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- EMDB-34713: Cryo-EM structure of nucleotide-bound ComA at outward-facing stat... -

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Basic information

Entry
Database: EMDB / ID: EMD-34713
TitleCryo-EM structure of nucleotide-bound ComA at outward-facing state with EC gate open conformation
Map data
Sample
  • Complex: ComA
    • Protein or peptide: Transport/processing ATP-binding protein ComA
  • Ligand: PHOSPHOTHIOPHOSPHORIC ACID-ADENYLATE ESTER
  • Ligand: MAGNESIUM ION
KeywordsABC transport / PCAT / MEMBRANE PROTEIN / TRANSPORT PROTEIN
Function / homology
Function and homology information


ABC-type bacteriocin transporter activity / Translocases; Catalysing the translocation of amino acids and peptides; Linked to the hydrolysis of a nucleoside triphosphate / establishment of competence for transformation / ATPase-coupled lipid transmembrane transporter activity / cysteine-type peptidase activity / transmembrane transport / Hydrolases; Acting on peptide bonds (peptidases); Cysteine endopeptidases / ATP hydrolysis activity / proteolysis / ATP binding / plasma membrane
Similarity search - Function
Peptidase C39, ABC-type bacteriocin transporter / Peptidase C39 family / Peptidase C39, bacteriocin processing / Peptidase family C39 domain profile. / Type 1 protein exporter / ABC transporter transmembrane region / ABC transporter type 1, transmembrane domain / ABC transporter integral membrane type-1 fused domain profile. / ABC transporter type 1, transmembrane domain superfamily / ABC transporter-like, conserved site ...Peptidase C39, ABC-type bacteriocin transporter / Peptidase C39 family / Peptidase C39, bacteriocin processing / Peptidase family C39 domain profile. / Type 1 protein exporter / ABC transporter transmembrane region / ABC transporter type 1, transmembrane domain / ABC transporter integral membrane type-1 fused domain profile. / ABC transporter type 1, transmembrane domain superfamily / 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/processing ATP-binding protein ComA
Similarity search - Component
Biological speciesStreptococcus pneumoniae D39 (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.9 Å
AuthorsYu L / Xin X / Min L
Funding support Singapore, 1 items
OrganizationGrant numberCountry
Ministry of Education (MoE, Singapore)A-0008412-00-00 Singapore
CitationJournal: Nat Commun / Year: 2023
Title: Structural basis of peptide secretion for Quorum sensing by ComA.
Authors: Lin Yu / Xin Xu / Wan-Zhen Chua / Hao Feng / Zheng Ser / Kai Shao / Jian Shi / Yumei Wang / Zongli Li / Radoslaw M Sobota / Lok-To Sham / Min Luo /
Abstract: Quorum sensing (QS) is a crucial regulatory mechanism controlling bacterial signalling and holds promise for novel therapies against antimicrobial resistance. In Gram-positive bacteria, such as ...Quorum sensing (QS) is a crucial regulatory mechanism controlling bacterial signalling and holds promise for novel therapies against antimicrobial resistance. In Gram-positive bacteria, such as Streptococcus pneumoniae, ComA is a conserved efflux pump responsible for the maturation and secretion of peptide signals, including the competence-stimulating peptide (CSP), yet its structure and function remain unclear. Here, we functionally characterize ComA as an ABC transporter with high ATP affinity and determined its cryo-EM structures in the presence or absence of CSP or nucleotides. Our findings reveal a network of strong electrostatic interactions unique to ComA at the intracellular gate, a putative binding pocket for two CSP molecules, and negatively charged residues facilitating CSP translocation. Mutations of these residues affect ComA's peptidase activity in-vitro and prevent CSP export in-vivo. We demonstrate that ATP-Mg triggers the outward-facing conformation of ComA for CSP release, rather than ATP alone. Our study provides molecular insights into the QS signal peptide secretion, highlighting potential targets for QS-targeting drugs.
History
DepositionNov 9, 2022-
Header (metadata) releaseOct 4, 2023-
Map releaseOct 4, 2023-
UpdateMar 6, 2024-
Current statusMar 6, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_34713.png

Downloads & links

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Map

FileDownload / File: emd_34713.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.86 Å/pix.
x 256 pix.
= 219.648 Å		0.86 Å/pix.
x 256 pix.
= 219.648 Å		0.86 Å/pix.
x 256 pix.
= 219.648 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.858 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.016
Minimum - Maximum-0.055454105 - 0.114698105
Average (Standard dev.)-0.00002010131 (±0.004240511)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-128-128-128
Dimensions256256256
Spacing256256256
CellA=B=C: 219.648 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #2

Fileemd_34713_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_34713_half_map_2.map
Projections & Slices
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Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : ComA

EntireName: ComA
Components
  • Complex: ComA
    • Protein or peptide: Transport/processing ATP-binding protein ComA
  • Ligand: PHOSPHOTHIOPHOSPHORIC ACID-ADENYLATE ESTER
  • Ligand: MAGNESIUM ION

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Supramolecule #1: ComA

SupramoleculeName: ComA / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: Streptococcus pneumoniae D39 (bacteria)

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Macromolecule #1: Transport/processing ATP-binding protein ComA

MacromoleculeName: Transport/processing ATP-binding protein ComA / type: protein_or_peptide / ID: 1 / Number of copies: 2 / Enantiomer: LEVO
EC number: Hydrolases; Acting on peptide bonds (peptidases); Cysteine endopeptidases
Source (natural)Organism: Streptococcus pneumoniae D39 (bacteria)
Molecular weightTheoretical: 80.433031 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MKFGKRHYRP QVDQMDCGVA SLAMVFGYYG SYYFLAHLRE LAKTTMDGTT ALGLVKVAEE IGFETRAIKA DMTLFDLPDL TFPFVAHVL KEGKLLHYYV VTGQDKDSIH IADPDPGVKL TKLPRERFEE EWTGVTLFMA PSPDYKPHKE QKNGLLSFIP I LVKQRGLI ...String:
MKFGKRHYRP QVDQMDCGVA SLAMVFGYYG SYYFLAHLRE LAKTTMDGTT ALGLVKVAEE IGFETRAIKA DMTLFDLPDL TFPFVAHVL KEGKLLHYYV VTGQDKDSIH IADPDPGVKL TKLPRERFEE EWTGVTLFMA PSPDYKPHKE QKNGLLSFIP I LVKQRGLI ANIVLATLLV TVINIVGSYY LQSIIDTYVP DQMRSTLGII SIGLVIVYIL QQILSYAQEY LLLVLGQRLS ID VILSYIK HVFHLPMSFF ATRRTGEIVS RFTDANSIID ALASTILSIF LDVSTVVIIS LVLFSQNTNL FFMTLLALPI YTV IIFAFM KPFEKMNRDT MEANAVLSSS IIEDINGIET IKSLTSESQR YQKIDKEFVD YLKKSFTYSR AESQQKALKK VAHL LLNVG ILWMGAVLVM DGKMSLGQLI TYNTLLVYFT NPLENIINLQ TKLQTAQVAN NRLNEVYLVA SEFEEKKTVE DLSLM KGDM TFKQVHYKYG YGRDVLSDIN LTVPQGSKVA FVGISGSGKT TLAKMMVNFY DPSQGEISLG GVNLNQIDKK ALRQYI NYL PQQPYVFNGT ILENLLLGAK EGTTQEDILR AVELAEIRED IERMPLNYQT ELTSDGAGIS GGQRQRIALA RALLTDA PV IILDEATSSL DILTEKRIVD NLIALDKTLI FIAHRLTIAE RTEKVVVLDQ GKIVEEGKHA DLLAQGGFYA HLVNS

UniProtKB: Transport/processing ATP-binding protein ComA

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Macromolecule #2: PHOSPHOTHIOPHOSPHORIC ACID-ADENYLATE ESTER

MacromoleculeName: PHOSPHOTHIOPHOSPHORIC ACID-ADENYLATE ESTER / type: ligand / ID: 2 / Number of copies: 2 / Formula: AGS
Molecular weightTheoretical: 523.247 Da
Chemical component information

ChemComp-AGS:
PHOSPHOTHIOPHOSPHORIC ACID-ADENYLATE ESTER / ATP-gamma-S, energy-carrying molecule analogue*YM

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Macromolecule #3: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 3 / Number of copies: 2 / 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

Concentration5 mg/mL
BufferpH: 7.5 / Details: 25 mM Tris, pH 7.5, 150 mM NaCl, 2 mM DTT
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 293.15 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average electron dose: 1.2 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.5 µm / Nominal defocus min: 1.0 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: NONE
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 2.9 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 135756
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD

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