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Yorodumi- PDB-7t54: Cryo-EM structure of ATP-bound PCAT1 in the outward-facing confor... -
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-Basic information
Entry | Database: PDB / ID: 7t54 | ||||||
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Title | Cryo-EM structure of ATP-bound PCAT1 in the outward-facing conformation | ||||||
Components | ABC-type bacteriocin transporter | ||||||
Keywords | TRANSPORT PROTEIN / ATP-binding cassette / ABC transporter / protein transport | ||||||
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 | ||||||
Biological species | Acetivibrio thermocellus (bacteria) | ||||||
Method | ELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4.5 Å | ||||||
Authors | Kieuvongngam, V. / Chen, J. | ||||||
Funding support | United States, 1items
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Citation | Journal: Proc Natl Acad Sci U S A / Year: 2022 Title: Structures of the peptidase-containing ABC transporter PCAT1 under equilibrium and nonequilibrium conditions. Authors: Virapat Kieuvongngam / Jue Chen / Abstract: ATP-binding cassette (ABC) transporters are ubiquitous molecular pumps that transport a broad range of substrates across biological membranes. Although the structure and function of ABC transporters ...ATP-binding cassette (ABC) transporters are ubiquitous molecular pumps that transport a broad range of substrates across biological membranes. Although the structure and function of ABC transporters has been studied extensively, our understanding of their energetics and dynamics remains limited. Here, we present studies of the peptidase-containing ABC transporter 1 (PCAT1), a polypeptide processing and secretion ABC transporter that functions via the classic alternating access mechanism. PCAT1 is a homodimer containing two peptidase (PEP) domains, two transmembrane domains, and two nucleotide-binding domains (NBDs). Using cryo-electron microscopy, we analyzed the structures of wild-type PCAT1 under conditions that either prevent or permit ATP hydrolysis and observed two completely different conformational distributions. In the presence of ATP but absence of Mg, PCAT1 adopts an NBD-dimerized, outward-facing conformation. The two PEP domains are dissociated from the transporter core, preventing uncoupled substrate cleavage. The addition of Mg to promote ATP hydrolysis shifts the majority of the particles into NBD-separated, inward-facing conformations. Under this ATP turnover condition, only a small fraction of PCAT1 adopts the NBD-dimerized conformation. These data give rise to two mechanistic conclusions: 1) the ATP-bound, NBD-dimerized conformation is the lowest energy state, and 2) the rate-limiting step in the PCAT1 transport cycle is the formation of the NBD dimer. The thermodynamic conclusion is likely a general property shared by many ABC transporters. The kinetic bottleneck, however, varies from transporter to transporter. | ||||||
History |
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-Structure visualization
Movie |
Movie viewer |
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Structure viewer | Molecule: MolmilJmol/JSmol |
-Downloads & links
-Download
PDBx/mmCIF format | 7t54.cif.gz | 222.5 KB | Display | PDBx/mmCIF format |
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PDB format | pdb7t54.ent.gz | 163.5 KB | Display | PDB format |
PDBx/mmJSON format | 7t54.json.gz | Tree view | PDBx/mmJSON format | |
Others | Other downloads |
-Validation report
Summary document | 7t54_validation.pdf.gz | 1.6 MB | Display | wwPDB validaton report |
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Full document | 7t54_full_validation.pdf.gz | 1.6 MB | Display | |
Data in XML | 7t54_validation.xml.gz | 50.5 KB | Display | |
Data in CIF | 7t54_validation.cif.gz | 76.3 KB | Display | |
Arichive directory | https://data.pdbj.org/pub/pdb/validation_reports/t5/7t54 ftp://data.pdbj.org/pub/pdb/validation_reports/t5/7t54 | HTTPS FTP |
-Related structure data
Related structure data | 25694MC 7t55C 7t56C 7t57C M: map data used to model this data C: citing same article (ref.) |
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Similar structure data | |
EM raw data | EMPIAR-11009 (Title: Cryo-electron microscopy reconstruction of ATP-bound PCAT1 in an outward-facing conformation in a Mg2+ free condition Data size: 2.6 TB Data #1: Unaligned and uncorrected multiframe movies of ATP-bound PCAT1 in an outward-facing conformation from a Mg2+ free condition [micrographs - multiframe]) |
-Links
-Assembly
Deposited unit |
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-Components
#1: Protein | Mass: 81180.805 Da / Num. of mol.: 2 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Acetivibrio thermocellus (bacteria) Strain: ATCC 27405 / DSM 1237 / JCM 9322 / NBRC 103400 / NCIMB 10682 / NRRL B-4536 / VPI 7372 Gene: Cthe_0534 / Production host: Escherichia coli BL21(DE3) (bacteria) / Variant (production host): RIL / References: UniProt: A3DCU1 #2: Chemical | Has ligand of interest | Y | |
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-Experimental details
-Experiment
Experiment | Method: ELECTRON MICROSCOPY |
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EM experiment | Aggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction |
-Sample preparation
Component | Name: PCAT1 homodimer / Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT | ||||||||||||||||||||
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Molecular weight | Value: 0.162176 MDa / Experimental value: YES | ||||||||||||||||||||
Source (natural) | Organism: Acetivibrio thermocellus (bacteria) | ||||||||||||||||||||
Source (recombinant) | Organism: Escherichia coli (E. coli) / Strain: BL21 (DE3) RIL / Plasmid: pMCSG20 | ||||||||||||||||||||
Buffer solution | pH: 7 | ||||||||||||||||||||
Buffer component |
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Specimen | Conc.: 5 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES | ||||||||||||||||||||
Specimen support | Grid material: GOLD / Grid mesh size: 400 divisions/in. / Grid type: Quantifoil R1.2/1.3 | ||||||||||||||||||||
Vitrification | Instrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 295 K |
-Electron microscopy imaging
Experimental equipment | Model: Titan Krios / Image courtesy: FEI Company |
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Microscopy | Model: FEI TITAN KRIOS |
Electron gun | Electron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM |
Electron lens | Mode: BRIGHT FIELD / Nominal defocus max: 2200 nm / Nominal defocus min: 800 nm / Cs: 2.7 mm |
Specimen holder | Cryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Temperature (min): 100 K |
Image recording | Average exposure time: 0.2 sec. / Electron dose: 1.5 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: 5994 |
Image scans | Movie frames/image: 50 / Used frames/image: 1-50 |
-Processing
Software | Name: PHENIX / Version: 1.19.2_4158: / Classification: refinement | |||||||||||||||||||||||||||||||||||||||||||||
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EM software |
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CTF correction | Type: PHASE FLIPPING ONLY | |||||||||||||||||||||||||||||||||||||||||||||
Particle selection | Num. of particles selected: 1500000 | |||||||||||||||||||||||||||||||||||||||||||||
Symmetry | Point symmetry: C1 (asymmetric) | |||||||||||||||||||||||||||||||||||||||||||||
3D reconstruction | Resolution: 4.5 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 98523 / Num. of class averages: 1 / Symmetry type: POINT | |||||||||||||||||||||||||||||||||||||||||||||
Atomic model building | B value: 80 / Space: RECIPROCAL | |||||||||||||||||||||||||||||||||||||||||||||
Atomic model building | PDB-ID: 4RY2 Accession code: 4RY2 / Source name: PDB / Type: experimental model | |||||||||||||||||||||||||||||||||||||||||||||
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