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- PDB-9azl: Cryo-EM structure of the ABC transporter PCAT1 bound with MgADP class1 -

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Basic information

Entry
Database: PDB / ID: 9azl
TitleCryo-EM structure of the ABC transporter PCAT1 bound with MgADP class1
ComponentsABC-type bacteriocin transporter
KeywordsTRANSPORT PROTEIN / ABC transporter / Nucleotide / Membrane Protein
Function / homology
Function and homology information


ABC-type bacteriocin transporter activity / ABC-type oligopeptide transporter activity / cysteine-type peptidase activity / ATP hydrolysis activity / proteolysis / ATP binding / plasma membrane
Similarity search - Function
Peptidase C39, ABC-type bacteriocin transporter / Peptidase family C39 domain profile. / Peptidase C39 family / Peptidase C39, bacteriocin processing / 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 ...Peptidase C39, ABC-type bacteriocin transporter / Peptidase family C39 domain profile. / Peptidase C39 family / Peptidase C39, bacteriocin processing / 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 / 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 / Chem-POV / ABC-type bacteriocin transporter
Similarity search - Component
Biological speciesAcetivibrio thermocellus ATCC 27405 (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / Resolution: 2.99 Å
AuthorsZhang, R. / Jagessar, K.L. / Polasa, A. / Brownd, M. / Stein, R. / Moradi, M. / Karakas, E. / Mchaourab, H.S.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01 128087 United States
Citation
Journal: Nat Commun / Year: 2024
Title: Conformational cycle of a protease-containing ABC transporter in lipid nanodiscs reveals the mechanism of cargo-protein coupling.
Authors: Ruojing Zhang / Kevin L Jagessar / Matthew Brownd / Adithya Polasa / Richard A Stein / Mahmoud Moradi / Erkan Karakas / Hassane S Mchaourab /
Abstract: Protease-containing ABC transporters (PCATs) couple the energy of ATP hydrolysis to the processing and export of diverse cargo proteins across cell membranes to mediate antimicrobial resistance and ...Protease-containing ABC transporters (PCATs) couple the energy of ATP hydrolysis to the processing and export of diverse cargo proteins across cell membranes to mediate antimicrobial resistance and quorum sensing. Here, we combine biochemical analysis, single particle cryoEM, and DEER spectroscopy in lipid bilayers along with computational analysis to illuminate the structural and energetic underpinnings of coupled cargo protein export. Our integrated investigation uncovers competitive interplay between nucleotides and cargo protein binding that ensures the latter's orderly processing and subsequent transport. The energetics of cryoEM structures in lipid bilayers are congruent with the inferred mechanism from ATP turnover analysis and reveal a snapshot of a high-energy outward-facing conformation that provides an exit pathway into the lipid bilayer and/or the extracellular side. DEER investigation of the core ABC transporter suggests evolutionary tuning of the energetic landscape to fulfill the function of substrate processing prior to export.
#1: Journal: J Gen Virol / Year: 1984
Title: Investigation of varicella-zoster virus-infected cell proteins that elicit antibody production during primary varicella using the immune transfer method.
Abstract: The varicella-zoster virus-infected cell proteins (VZV-ICPs) against which IgG, IgM and IgA antibodies were made in the course of primary varicella-zoster virus (VZV) infection were analysed by the ...The varicella-zoster virus-infected cell proteins (VZV-ICPs) against which IgG, IgM and IgA antibodies were made in the course of primary varicella-zoster virus (VZV) infection were analysed by the immune transfer method. IgG antibodies were made against one or more of 18 VZV-ICPs by patients with varicella. IgM antibodies were produced which reacted with 21 VZV-ICPs. The spectrum of IgG antibody production during the first week after the onset of infection was limited to an average of three VZV-ICPs while IgM antibodies which reacted with an average of seven VZV-ICPs were detectable in the acute phase of varicella. Equivalent VZV IgG or IgM antibody titres by radioimmunoassay did not correlate with a similar pattern of antibody specificity for VZV-ICPs by immune transfer. A detectable immune response to all VZV-ICPs was not required for the recovery of individual patients from primary VZV infection.
History
DepositionMar 11, 2024Deposition site: RCSB / Processing site: RCSB
Revision 1.0Oct 30, 2024Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: ABC-type bacteriocin transporter
B: ABC-type bacteriocin transporter
hetero molecules


Theoretical massNumber of molelcules
Total (without water)169,0407
Polymers167,3772
Non-polymers1,6635
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

#1: Protein ABC-type bacteriocin transporter


Mass: 83688.461 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Acetivibrio thermocellus ATCC 27405 (bacteria)
Gene: Cthe_0534 / Production host: Escherichia coli BL21(DE3) (bacteria) / Variant (production host): C43 / References: UniProt: A3DCU1
#2: 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
#3: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Mg / Feature type: SUBJECT OF INVESTIGATION
#4: Chemical ChemComp-POV / (2S)-3-(hexadecanoyloxy)-2-[(9Z)-octadec-9-enoyloxy]propyl 2-(trimethylammonio)ethyl phosphate / POPC


Mass: 760.076 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C42H82NO8P / Comment: phospholipid*YM
Has ligand of interestY
Has protein modificationN

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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: PCAT1 / Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT
Molecular weightValue: 1.62 MDa / Experimental value: NO
Source (natural)Organism: Acetivibrio thermocellus ATCC 27405 (bacteria)
Source (recombinant)Organism: Escherichia coli (E. coli) / Strain: C43
Buffer solutionpH: 7.4 / Details: 50mM MOPS, 50mM NaCl, PH 7.4
Buffer component
IDConc.NameFormulaBuffer-ID
150 mM3-(N-morpholino)propanesulfonic acidMOPS1
250 mMSodium chlorideNaCl1
SpecimenConc.: 0.5 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: NO
Specimen supportGrid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R1.2/1.3

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: TFS KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: SPOT SCAN
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 2100 nm / Nominal defocus min: 900 nm
Image recordingElectron dose: 1.09948 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 13051

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Processing

EM software
IDNameVersionCategory
1cryoSPARC4.2.1particle selection
4cryoSPARC4.2.1CTF correction
9PHENIX1.20.1-4487model refinement
12cryoSPARC4.2.1classification
13cryoSPARC4.2.13D reconstruction
CTF correctionType: NONE
Particle selectionNum. of particles selected: 1926956
3D reconstructionResolution: 2.99 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 177860 / Num. of class averages: 1 / Symmetry type: POINT
RefinementCross valid method: NONE

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