides thetaiotaomicron VPI-5482 (bacteria)
Molecular weightTheoretical: 41.165793 KDa
SequenceString: MKRILLSVLF IVFCLTAFVG CMKWDYGKME PFRATGDGLF IMNEGNFQYG NATLSYYDPE TKKVENEIFY RANAMKLGDV AQSMIVRDT IGWVVVNNSH VIFAISTNTF KEVGRITGLT SPRYIHFISD EKAYITQIWD YRIFIVNPKT YQITGYIECP D MTMETGST ...String:
MKRILLSVLF IVFCLTAFVG CMKWDYGKME PFRATGDGLF IMNEGNFQYG NATLSYYDPE TKKVENEIFY RANAMKLGDV AQSMIVRDT IGWVVVNNSH VIFAISTNTF KEVGRITGLT SPRYIHFISD EKAYITQIWD YRIFIVNPKT YQITGYIECP D MTMETGST EQMVQYGKYV YVNCWSYQNR ILKIDTTTDK VVDQLTVGIQ PTSLVMDKNF KMWTITDGGY KGSPYGYEEP SL YRIDAET FKIEKQFKFQ LGDAPSEVQL NGAGDELYWI NKDIWRMSVD EERVPVRPFL KYRDTKYYGL TVSPKNGDVY VAD AIDYQQ QGMIYRYTED GELVDEFYVG IIPGAFCWK

UniProtKB: Surface layer protein

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Macromolecule #3: CYANOCOBALAMIN

MacromoleculeName: CYANOCOBALAMIN / type: ligand / ID: 3 / Number of copies: 1 / Formula: CNC
Molecular weightTheoretical: 1.356373 KDa
Chemical component information

ChemComp-CNC:
CYANOCOBALAMIN / Cyanocobalamin

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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

Concentration4 mg/mL
BufferpH: 7.5
Component:
ConcentrationNameFormula
10.0 mMHEPES
100.0 mMsodium chlorideNaClSodium chloride
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / Support film - Material: CARBON / Support film - topology: HOLEY ARRAY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Atmosphere: AIR
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV
DetailsThe protein complex was purified in lauryl maltose neopentyl glycol (LMNG). Prior to making grids, the sample was subjected to size exclusion chromatography without LMNG in the mobile phase to separate protein-LMNG complexes from free LMNG.

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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: 2.3000000000000003 µm / Nominal defocus min: 0.8 µm / Nominal magnification: 165000
Specialist opticsEnergy filter - Name: TFS Selectris X / Energy filter - Slit width: 10 eV
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingFilm or detector model: FEI FALCON IV (4k x 4k) / Number grids imaged: 1 / Number real images: 9826 / Average electron dose: 35.6 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 3311038
Startup modelType of model: INSILICO MODEL
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 4.1.2)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 4.1.2)
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 2.97 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 4.1.2) / Number images used: 66318
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial model
PDB IDChain

8okv

chain_id: B, source_name: PDB, initial_model_type: experimental model
chain_id: A, source_name: AlphaFold, initial_model_type: in silico model
RefinementSpace: REAL / Protocol: RIGID BODY FIT
Output model

PDB-8p98:
BtuB3G3 bound to cyanocobalamin with ordered EL8

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