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- PDB-8et3: Cryo-EM structure of a delivery complex containing the SspB adapt... -

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Basic information

Entry
Database: PDB / ID: 8et3
TitleCryo-EM structure of a delivery complex containing the SspB adaptor, an ssrA-tagged substrate, and the AAA+ ClpXP protease
Components
  • (ATP-dependent Clp protease ...) x 2
  • Green fluorescent protein
  • Stringent starvation protein B
KeywordsCHAPERONE / AAA+ protease / ClpXP / SspB adaptor / HYDROLASE
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
ADENOSINE-5'-DIPHOSPHATE / PHOSPHOTHIOPHOSPHORIC ACID-ADENYLATE ESTER / 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)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.7 Å
AuthorsGhanbarpour, A. / Fei, X. / Davis, J.H. / Sauer, R.T.
Funding support United States, 1items
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, 2022Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jan 25, 2023Provider: repository / Type: Initial release
Revision 1.1Aug 9, 2023Group: Database references / Category: citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_ASTM / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year / _citation_author.identifier_ORCID

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: ATP-dependent Clp protease ATP-binding subunit ClpX
B: ATP-dependent Clp protease ATP-binding subunit ClpX
C: ATP-dependent Clp protease ATP-binding subunit ClpX
D: ATP-dependent Clp protease ATP-binding subunit ClpX
E: ATP-dependent Clp protease ATP-binding subunit ClpX
F: ATP-dependent Clp protease ATP-binding subunit ClpX
H: ATP-dependent Clp protease proteolytic subunit
I: ATP-dependent Clp protease proteolytic subunit
J: ATP-dependent Clp protease proteolytic subunit
K: ATP-dependent Clp protease proteolytic subunit
L: ATP-dependent Clp protease proteolytic subunit
M: ATP-dependent Clp protease proteolytic subunit
N: ATP-dependent Clp protease proteolytic subunit
S: Green fluorescent protein
Y: Stringent starvation protein B
Z: Stringent starvation protein B
hetero molecules


Theoretical massNumber of molelcules
Total (without water)513,13322
Polymers510,18516
Non-polymers2,9476
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: microscopy
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

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ATP-dependent Clp protease ... , 2 types, 13 molecules ABCDEFHIJKLMN

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


Mass: 46414.848 Da / Num. of mol.: 6
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (E. coli) / Gene: clpX / Production host: Escherichia coli (E. coli) / References: UniProt: C3TLS7
#2: Protein
ATP-dependent Clp protease proteolytic subunit / / Caseinolytic protease / Endopeptidase Clp / Heat shock protein F21.5 / Protease Ti


Mass: 23468.869 Da / Num. of mol.: 7 / Fragment: UNP residues 16-207
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (E. coli) / Gene: clpP, lopP, b0437, JW0427 / Production host: Escherichia coli (E. coli) / References: UniProt: P0A6G7, endopeptidase Clp

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Protein , 2 types, 3 molecules SYZ

#3: Protein Green fluorescent protein /


Mass: 30855.428 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (E. coli) / Gene: GFP / Production host: Escherichia coli (E. coli) / References: UniProt: P42212
#4: Protein Stringent starvation protein B


Mass: 18279.352 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (E. coli) / Production host: Escherichia coli (E. coli) / References: UniProt: P0AFZ4

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Non-polymers , 2 types, 6 molecules

#5: Chemical
ChemComp-AGS / PHOSPHOTHIOPHOSPHORIC ACID-ADENYLATE ESTER / ATP-GAMMA-S / ADENOSINE 5'-(3-THIOTRIPHOSPHATE) / ADENOSINE 5'-(GAMMA-THIOTRIPHOSPHATE) / ADENOSINE-5'-DIPHOSPHATE MONOTHIOPHOSPHATE


Mass: 523.247 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: C10H16N5O12P3S / Feature type: SUBJECT OF INVESTIGATION / Comment: ATP-gamma-S, energy-carrying molecule analogue*YM
#6: Chemical ChemComp-ADP / ADENOSINE-5'-DIPHOSPHATE / Adenosine diphosphate


Mass: 427.201 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C10H15N5O10P2 / Feature type: SUBJECT OF INVESTIGATION / Comment: ADP, energy-carrying molecule*YM

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Details

Has ligand of interestY

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: ClpXP AAA protease complex bound with SspB and GFP-ssrA
Type: COMPLEX / Entity ID: #1-#4 / Source: RECOMBINANT
Source (natural)Organism: Escherichia coli (E. coli)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 7.5
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R2/1
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 2500 nm / Nominal defocus min: 1000 nm / Cs: 2.7 mm / C2 aperture diameter: 50 µm
Image recordingElectron dose: 75.98 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k)

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Processing

EM software
IDNameVersionCategory
1RELION3.1particle selection
4CTFFIND4CTF correction
7Coot9.4model fitting
9PHENIXmodel refinement
10RELION3.1initial Euler assignment
11RELION3.1final Euler assignment
CTF correctionType: PHASE FLIPPING ONLY
3D reconstructionResolution: 3.7 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 236728 / Symmetry type: POINT
Atomic model buildingProtocol: RIGID BODY FIT / Space: REAL
Atomic model buildingPDB-ID: 6WRF

6wrf

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