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- EMDB-25697: Cryo-EM structure of PCAT1 in the inward-facing narrow conformati... -

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Basic information

Entry
Database: EMDB / ID: EMD-25697
TitleCryo-EM structure of PCAT1 in the inward-facing narrow conformation under ATP turnover condition
Map datafull map
Sample
  • Complex: Ternary complex of PCAT1 with its cleaved peptide substrate and ATP-Mg
    • Protein or peptide: ABC-type bacteriocin transporter
    • Protein or peptide: PCAT1 peptide substrate
  • Ligand: ADENOSINE-5'-TRIPHOSPHATE
  • Ligand: MAGNESIUM ION
KeywordsATP-binding cassette / ABC transporter / protein transport / TRANSPORT 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 C39 family / Peptidase C39, bacteriocin processing / Peptidase family C39 domain profile. / 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 C39 family / Peptidase C39, bacteriocin processing / Peptidase family C39 domain profile. / 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
ABC-type bacteriocin transporter / Bacteriocin-type signal sequence-containing protein
Similarity search - Component
Biological speciesAcetivibrio thermocellus (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.7 Å
AuthorsKieuvongngam V / Chen J
Funding support United States, 1 items
OrganizationGrant numberCountry
Howard Hughes Medical Institute (HHMI) United States
CitationJournal: 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
DepositionDec 11, 2021-
Header (metadata) releaseFeb 2, 2022-
Map releaseFeb 2, 2022-
UpdateFeb 28, 2024-
Current statusFeb 28, 2024Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.25
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.25
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-7t57
  • Surface level: 0.25
  • Imaged by UCSF Chimera
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  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-7t57
  • Imaged by Jmol
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_25697.png

Downloads & links

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Map

FileDownload / File: emd_25697.map.gz / Format: CCP4 / Size: 103 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationfull map
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.03 Å/pix.
x 300 pix.
= 309. Å		1.03 Å/pix.
x 300 pix.
= 309. Å		1.03 Å/pix.
x 300 pix.
= 309. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.03 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.25 / Movie #1: 0.25
Minimum - Maximum-0.6939052 - 1.1965389
Average (Standard dev.)-0.000053502958 (±0.032149542)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions300300300
Spacing300300300
CellA=B=C: 309.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.031.031.03
M x/y/z300300300
origin x/y/z0.0000.0000.000
length x/y/z309.000309.000309.000
α/β/γ90.00090.00090.000
start NX/NY/NZ154162115
NX/NY/NZ9384171
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS300300300
D min/max/mean-0.6941.197-0.000

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Supplemental data

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Half map: half map A

Fileemd_25697_half_map_1.map
Annotationhalf map A
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: half map B

Fileemd_25697_half_map_2.map
Annotationhalf map B
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Ternary complex of PCAT1 with its cleaved peptide substrate and ATP-Mg

EntireName: Ternary complex of PCAT1 with its cleaved peptide substrate and ATP-Mg
Components
  • Complex: Ternary complex of PCAT1 with its cleaved peptide substrate and ATP-Mg
    • Protein or peptide: ABC-type bacteriocin transporter
    • Protein or peptide: PCAT1 peptide substrate
  • Ligand: ADENOSINE-5'-TRIPHOSPHATE
  • Ligand: MAGNESIUM ION

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Supramolecule #1: Ternary complex of PCAT1 with its cleaved peptide substrate and ATP-Mg

SupramoleculeName: Ternary complex of PCAT1 with its cleaved peptide substrate and ATP-Mg
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#2
Source (natural)Organism: Acetivibrio thermocellus (bacteria)
Molecular weightTheoretical: 162.176 KDa

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Macromolecule #1: ABC-type bacteriocin transporter

MacromoleculeName: ABC-type bacteriocin transporter / type: protein_or_peptide / ID: 1 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Acetivibrio thermocellus (bacteria)
Strain: ATCC 27405 / DSM 1237 / JCM 9322 / NBRC 103400 / NCIMB 10682 / NRRL B-4536 / VPI 7372
Molecular weightTheoretical: 81.180805 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: 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

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Macromolecule #2: PCAT1 peptide substrate

MacromoleculeName: PCAT1 peptide substrate / type: protein_or_peptide / ID: 2 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Acetivibrio thermocellus (bacteria)
Strain: ATCC 27405 / DSM 1237 / JCM 9322 / NBRC 103400 / NCIMB 10682 / NRRL B-4536 / VPI 7372
Molecular weightTheoretical: 10.217867 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
SNAMSEAKKL NIGRELTDEE LMEMTGGSTF SIQCQKDYTY KPSLPVVKYG VVIDEPEVVI KYGVGPIVGI KYGVEPIGPI QPMYGIKPV ETLK

UniProtKB: Bacteriocin-type signal sequence-containing protein

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Macromolecule #3: ADENOSINE-5'-TRIPHOSPHATE

MacromoleculeName: ADENOSINE-5'-TRIPHOSPHATE / type: ligand / ID: 3 / Number of copies: 2 / Formula: ATP
Molecular weightTheoretical: 507.181 Da
Chemical component information

ChemComp-ATP:
ADENOSINE-5'-TRIPHOSPHATE / ATP, energy-carrying molecule*YM

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Macromolecule #4: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 4 / Number of copies: 2 / Formula: MG
Molecular weightTheoretical: 24.305 Da

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration7 mg/mL
BufferpH: 7
Component:
ConcentrationName
50.0 mMNaCl
150.0 mMTris-HCl
5.0 mMDTT
2.0 mMUDM
GridModel: 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
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 295 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
TemperatureMin: 100.0 K
Image recordingFilm 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 beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 2.2 µm / Nominal defocus min: 0.8 µm
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 2800000
Startup modelType of model: PDB ENTRY
PDB model - PDB ID:

4ry2

Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 3.7 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 3.0) / Number images used: 80156
Initial angle assignmentType: ANGULAR RECONSTITUTION / Software - Name: RELION (ver. 3.0)
Final angle assignmentType: ANGULAR RECONSTITUTION / Software - Name: cryoSPARC (ver. 3.0)
Final 3D classificationNumber classes: 7 / Software - Name: RELION (ver. 3.0)
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

4ry2


Chain - Source name: PDB / Chain - Initial model type: experimental model
RefinementSpace: RECIPROCAL / Protocol: OTHER / Overall B value: 80
Output model

PDB-7t57:
Cryo-EM structure of PCAT1 in the inward-facing narrow conformation under ATP turnover condition

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