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- PDB-6v9z: Cryo-EM structure of PCAT1 bound to its CtA peptide substrate -

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Basic information

Entry
Database: PDB / ID: 6v9z
TitleCryo-EM structure of PCAT1 bound to its CtA peptide substrate
Components
  • ABC-type bacteriocin transporter
  • CtA
KeywordsPROTEIN TRANSPORT / ATP-Binding Cassette
Function / homology
Function and homology information


ABC-type bacteriocin transporter activity / ATPase-coupled lipid transmembrane transporter activity / cysteine-type peptidase activity / ATP hydrolysis activity / proteolysis / ATP binding / membrane
Similarity search - Function
ABC transporter transmembrane region fold / ABC transporter type 1, transmembrane domain / Peptidase C39, ABC-type bacteriocin transporter / Peptidase C39 family / Peptidase C39, bacteriocin processing / Peptidase family C39 domain profile. / Cysteine proteinases / Type 1 protein exporter / Cathepsin B; Chain A / ABC transporter transmembrane region ...ABC transporter transmembrane region fold / ABC transporter type 1, transmembrane domain / Peptidase C39, ABC-type bacteriocin transporter / Peptidase C39 family / Peptidase C39, bacteriocin processing / Peptidase family C39 domain profile. / Cysteine proteinases / Type 1 protein exporter / Cathepsin B; Chain A / 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. / P-loop containing nucleotide triphosphate hydrolases / ATPases associated with a variety of cellular activities / AAA+ ATPase domain / Alpha-Beta Complex / Up-down Bundle / Rossmann fold / P-loop containing nucleoside triphosphate hydrolase / 3-Layer(aba) Sandwich / Mainly Alpha / Alpha Beta
Similarity search - Domain/homology
ABC-type bacteriocin transporter / Bacteriocin-type signal sequence-containing protein
Similarity search - Component
Biological speciesHungateiclostridium thermocellum (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.35 Å
AuthorsKieuvongngam, V. / Oldham, M.L. / Chen, J.
Funding support United States, 1items
OrganizationGrant numberCountry
Howard Hughes Medical Institute (HHMI) United States
CitationJournal: Elife / Year: 2020
Title: Structural basis of substrate recognition by a polypeptide processing and secretion transporter.
Authors: Virapat Kieuvongngam / Paul Dominic B Olinares / Anthony Palillo / Michael L Oldham / Brian T Chait / Jue Chen /
Abstract: The peptidase-containing ATP-binding cassette transporters (PCATs) are unique members of the ABC transporter family that proteolytically process and export peptides and proteins. Each PCAT contains ...The peptidase-containing ATP-binding cassette transporters (PCATs) are unique members of the ABC transporter family that proteolytically process and export peptides and proteins. Each PCAT contains two peptidase domains that cleave off the secretion signal, two transmembrane domains forming a translocation pathway, and two nucleotide-binding domains that hydrolyze ATP. Previously the crystal structures of a PCAT from (PCAT1) were determined in the absence and presence of ATP, revealing how ATP binding regulates the protease activity and access to the translocation pathway. However, how the substrate CtA, a 90-residue polypeptide, is recognized by PCAT1 remained elusive. To address this question, we determined the structure of the PCAT1-CtA complex by electron cryo-microscopy (cryo-EM) to 3.4 Å resolution. The structure shows that two CtAs are bound via their N-terminal leader peptides, but only one is positioned for cleavage and translocation. Based on these results, we propose a model of how substrate cleavage, ATP hydrolysis, and substrate translocation are coordinated in a transport cycle.
History
DepositionDec 16, 2019Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jan 22, 2020Provider: repository / Type: Initial release
Revision 1.1Jan 29, 2020Group: Database references / Category: citation / citation_author
Item: _citation.journal_abbrev / _citation.journal_id_CSD ..._citation.journal_abbrev / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.journal_volume / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year / _citation_author.identifier_ORCID
Revision 1.2Mar 6, 2024Group: Data collection / Database references / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / em_3d_fitting_list / pdbx_initial_refinement_model
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id / _em_3d_fitting_list.source_name / _em_3d_fitting_list.type

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Structure visualization

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Assembly

Deposited unit
A: ABC-type bacteriocin transporter
C: CtA
B: ABC-type bacteriocin transporter
D: CtA


Theoretical massNumber of molelcules
Total (without water)182,7334
Polymers182,7334
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: mass spectrometry, MS identified the full complex speciesat 182558 kDa
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein ABC-type bacteriocin transporter


Mass: 81148.742 Da / Num. of mol.: 2 / Mutation: C21A
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Hungateiclostridium thermocellum (strain ATCC 27405 / DSM 1237 / JCM 9322 / NBRC 103400 / NCIMB 10682 / NRRL B-4536 / VPI 7372) (bacteria)
Strain: ATCC 27405 / DSM 1237 / JCM 9322 / NBRC 103400 / NCIMB 10682 / NRRL B-4536 / VPI 7372
Gene: Cthe_0534 / Plasmid: pMCSG20 / Production host: Escherichia coli BL21(DE3) (bacteria) / Variant (production host): RIL / References: UniProt: A3DCU1
#2: Protein CtA


Mass: 10217.867 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Hungateiclostridium thermocellum (strain ATCC 27405 / DSM 1237 / JCM 9322 / NBRC 103400 / NCIMB 10682 / NRRL B-4536 / VPI 7372) (bacteria)
Strain: ATCC 27405 / DSM 1237 / JCM 9322 / NBRC 103400 / NCIMB 10682 / NRRL B-4536 / VPI 7372
Gene: Cthe_0535 / Plasmid: pMCSG7 / Production host: Escherichia coli BL21(DE3) (bacteria) / Variant (production host): RIL / References: UniProt: A3DCU2

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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: Ternary complex of a homodimeric PCAT1 ABC transporter with two copies of bound peptide substrate
Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Molecular weightValue: 0.182558 MDa / Experimental value: YES
Source (natural)Organism: Hungateiclostridium thermocellum (bacteria)
Source (recombinant)Organism: Escherichia coli (E. coli) / Strain: BL21(DE3) RIL / Plasmid: pMCSG20 and pMCSG7
Buffer solutionpH: 7
Buffer component
IDConc.NameBuffer-ID
150 mMTris-HCl1
2150 mMNaCl1
32 mMUDM1
45 mMDTT1
SpecimenConc.: 5 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GOLD / Grid mesh size: 400 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 295 K

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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: 2200 nm / Nominal defocus min: 700 nm / Cs: 0 mm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Temperature (min): 100 K
Image recordingAverage exposure time: 0.2 sec. / Electron dose: 1.33 e/Å2 / Detector mode: SUPER-RESOLUTION / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of grids imaged: 2 / Num. of real images: 3478
EM imaging opticsEnergyfilter slit width: 20 eV
Spherical aberration corrector: Microscope was modified with a Cs corrector
Image scansMovie frames/image: 60 / Used frames/image: 1-60

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Processing

EM software
IDNameVersionCategoryDetails
1RELION3particle selectionautopick
2Leginonimage acquisition
4Gctf1.06CTF correction
7REFMACccp4i version 7.0model fitting
9RELION3initial Euler assignment
10cryoSPARC0.6.5final Euler assignment
11RELION3classification
12cryoSPARCv.0.6.53D reconstruction
13Coot0.8.9.2model refinement
CTF correctionType: PHASE FLIPPING ONLY
Particle selectionNum. of particles selected: 572800
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.35 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 133698 / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingB value: 75 / Protocol: OTHER / Space: RECIPROCAL
Atomic model buildingPDB-ID: 4RY2

4ry2


Accession code: 4RY2 / Source name: PDB / Type: experimental model

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