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- EMDB-41595: S. thermodepolymerans KpsM-KpsE in Apo 2 state with rigid body fi... -

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Basic information

Entry
Database: EMDB / ID: EMD-41595
TitleS. thermodepolymerans KpsM-KpsE in Apo 2 state with rigid body fitted KpsT
Map data
Sample
  • Complex: ABC transporter KpsMT in complex with polysaccharide co-polymerase KpsE in Apo 2 state
    • Protein or peptide: Transport permease protein
    • Protein or peptide: Capsular biosynthesis protein
    • Protein or peptide: ABC transporter ATP-binding protein
KeywordsABC transporter / Capsular polysaccharide / MEMBRANE PROTEIN
Function / homology
Function and homology information


ABC-type transporter activity / ATP-binding cassette (ABC) transporter complex / ATP hydrolysis activity / ATP binding / membrane / plasma membrane
Similarity search - Function
ABC-2 transporter / : / ABC transporter integral membrane type-2 domain profile. / ABC transporter, teichoic acids export TagH-like / ABC-2 type transporter / ABC-2 type transporter / ABC transporter-like, conserved site / ABC transporters family signature. / ABC transporter / ABC transporter-like, ATP-binding domain ...ABC-2 transporter / : / ABC transporter integral membrane type-2 domain profile. / ABC transporter, teichoic acids export TagH-like / ABC-2 type transporter / ABC-2 type transporter / ABC transporter-like, conserved site / ABC transporters family signature. / ABC transporter / ABC transporter-like, ATP-binding domain / ATP-binding cassette, ABC transporter-type domain profile. / ATPases associated with a variety of cellular activities / AAA+ ATPase domain / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
Transport permease protein / Capsular biosynthesis protein / ABC transporter ATP-binding protein
Similarity search - Component
Biological speciesCaldimonas thermodepolymerans (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.4 Å
AuthorsKuklewicz J / Zimmer J
Funding support United States, 1 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R35GM144130 United States
CitationJournal: Nature / Year: 2024
Title: Molecular insights into capsular polysaccharide secretion.
Authors: Jeremi Kuklewicz / Jochen Zimmer /
Abstract: Capsular polysaccharides (CPSs) fortify the cell boundaries of many commensal and pathogenic bacteria. Through the ABC-transporter-dependent biosynthesis pathway, CPSs are synthesized intracellularly ...Capsular polysaccharides (CPSs) fortify the cell boundaries of many commensal and pathogenic bacteria. Through the ABC-transporter-dependent biosynthesis pathway, CPSs are synthesized intracellularly on a lipid anchor and secreted across the cell envelope by the KpsMT ABC transporter associated with the KpsE and KpsD subunits. Here we use structural and functional studies to uncover crucial steps of CPS secretion in Gram-negative bacteria. We show that KpsMT has broad substrate specificity and is sufficient for the translocation of CPSs across the inner bacterial membrane, and we determine the cell surface organization and localization of CPSs using super-resolution fluorescence microscopy. Cryo-electron microscopy analyses of the KpsMT-KpsE complex in six different states reveal a KpsE-encaged ABC transporter, rigid-body conformational rearrangements of KpsMT during ATP hydrolysis and recognition of a glycolipid inside a membrane-exposed electropositive canyon. In vivo CPS secretion assays underscore the functional importance of canyon-lining basic residues. Combined, our analyses suggest a molecular model of CPS secretion by ABC transporters.
History
DepositionAug 11, 2023-
Header (metadata) releaseApr 17, 2024-
Map releaseApr 17, 2024-
UpdateApr 17, 2024-
Current statusApr 17, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_41595.png

Downloads & links

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Map

FileDownload / File: emd_41595.map.gz / Format: CCP4 / Size: 178 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesX (Sec.)Y (Row.)Z (Col.)
1.08 Å/pix.
x 360 pix.
= 388.8 Å		1.08 Å/pix.
x 360 pix.
= 388.8 Å		1.08 Å/pix.
x 360 pix.
= 388.8 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.08 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 4.0
Minimum - Maximum-24.826989999999999 - 47.900770000000001
Average (Standard dev.)-0.000000000005598 (±1.0)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderZYX
Origin000
Dimensions360360360
Spacing360360360
CellA=B=C: 388.80002 Å
α=β=γ: 90.0 °

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Supplemental data

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Sample components

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Entire : ABC transporter KpsMT in complex with polysaccharide co-polymeras...

EntireName: ABC transporter KpsMT in complex with polysaccharide co-polymerase KpsE in Apo 2 state
Components
  • Complex: ABC transporter KpsMT in complex with polysaccharide co-polymerase KpsE in Apo 2 state
    • Protein or peptide: Transport permease protein
    • Protein or peptide: Capsular biosynthesis protein
    • Protein or peptide: ABC transporter ATP-binding protein

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Supramolecule #1: ABC transporter KpsMT in complex with polysaccharide co-polymeras...

SupramoleculeName: ABC transporter KpsMT in complex with polysaccharide co-polymerase KpsE in Apo 2 state
type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Caldimonas thermodepolymerans (bacteria)

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Macromolecule #1: Transport permease protein

MacromoleculeName: Transport permease protein / type: protein_or_peptide / ID: 1 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Caldimonas thermodepolymerans (bacteria)
Molecular weightTheoretical: 30.800959 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MGKIHLAVSE RSPRVKRSPW QIQQAVLFAL FLRELKTRLG GRWLGVFWVL LEPVAHIAVM TTLFSLAHRA AMPSIEYPVF LITGLIPFF MFRGLVTRLM EAIDSNRGLF AYRQVKPIDT VIARAMLEIS LQSIVYLIAL GTLGWLGFHF LPVRALELAG V SAVLIMLG ...String:
MGKIHLAVSE RSPRVKRSPW QIQQAVLFAL FLRELKTRLG GRWLGVFWVL LEPVAHIAVM TTLFSLAHRA AMPSIEYPVF LITGLIPFF MFRGLVTRLM EAIDSNRGLF AYRQVKPIDT VIARAMLEIS LQSIVYLIAL GTLGWLGFHF LPVRALELAG V SAVLIMLG ASLGLFFAVV TNEIPQARAI VRISLLPLYF VSGVIFPVHT IPPQYLPLLQ LNPVLHLIEL SRASFFPQYR VL QGINLAY PAGFALLSLF LALMLYRLRR HQLASVV

UniProtKB: Transport permease protein

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Macromolecule #2: Capsular biosynthesis protein

MacromoleculeName: Capsular biosynthesis protein / type: protein_or_peptide / ID: 2 / Number of copies: 8 / Enantiomer: LEVO
Source (natural)Organism: Caldimonas thermodepolymerans (bacteria)
Molecular weightTheoretical: 44.057266 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MGKIHMKLVS RLTAKRLQWA LVYLPMLVAT VYFLVFSADR YVSESVITVR QTSSNAPTGG MSGAALLLAG LTPASREDTC YLQTYIHSM GLLQKLDQQL KLREHFGTPL RDPLFRLWGG TSQEWFLEYY RSRVEVLMDD ICGLLTVRVQ GFEPEFAQAL N RAILEESE ...String:
MGKIHMKLVS RLTAKRLQWA LVYLPMLVAT VYFLVFSADR YVSESVITVR QTSSNAPTGG MSGAALLLAG LTPASREDTC YLQTYIHSM GLLQKLDQQL KLREHFGTPL RDPLFRLWGG TSQEWFLEYY RSRVEVLMDD ICGLLTVRVQ GFEPEFAQAL N RAILEESE RFVNELSHRM AREQGQFAEA ELERATARLQ EAKRQLIAFQ AKHKLLDPLA QAQATGTLTA ELQAALTRQE AE LRNALTY LNEDSYQVKA LRSQINALRQ QIDEERLRAT AGKNGDRINA VAAEFHDLQL QVGFAEDAYK LALAAVESAR IEA TRKLKS LVVVEPPVLP EIAEYPRRWY NLATLLVVCC LIYGVVSLVV ATIRDHQDGS GSGSHHHHHH HHHH

UniProtKB: Capsular biosynthesis protein

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Macromolecule #3: ABC transporter ATP-binding protein

MacromoleculeName: ABC transporter ATP-binding protein / type: protein_or_peptide / ID: 3 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Caldimonas thermodepolymerans (bacteria)
Molecular weightTheoretical: 26.224729 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MIELRNLTKW YPTPHGRRYV FRNLNFRFPD DVSIGLIGRN GAGKSTLMRL LGGIEAPNEG EVVTDVSISW PVGLSGGFQG SLTARENVK FVCRIYGTSH EDMLRKVRFV EEFAEIGEHF DLPMKTYSSG MRSRVAFGLS MAFDFDYYLI DEAMAVGDAQ F RAKSRAVF ...String:
MIELRNLTKW YPTPHGRRYV FRNLNFRFPD DVSIGLIGRN GAGKSTLMRL LGGIEAPNEG EVVTDVSISW PVGLSGGFQG SLTARENVK FVCRIYGTSH EDMLRKVRFV EEFAEIGEHF DLPMKTYSSG MRSRVAFGLS MAFDFDYYLI DEAMAVGDAQ F RAKSRAVF DSRVGQANMI LVSHNMNDIK EYCDVVVLVD QGQATLYEDV EAGIAAYQGS LKKAAAKPDY KDDDDK

UniProtKB: ABC transporter ATP-binding protein

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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

BufferpH: 7.5
VitrificationCryogen name: ETHANE

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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: 1.8 µm / Nominal defocus min: 1.0 µm
Image recordingFilm or detector model: FEI FALCON III (4k x 4k) / Average electron dose: 50.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: INSILICO MODEL / Details: AlphaFold 2
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.4 Å / Resolution method: FSC 0.5 CUT-OFF / Number images used: 46064

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