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- EMDB-25696: Cryo-EM structure of PCAT1 in the inward-facing intermediate conf... -
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Open data
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Basic information
Entry | Database: EMDB / ID: EMD-25696 | |||||||||
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Title | Cryo-EM structure of PCAT1 in the inward-facing intermediate conformation under ATP turnover condition | |||||||||
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![]() | ATP-binding cassette / ABC transporter / protein transport / TRANSPORT PROTEIN | |||||||||
Function / homology | ![]() 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 | ![]() | |||||||||
Method | single particle reconstruction / cryo EM / Resolution: 3.7 Å | |||||||||
![]() | Kieuvongngam V / Chen J | |||||||||
Funding support | ![]()
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![]() | ![]() 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 |
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Structure viewer | EM map: ![]() ![]() ![]() |
Supplemental images |
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Downloads & links
-EMDB archive
Map data | ![]() | 51.8 MB | ![]() | |
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Header (meta data) | ![]() ![]() | 20.4 KB 20.4 KB | Display Display | ![]() |
FSC (resolution estimation) | ![]() | 10.4 KB | Display | ![]() |
Images | ![]() | 53.1 KB | ||
Filedesc metadata | ![]() | 6.6 KB | ||
Others | ![]() ![]() | 95.4 MB 95.4 MB | ||
Archive directory | ![]() ![]() | HTTPS FTP |
-Related structure data
Related structure data | ![]() 7t56MC ![]() 7t54C ![]() 7t55C ![]() 7t57C M: atomic model generated by this map C: citing same article ( |
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Similar structure data | |
EM raw data | ![]() Data size: 5.2 TB Data #1: Unaligned, uncorrected movies of PCAT1 in ATP active turnover condition [micrographs - multiframe]) |
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Links
EMDB pages | ![]() ![]() |
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Related items in Molecule of the Month |
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Map
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Annotation | full map | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Voxel size | X=Y=Z: 1.03 Å | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Details | EMDB XML:
CCP4 map header:
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-Supplemental data
-Half map: half map A
File | emd_25696_half_map_1.map | ||||||||||||
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Annotation | half map A | ||||||||||||
Projections & Slices |
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Density Histograms |
-Half map: half map B
File | emd_25696_half_map_2.map | ||||||||||||
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Annotation | half map B | ||||||||||||
Projections & Slices |
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Density Histograms |
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Sample components
-Entire : Ternary complex of PCAT1 with its cleaved peptide substrate and ATP-Mg
Entire | Name: Ternary complex of PCAT1 with its cleaved peptide substrate and ATP-Mg |
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Components |
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-Supramolecule #1: Ternary complex of PCAT1 with its cleaved peptide substrate and ATP-Mg
Supramolecule | Name: Ternary complex of PCAT1 with its cleaved peptide substrate and ATP-Mg type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#2 |
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Source (natural) | Organism: ![]() |
Molecular weight | Theoretical: 162.176 KDa |
-Macromolecule #1: ABC-type bacteriocin transporter
Macromolecule | Name: ABC-type bacteriocin transporter / type: protein_or_peptide / ID: 1 / Number of copies: 2 / Enantiomer: LEVO |
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Source (natural) | Organism: ![]() Strain: ATCC 27405 / DSM 1237 / JCM 9322 / NBRC 103400 / NCIMB 10682 / NRRL B-4536 / VPI 7372 |
Molecular weight | Theoretical: 81.180805 KDa |
Recombinant expression | Organism: ![]() ![]() |
Sequence | String: SNAMLRRLFK KKYVCVRQYD LTDCGAACLS SIAQYYGLKM SLAKIREMTG TDTQGTNAYG LIHAAKQLGF SAKGVKASKE DLLKDFRLP AIANVIVDNR LAHFVVIYSI KNRIITVADP GKGIVRYSMD DFCSIWTGGL VLLEPGEAFQ KGDYTQNMMV K FAGFLKPL ...String: SNAMLRRLFK KKYVCVRQYD LTDCGAACLS SIAQYYGLKM SLAKIREMTG TDTQGTNAYG LIHAAKQLGF SAKGVKASKE DLLKDFRLP AIANVIVDNR LAHFVVIYSI KNRIITVADP GKGIVRYSMD DFCSIWTGGL VLLEPGEAFQ KGDYTQNMMV K FAGFLKPL KKTVLCIFLA SLLYTALGIA GSFYIKFLFD DLIKFEKLND LHIISAGFAV IFLLQIFLNY YRSILVTKLG MS IDKSIMM EYYSHVLKLP MNFFNSRKVG EIISRFMDAS KIRQAISGAT LTIMIDTIMA VIGGILLYIQ NSSLFFISFI IIL LYGIIV TVFNKPIQNA NRQIMEDNAK LTSALVESVK GIETIKSFGA EEQTEKSTRD KIETVMKSSF KEGMLYINLS SLTG IVAGL GGIVILWAGA YNVIKGNMSG GQLLAFNALL AYFLTPVKNL IDLQPLIQTA VVASNRLGEI LELATEKELR EDSDD FVIS LKGDIEFRNV DFRYGLRKPV LKNINLTIPK GKTVAIVGES GSGKTTLAKL LMNFYSPEKG DILINGHSIK NISLEL IRK KIAFVSQDVF IFSGTVKENL CLGNENVDMD EIIKAAKMAN AHDFIEKLPL KYDTFLNESG ANLSEGQKQR LAIARAL LK KPDILILDEA TSNLDSITEN HIKDAIYGLE DDVTVIIIAH RLSTIVNCDK IYLLKDGEIV ESGSHTELIA LKGCYFKM W KQTENTLAS UniProtKB: ABC-type bacteriocin transporter |
-Macromolecule #2: PCAT1 peptide substrate
Macromolecule | Name: PCAT1 peptide substrate / type: protein_or_peptide / ID: 2 / Number of copies: 2 / Enantiomer: LEVO |
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Source (natural) | Organism: ![]() Strain: ATCC 27405 / DSM 1237 / JCM 9322 / NBRC 103400 / NCIMB 10682 / NRRL B-4536 / VPI 7372 |
Molecular weight | Theoretical: 10.217867 KDa |
Recombinant expression | Organism: ![]() ![]() |
Sequence | String: SNAMSEAKKL NIGRELTDEE LMEMTGGSTF SIQCQKDYTY KPSLPVVKYG VVIDEPEVVI KYGVGPIVGI KYGVEPIGPI QPMYGIKPV ETLK UniProtKB: Bacteriocin-type signal sequence-containing protein |
-Macromolecule #3: ADENOSINE-5'-TRIPHOSPHATE
Macromolecule | Name: ADENOSINE-5'-TRIPHOSPHATE / type: ligand / ID: 3 / Number of copies: 2 / Formula: ATP |
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Molecular weight | Theoretical: 507.181 Da |
Chemical component information | ![]() ChemComp-ATP: |
-Macromolecule #4: MAGNESIUM ION
Macromolecule | Name: MAGNESIUM ION / type: ligand / ID: 4 / Number of copies: 2 / Formula: MG |
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Molecular weight | Theoretical: 24.305 Da |
-Experimental details
-Structure determination
Method | cryo EM |
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![]() | single particle reconstruction |
Aggregation state | particle |
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Sample preparation
Concentration | 7 mg/mL | ||||||||||
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Buffer | pH: 7 Component:
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Grid | Model: Quantifoil R1.2/1.3 / Material: GOLD / Mesh: 400 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 20 sec. / Pretreatment - Atmosphere: AIR | ||||||||||
Vitrification | Cryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 295 K / Instrument: FEI VITROBOT MARK IV |
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Electron microscopy
Microscope | FEI TITAN KRIOS |
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Temperature | Min: 100.0 K |
Image recording | Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: SUPER-RESOLUTION / Digitization - Frames/image: 1-50 / Number grids imaged: 2 / Number real images: 11966 / Average exposure time: 0.2 sec. / Average electron dose: 1.5 e/Å2 |
Electron beam | Acceleration voltage: 300 kV / Electron source: ![]() |
Electron optics | Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 2.2 µm / Nominal defocus min: 0.8 µm |
Sample stage | Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN |
Experimental equipment | ![]() Model: Titan Krios / Image courtesy: FEI Company |