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Yorodumi- EMDB-25694: Cryo-EM structure of ATP-bound PCAT1 in the outward-facing confor... -
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-Basic information
Entry | Database: EMDB / ID: EMD-25694 | |||||||||
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Title | Cryo-EM structure of ATP-bound PCAT1 in the outward-facing conformation | |||||||||
Map data | full map | |||||||||
Sample |
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Keywords | ATP-binding cassette / ABC transporter / protein transport / TRANSPORT PROTEIN | |||||||||
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 | single particle reconstruction / cryo EM / Resolution: 4.5 Å | |||||||||
Authors | Kieuvongngam V / Chen J | |||||||||
Funding support | United States, 1 items
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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 | EM map: SurfViewMolmilJmol/JSmol |
Supplemental images |
-Downloads & links
-EMDB archive
Map data | emd_25694.map.gz | 50.9 MB | EMDB map data format | |
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Header (meta data) | emd-25694-v30.xml emd-25694.xml | 18.7 KB 18.7 KB | Display Display | EMDB header |
FSC (resolution estimation) | emd_25694_fsc.xml | 10.5 KB | Display | FSC data file |
Images | emd_25694.png | 42 KB | ||
Filedesc metadata | emd-25694.cif.gz | 6.4 KB | ||
Others | emd_25694_half_map_1.map.gz emd_25694_half_map_2.map.gz | 95.4 MB 95.4 MB | ||
Archive directory | http://ftp.pdbj.org/pub/emdb/structures/EMD-25694 ftp://ftp.pdbj.org/pub/emdb/structures/EMD-25694 | HTTPS FTP |
-Validation report
Summary document | emd_25694_validation.pdf.gz | 1.1 MB | Display | EMDB validaton report |
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Full document | emd_25694_full_validation.pdf.gz | 1.1 MB | Display | |
Data in XML | emd_25694_validation.xml.gz | 18.3 KB | Display | |
Data in CIF | emd_25694_validation.cif.gz | 23.5 KB | Display | |
Arichive directory | https://ftp.pdbj.org/pub/emdb/validation_reports/EMD-25694 ftp://ftp.pdbj.org/pub/emdb/validation_reports/EMD-25694 | HTTPS FTP |
-Related structure data
Related structure data | 7t54MC 7t55C 7t56C 7t57C M: atomic model generated by this map 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
EMDB pages | EMDB (EBI/PDBe) / EMDataResource |
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Related items in Molecule of the Month |
-Map
File | Download / File: emd_25694.map.gz / Format: CCP4 / Size: 103 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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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_25694_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_25694_half_map_2.map | ||||||||||||
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Annotation | half map B | ||||||||||||
Projections & Slices |
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Density Histograms |
-Sample components
-Entire : PCAT1 homodimer
Entire | Name: PCAT1 homodimer |
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Components |
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-Supramolecule #1: PCAT1 homodimer
Supramolecule | Name: PCAT1 homodimer / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1 |
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Source (natural) | Organism: Acetivibrio thermocellus (bacteria) |
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: Acetivibrio thermocellus (bacteria) 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: Escherichia coli BL21(DE3) (bacteria) |
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: ADENOSINE-5'-TRIPHOSPHATE
Macromolecule | Name: ADENOSINE-5'-TRIPHOSPHATE / type: ligand / ID: 2 / Number of copies: 2 / Formula: ATP |
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Molecular weight | Theoretical: 507.181 Da |
Chemical component information | ChemComp-ATP: |
-Experimental details
-Structure determination
Method | cryo EM |
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Processing | single particle reconstruction |
Aggregation state | particle |
-Sample preparation
Concentration | 5 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 |
-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: 5994 / Average exposure time: 0.2 sec. / Average electron dose: 1.5 e/Å2 |
Electron beam | Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN |
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 |