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- EMDB-21132: Cryo-EM structure of PCAT1 bound to its CtA peptide substrate -

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Basic information

Entry
Database: EMDB / ID: EMD-21132
TitleCryo-EM structure of PCAT1 bound to its CtA peptide substrate
Map datafull map from cryosparc
Sample
  • Complex: Ternary complex of a homodimeric PCAT1 ABC transporter with two copies of bound peptide substrate
    • Protein or peptide: ABC-type bacteriocin transporter
    • Protein or peptide: CtA
KeywordsATP-Binding Cassette / PROTEIN TRANSPORT
Function / homology
Function and homology information


ABC-type bacteriocin transporter activity / cysteine-type peptidase activity / ATP hydrolysis activity / proteolysis / ATP binding / 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 speciesHungateiclostridium thermocellum (bacteria) / Hungateiclostridium thermocellum (strain ATCC 27405 / DSM 1237 / JCM 9322 / NBRC 103400 / NCIMB 10682 / NRRL B-4536 / VPI 7372) (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.35 Å
AuthorsKieuvongngam V / Oldham ML
Funding support United States, 1 items
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, 2019-
Header (metadata) releaseJan 15, 2020-
Map releaseJan 22, 2020-
UpdateMar 6, 2024-
Current statusMar 6, 2024Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.55
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 0.55
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6v9z
  • Surface level: 0.55
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_21132.png

Downloads & links

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Map

FileDownload / File: emd_21132.map.gz / Format: CCP4 / Size: 103 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationfull map from cryosparc
Voxel sizeX=Y=Z: 1.09 Å
Density
Contour LevelBy AUTHOR: 0.55 / Movie #1: 0.55
Minimum - Maximum-0.5329742 - 1.5096163
Average (Standard dev.)0.0024543286 (±0.045661163)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions300300300
Spacing300300300
CellA=B=C: 327.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.091.091.09
M x/y/z300300300
origin x/y/z0.0000.0000.000
length x/y/z327.000327.000327.000
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS300300300
D min/max/mean-0.5331.5100.002

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Supplemental data

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Additional map: sharpened map (bfactor -75 Angstrom^2) from cryosparc

Fileemd_21132_additional.map
Annotationsharpened map (bfactor -75 Angstrom^2) from cryosparc
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: half map2 from cryosparc

Fileemd_21132_half_map_1.map
Annotationhalf map2 from cryosparc
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: half map1 from cryosparc

Fileemd_21132_half_map_2.map
Annotationhalf map1 from cryosparc
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 a homodimeric PCAT1 ABC transporter with two c...

EntireName: Ternary complex of a homodimeric PCAT1 ABC transporter with two copies of bound peptide substrate
Components
  • Complex: Ternary complex of a homodimeric PCAT1 ABC transporter with two copies of bound peptide substrate
    • Protein or peptide: ABC-type bacteriocin transporter
    • Protein or peptide: CtA

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Supramolecule #1: Ternary complex of a homodimeric PCAT1 ABC transporter with two c...

SupramoleculeName: Ternary complex of a homodimeric PCAT1 ABC transporter with two copies of bound peptide substrate
type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Hungateiclostridium thermocellum (bacteria)
Molecular weightTheoretical: 182.558 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: 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
Molecular weightTheoretical: 81.148742 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: SNAMLRRLFK KKYVCVRQYD LTDAGAACLS SIAQYYGLKM SLAKIREMTG TDTQGTNAYG LIHAAKQLGF SAKGVKASKE DLLKDFRLP AIANVIVDNR LAHFVVIYSI KNRIITVADP GKGIVRYSMD DFCSIWTGGL VLLEPGEAFQ KGDYTQNMMV K FAGFLKPL ...String:
SNAMLRRLFK KKYVCVRQYD LTDAGAACLS 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: CtA

MacromoleculeName: CtA / type: protein_or_peptide / ID: 2 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: 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
Molecular weightTheoretical: 10.217867 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString:
SNAMSEAKKL NIGRELTDEE LMEMTGGSTF SIQCQKDYTY KPSLPVVKYG VVIDEPEVVI KYGVGPIVGI KYGVEPIGPI QPMYGIKPV ETLK

UniProtKB: Bacteriocin-type signal sequence-containing protein

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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

Concentration5 mg/mL
BufferpH: 7
Component:
ConcentrationName
50.0 mMTris-HClTris
150.0 mMNaClSodium chloride
2.0 mMUDM
5.0 mMDTT
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
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 0.0 mm / Nominal defocus max: 2.2 µm / Nominal defocus min: 0.7000000000000001 µm
Specialist opticsSpherical aberration corrector: Microscope was modified with a Cs corrector
Energy filter - Slit width: 20 eV
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
TemperatureMin: 100.0 K
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: SUPER-RESOLUTION / Digitization - Frames/image: 1-60 / Number grids imaged: 2 / Number real images: 3478 / Average exposure time: 0.2 sec. / Average electron dose: 1.33 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 572800
Startup modelType of model: INSILICO MODEL / In silico model: Ab initio model was generated in cryosparc
Initial angle assignmentType: ANGULAR RECONSTITUTION / Software - Name: RELION (ver. 3.0)
Final 3D classificationNumber classes: 4 / Software - Name: RELION (ver. 3.0)
Final angle assignmentType: ANGULAR RECONSTITUTION / Software - Name: cryoSPARC (ver. 0.6.5)
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 3.35 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. v.0.6.5) / Number images used: 133698

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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: 75
Output model

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

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