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- PDB-8tsi: S. thermodepolymerans KpsMT-KpsE in complex with ADP:AlF4- -

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Basic information

Entry
Database: PDB / ID: 8tsi
TitleS. thermodepolymerans KpsMT-KpsE in complex with ADP:AlF4-
Components
  • ABC transporter ATP-binding protein
  • Capsular biosynthesis protein
  • Transport permease protein
KeywordsMEMBRANE PROTEIN / ABC transporter / Capsular polysaccharide
Function / homology
Function and homology information


lipopolysaccharide transport / ABC-type transporter activity / ATP-binding cassette (ABC) transporter complex / protein tyrosine kinase activity / ATP hydrolysis activity / ATP binding / plasma membrane
Similarity search - Function
ABC-2 transporter / ABC transporter, teichoic acids export TagH-like / : / : / ABC transporter integral membrane type-2 domain profile. / : / ABC-2 type transporter / ABC-2 type transporter / ABC transporter-like, conserved site / ABC transporters family signature. ...ABC-2 transporter / ABC transporter, teichoic acids export TagH-like / : / : / ABC transporter integral membrane type-2 domain profile. / : / 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
ADENOSINE-5'-DIPHOSPHATE / TETRAFLUOROALUMINATE ION / Transport permease protein / Capsular biosynthesis protein / ABC transporter ATP-binding protein
Similarity search - Component
Biological speciesCaldimonas thermodepolymerans (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4.4 Å
AuthorsKuklewicz, J. / Zimmer, J.
Funding support United States, 1items
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, 2023Deposition site: RCSB / Processing site: RCSB
Revision 1.0Apr 17, 2024Provider: repository / Type: Initial release
Revision 1.1May 8, 2024Group: Database references / Category: citation / citation_author
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation_author.identifier_ORCID
Revision 1.2Oct 16, 2024Group: Data collection / Structure summary
Category: em_admin / pdbx_entry_details / pdbx_modification_feature
Item: _em_admin.last_update / _pdbx_entry_details.has_protein_modification

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: ABC transporter ATP-binding protein
C: Transport permease protein
E: Capsular biosynthesis protein
F: Capsular biosynthesis protein
G: Capsular biosynthesis protein
H: Capsular biosynthesis protein
I: Capsular biosynthesis protein
J: Capsular biosynthesis protein
K: Capsular biosynthesis protein
L: Capsular biosynthesis protein
D: Transport permease protein
B: ABC transporter ATP-binding protein
hetero molecules


Theoretical massNumber of molelcules
Total (without water)467,57016
Polymers466,51012
Non-polymers1,0604
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein ABC transporter ATP-binding protein


Mass: 26224.729 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Caldimonas thermodepolymerans (bacteria)
Gene: C1702_11080 / Production host: Escherichia coli (E. coli) / References: UniProt: A0A2S5T4B3
#2: Protein Transport permease protein


Mass: 30800.959 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Caldimonas thermodepolymerans (bacteria)
Gene: C1702_11075 / Production host: Escherichia coli (E. coli) / References: UniProt: A0A2S5T447
#3: Protein
Capsular biosynthesis protein


Mass: 44057.266 Da / Num. of mol.: 8
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Caldimonas thermodepolymerans (bacteria)
Gene: C1702_11085 / Production host: Escherichia coli (E. coli) / References: UniProt: A0A2S5T4A0
#4: Chemical ChemComp-ADP / ADENOSINE-5'-DIPHOSPHATE


Mass: 427.201 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C10H15N5O10P2 / Feature type: SUBJECT OF INVESTIGATION / Comment: ADP, energy-carrying molecule*YM
#5: Chemical ChemComp-ALF / TETRAFLUOROALUMINATE ION


Mass: 102.975 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: AlF4 / Feature type: SUBJECT OF INVESTIGATION
Has ligand of interestY
Has protein modificationY

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: ABC transporter KpsMT in complex with polysaccharide co-polymerase KpsE in ADP:AlF4- -bound state
Type: COMPLEX / Entity ID: #1-#3 / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Caldimonas thermodepolymerans (bacteria)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 7.5
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 1800 nm / Nominal defocus min: 1000 nm
Image recordingElectron dose: 50 e/Å2 / Film or detector model: FEI FALCON III (4k x 4k)

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Processing

CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 4.4 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 58165 / Symmetry type: POINT

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