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- EMDB-28585: Cryo-EM structure of a delivery complex containing the SspB adapt... -

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Basic information

Entry
Database: EMDB / ID: EMD-28585
TitleCryo-EM structure of a delivery complex containing the SspB adaptor, an ssrA-tagged substrate, and the AAA+ ClpXP protease
Map data
Sample
  • Complex: ClpXP AAA protease complex bound with SspB and GFP-ssrA
    • Protein or peptide: ATP-dependent Clp protease ATP-binding subunit ClpX
    • Protein or peptide: ATP-dependent Clp protease proteolytic subunit
    • Protein or peptide: Green fluorescent protein
    • Protein or peptide: Stringent starvation protein B
  • Ligand: PHOSPHOTHIOPHOSPHORIC ACID-ADENYLATE ESTER
  • Ligand: ADENOSINE-5'-DIPHOSPHATE
KeywordsAAA+ protease / ClpXP / SspB adaptor / HYDROLASE / CHAPERONE
Function / homology
Function and homology information


HslUV protease complex / endopeptidase Clp complex / endopeptidase Clp / positive regulation of programmed cell death / response to temperature stimulus / ATP-dependent peptidase activity / protein quality control for misfolded or incompletely synthesized proteins / serine-type peptidase activity / bioluminescence / generation of precursor metabolites and energy ...HslUV protease complex / endopeptidase Clp complex / endopeptidase Clp / positive regulation of programmed cell death / response to temperature stimulus / ATP-dependent peptidase activity / protein quality control for misfolded or incompletely synthesized proteins / serine-type peptidase activity / bioluminescence / generation of precursor metabolites and energy / proteasomal protein catabolic process / ATP-dependent protein folding chaperone / response to radiation / unfolded protein binding / peptidase activity / ATPase binding / response to heat / protein dimerization activity / serine-type endopeptidase activity / ATP hydrolysis activity / proteolysis / zinc ion binding / ATP binding / membrane / identical protein binding / cytosol
Similarity search - Function
Stringent starvation protein B / SspB-like superfamily / Stringent starvation protein B / Clp protease, ATP-binding subunit ClpX, bacteria / Zinc finger, ClpX C4-type superfamily / ClpX C4-type zinc finger / Clp protease, ATP-binding subunit ClpX / Zinc finger, ClpX C4-type / ClpX zinc binding (ZB) domain profile. / ClpX C4-type zinc finger ...Stringent starvation protein B / SspB-like superfamily / Stringent starvation protein B / Clp protease, ATP-binding subunit ClpX, bacteria / Zinc finger, ClpX C4-type superfamily / ClpX C4-type zinc finger / Clp protease, ATP-binding subunit ClpX / Zinc finger, ClpX C4-type / ClpX zinc binding (ZB) domain profile. / ClpX C4-type zinc finger / ClpP, Ser active site / Endopeptidase Clp serine active site. / ClpP, histidine active site / Endopeptidase Clp histidine active site. / ATP-dependent Clp protease proteolytic subunit / Clp protease proteolytic subunit /Translocation-enhancing protein TepA / Clp protease / Clp ATPase, C-terminal / AAA domain (Cdc48 subfamily) / C-terminal, D2-small domain, of ClpB protein / C-terminal, D2-small domain, of ClpB protein / ClpP/crotonase-like domain superfamily / Green fluorescent protein, GFP / Green fluorescent protein-related / Green fluorescent protein / Green fluorescent protein / ATPase, AAA-type, core / ATPases associated with a variety of cellular activities / AAA+ ATPase domain / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
ATP-dependent Clp protease ATP-binding subunit ClpX / ATP-dependent Clp protease proteolytic subunit / Stringent starvation protein B / Green fluorescent protein
Similarity search - Component
Biological speciesEscherichia coli (E. coli)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.7 Å
AuthorsGhanbarpour A / Fei X / Davis JH / Sauer RT
Funding support United States, 1 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS) United States
CitationJournal: Proc Natl Acad Sci U S A / Year: 2023
Title: The SspB adaptor drives structural changes in the AAA+ ClpXP protease during ssrA-tagged substrate delivery.
Authors: Alireza Ghanbarpour / Xue Fei / Tania A Baker / Joseph H Davis / Robert T Sauer /
Abstract: Energy-dependent protein degradation by the AAA+ ClpXP protease helps maintain protein homeostasis in bacteria and eukaryotic organelles of bacterial origin. In  and many other proteobacteria, the ...Energy-dependent protein degradation by the AAA+ ClpXP protease helps maintain protein homeostasis in bacteria and eukaryotic organelles of bacterial origin. In  and many other proteobacteria, the SspB adaptor assists ClpXP in degrading ssrA-tagged polypeptides produced as a consequence of tmRNA-mediated ribosome rescue. By tethering these incomplete ssrA-tagged proteins to ClpXP, SspB facilitates their efficient degradation at low substrate concentrations. How this process occurs structurally is unknown. Here, we present a cryo-EM structure of the SspB adaptor bound to a GFP-ssrA substrate and to ClpXP. This structure provides evidence for simultaneous contacts of SspB and ClpX with the ssrA tag within the tethering complex, allowing direct substrate handoff concomitant with the initiation of substrate translocation. Furthermore, our structure reveals that binding of the substrate·adaptor complex induces unexpected conformational changes within the spiral structure of the AAA+ ClpX hexamer and its interaction with the ClpP tetradecamer.
History
DepositionOct 16, 2022-
Header (metadata) releaseJan 25, 2023-
Map releaseJan 25, 2023-
UpdateAug 9, 2023-
Current statusAug 9, 2023Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_28585.png

Downloads & links

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Map

FileDownload / File: emd_28585.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 0.832 Å
Density
Contour LevelBy AUTHOR: 0.00495
Minimum - Maximum-0.010238585 - 0.035946075
Average (Standard dev.)0.0004262982 (±0.0021284996)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 212.992 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_28585_msk_1.map
Projections & Slices
AxesZYX

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Half map: #1

Fileemd_28585_half_map_1.map
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Half map: #2

Fileemd_28585_half_map_2.map
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Sample components

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Entire : ClpXP AAA protease complex bound with SspB and GFP-ssrA

EntireName: ClpXP AAA protease complex bound with SspB and GFP-ssrA
Components
  • Complex: ClpXP AAA protease complex bound with SspB and GFP-ssrA
    • Protein or peptide: ATP-dependent Clp protease ATP-binding subunit ClpX
    • Protein or peptide: ATP-dependent Clp protease proteolytic subunit
    • Protein or peptide: Green fluorescent protein
    • Protein or peptide: Stringent starvation protein B
  • Ligand: PHOSPHOTHIOPHOSPHORIC ACID-ADENYLATE ESTER
  • Ligand: ADENOSINE-5'-DIPHOSPHATE

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Supramolecule #1: ClpXP AAA protease complex bound with SspB and GFP-ssrA

SupramoleculeName: ClpXP AAA protease complex bound with SspB and GFP-ssrA
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#4
Source (natural)Organism: Escherichia coli (E. coli)

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Macromolecule #1: ATP-dependent Clp protease ATP-binding subunit ClpX

MacromoleculeName: ATP-dependent Clp protease ATP-binding subunit ClpX / type: protein_or_peptide / ID: 1 / Number of copies: 6 / Enantiomer: LEVO
Source (natural)Organism: Escherichia coli (E. coli)
Molecular weightTheoretical: 46.414848 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MTDKRKDGSG KLLYCSFCGK SQHEVRKLIA GPSVYICDEC VDLCNDIIRE EIKEVAPHRE RSALPTPHEI RNHLDDYVIG QEQAKKVLA VAVYNHYKRL RNGDTSNGVE LGKSNILLIG PTGSGKTLLA ETLARLLDVP FTMADATTLT EAGYVGEDVE N IIQKLLQK ...String:
MTDKRKDGSG KLLYCSFCGK SQHEVRKLIA GPSVYICDEC VDLCNDIIRE EIKEVAPHRE RSALPTPHEI RNHLDDYVIG QEQAKKVLA VAVYNHYKRL RNGDTSNGVE LGKSNILLIG PTGSGKTLLA ETLARLLDVP FTMADATTLT EAGYVGEDVE N IIQKLLQK CDYDVQKAQR GIVYIDEIDK ISRKSDNPSI TRDVSGEGVQ QALLKLIEGT VAAVPPQGGR KHPQQEFLQV DT SKILFIC GGAFAGLDKV ISHRVETGSG IGFGATVKAK SDKASEGELL AQVEPEDLIK FGLIPEFIGR LPVVATLNEL SEE ALIQIL KEPKNALTKQ YQALFNLEGV DLEFRDEALD AIAKKAMARK TGARGLRSIV EAALLDTMYD LPSMEDVEKV VIDE SVIDG QSEPLLIYGK PEAQQASGE

UniProtKB: ATP-dependent Clp protease ATP-binding subunit ClpX

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Macromolecule #2: ATP-dependent Clp protease proteolytic subunit

MacromoleculeName: ATP-dependent Clp protease proteolytic subunit / type: protein_or_peptide / ID: 2 / Number of copies: 7 / Enantiomer: LEVO / EC number: endopeptidase Clp
Source (natural)Organism: Escherichia coli (E. coli)
Molecular weightTheoretical: 23.468869 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: LVPMVIEQTS RGERSFDIYS RLLKERVIFL TGQVEDHMAN LIVAQMLFLE AENPEKDIYL YINSPGGVIT AGMSIYDTMQ FIKPDVSTI CMGQAASMGA FLLTAGAKGK RFCLPNSRVM IHQPLGGYQG QATDIEIHAR EILKVKGRMN ELMALHTGQS L EQIERDTE ...String:
LVPMVIEQTS RGERSFDIYS RLLKERVIFL TGQVEDHMAN LIVAQMLFLE AENPEKDIYL YINSPGGVIT AGMSIYDTMQ FIKPDVSTI CMGQAASMGA FLLTAGAKGK RFCLPNSRVM IHQPLGGYQG QATDIEIHAR EILKVKGRMN ELMALHTGQS L EQIERDTE RDRFLSAPEA VEYGLVDSIL THRNENLYFQ SLEHHHHHH

UniProtKB: ATP-dependent Clp protease proteolytic subunit

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Macromolecule #3: Green fluorescent protein

MacromoleculeName: Green fluorescent protein / type: protein_or_peptide / ID: 3 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Escherichia coli (E. coli)
Molecular weightTheoretical: 30.855428 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MGSSHHHHHH DYDIPTTENL YFQGSRKGEE LFTGVVPILV ELDGDVNGHK FSVSGEGEGD ATYGKLTLKF ICTTGKLPVP WPTLVTTFG YGVQCFARYP DHMKQHDFFK SAMPEGYVQE RTIFFKDDGN YKTRAEVKFE GDTLVNRIEL KGIDFKEDGN I LGHKLEYN ...String:
MGSSHHHHHH DYDIPTTENL YFQGSRKGEE LFTGVVPILV ELDGDVNGHK FSVSGEGEGD ATYGKLTLKF ICTTGKLPVP WPTLVTTFG YGVQCFARYP DHMKQHDFFK SAMPEGYVQE RTIFFKDDGN YKTRAEVKFE GDTLVNRIEL KGIDFKEDGN I LGHKLEYN YNSHNVYIMA DKQKNGIKVN FKIRHNIEDG SVQLADHYQQ NTPIGDGPVL LPDNHYLSTQ SALSKDPNEK RD HMVLLEF VTAAGITHGM DELYKAANDE NYALAA

UniProtKB: Green fluorescent protein

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Macromolecule #4: Stringent starvation protein B

MacromoleculeName: Stringent starvation protein B / type: protein_or_peptide / ID: 4 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Escherichia coli (E. coli)
Molecular weightTheoretical: 18.279352 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
MDLSQLTPRR PYLLRAFYEW LLDNQLTPHL VVDVTLPGVQ VPMEYARDGQ IVLNIAPRAV GNLELANDEV RFNARFGGIP RQVSVPLAA VLAIYARENG AGTMFEPEAA YDEDTSIMND EEASADNETV MSVIDGDKPD HDDDTHPDDE PPQPPRGGRP A LRVVK

UniProtKB: Stringent starvation protein B

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Macromolecule #5: PHOSPHOTHIOPHOSPHORIC ACID-ADENYLATE ESTER

MacromoleculeName: PHOSPHOTHIOPHOSPHORIC ACID-ADENYLATE ESTER / type: ligand / ID: 5 / Number of copies: 4 / Formula: AGS
Molecular weightTheoretical: 523.247 Da
Chemical component information

ChemComp-AGS:
PHOSPHOTHIOPHOSPHORIC ACID-ADENYLATE ESTER / ATP-gamma-S, energy-carrying molecule analogue*YM

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Macromolecule #6: ADENOSINE-5'-DIPHOSPHATE

MacromoleculeName: ADENOSINE-5'-DIPHOSPHATE / type: ligand / ID: 6 / Number of copies: 2 / Formula: ADP
Molecular weightTheoretical: 427.201 Da
Chemical component information

ChemComp-ADP:
ADENOSINE-5'-DIPHOSPHATE / ADP, energy-carrying molecule*YM / Adenosine diphosphate

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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

BufferpH: 7.5
GridModel: Quantifoil R2/1 / Material: COPPER / Mesh: 300 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 60 sec.
VitrificationCryogen name: ETHANE / 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 opticsC2 aperture diameter: 50.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 2.5 µm / Nominal defocus min: 1.0 µm
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Average electron dose: 75.98 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: PDB ENTRY
PDB model - PDB ID:

6wrf

Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.1)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.1)
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.7 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 236728
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

6wrf

RefinementSpace: REAL / Protocol: RIGID BODY FIT
Output model

PDB-8et3:
Cryo-EM structure of a delivery complex containing the SspB adaptor, an ssrA-tagged substrate, and the AAA+ ClpXP protease

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