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- EMDB-0965: AAA+ ATPase, ClpL from Streptococcus pneumoniae - ATP bound -

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Basic information

Entry
Database: EMDB / ID: EMD-0965
TitleAAA+ ATPase, ClpL from Streptococcus pneumoniae - ATP bound
Map data
Sample
  • Organelle or cellular component: ClpL Trap(E193A/E526A):ATP-bound
    • Protein or peptide: ATP-dependent Clp protease, ATP-binding subunit
KeywordsAAA+ ATPase / Chaperone / Streptococcus pneumoniae
Function / homology
Function and homology information


peptidase activity / ATP hydrolysis activity / ATP binding
Similarity search - Function
UVR domain / UVR domain profile. / ClpA/B, conserved site 2 / Chaperonins clpA/B signature 2. / ClpA/ClpB, AAA lid domain / AAA lid domain / ClpA/B family / Clp ATPase, C-terminal / AAA domain (Cdc48 subfamily) / C-terminal, D2-small domain, of ClpB protein ...UVR domain / UVR domain profile. / ClpA/B, conserved site 2 / Chaperonins clpA/B signature 2. / ClpA/ClpB, AAA lid domain / AAA lid domain / ClpA/B family / Clp ATPase, C-terminal / AAA domain (Cdc48 subfamily) / C-terminal, D2-small domain, of ClpB protein / C-terminal, D2-small domain, of ClpB protein / ATPase family associated with various cellular activities (AAA) / 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
Similarity search - Component
Biological speciesStreptococcus pneumoniae (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 6.33 Å
AuthorsKim G / Lee SG
Funding support Korea, Republic Of, 4 items
OrganizationGrant numberCountry
National Research Foundation (NRF, Korea)NRF-2018R1A2B6004367 Korea, Republic Of
National Research Foundation (NRF, Korea)NRF-2017R1A5A1014560 Korea, Republic Of
Rural Development AdministrationPJ01367602 Korea, Republic Of
National Research Foundation (NRF, Korea)IBS-R030-C1 Korea, Republic Of
CitationJournal: FASEB J / Year: 2020
Title: ClpL is a functionally active tetradecameric AAA+ chaperone, distinct from hexameric/dodecameric ones.
Authors: Gyuhee Kim / Seong-Gyu Lee / Seungsu Han / Jaeeun Jung / Hyeong Seop Jeong / Jae-Kyung Hyun / Dong-Kwon Rhee / Ho Min Kim / Sangho Lee /
Abstract: AAA+ (ATPases associated with diverse cellular activities) chaperones are involved in a plethora of cellular activities to ensure protein homeostasis. The function of AAA+ chaperones is mostly ...AAA+ (ATPases associated with diverse cellular activities) chaperones are involved in a plethora of cellular activities to ensure protein homeostasis. The function of AAA+ chaperones is mostly modulated by their hexameric/dodecameric quaternary structures. Here we report the structural and biochemical characterizations of a tetradecameric AAA+ chaperone, ClpL from Streptococcus pneumoniae. ClpL exists as a tetradecamer in solution in the presence of ATP. The cryo-EM structure of ClpL at 4.5 Å resolution reveals a striking tetradecameric arrangement. Solution structures of ClpL derived from small-angle X-ray scattering data suggest that the tetradecameric ClpL could assume a spiral conformation found in active hexameric/dodecameric AAA+ chaperone structures. Vertical positioning of the middle domain accounts for the head-to-head arrangement of two heptameric rings. Biochemical activity assays with site-directed mutagenesis confirmed the critical roles of residues both in the integrity of the tetradecameric arrangement and activities of ClpL. Non-conserved Q321 and R670 are crucial in the heptameric ring assembly of ClpL. These results establish that ClpL is a functionally active tetradecamer, clearly distinct from hexameric/dodecameric AAA+ chaperones.
History
DepositionJan 20, 2020-
Header (metadata) releaseJan 27, 2021-
Map releaseJan 27, 2021-
UpdateMar 27, 2024-
Current statusMar 27, 2024Processing site: PDBj / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.2
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.2
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6lsy
  • Surface level: 0.2
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_0965.png

Downloads & links

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Map

FileDownload / File: emd_0965.map.gz / Format: CCP4 / Size: 52.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 1.4 Å
Density
Contour LevelBy AUTHOR: 0.2 / Movie #1: 0.2
Minimum - Maximum-1.5161932 - 2.406283
Average (Standard dev.)0.00456213 (±0.0966632)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions240240240
Spacing240240240
CellA=B=C: 336.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.41.41.4
M x/y/z240240240
origin x/y/z0.0000.0000.000
length x/y/z336.000336.000336.000
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS240240240
D min/max/mean-1.5162.4060.005

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Supplemental data

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Sample components

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Entire : ClpL Trap(E193A/E526A):ATP-bound

EntireName: ClpL Trap(E193A/E526A):ATP-bound
Components
  • Organelle or cellular component: ClpL Trap(E193A/E526A):ATP-bound
    • Protein or peptide: ATP-dependent Clp protease, ATP-binding subunit

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Supramolecule #1: ClpL Trap(E193A/E526A):ATP-bound

SupramoleculeName: ClpL Trap(E193A/E526A):ATP-bound / type: organelle_or_cellular_component / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Streptococcus pneumoniae (bacteria)
Molecular weightTheoretical: 1.1 MDa

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

MacromoleculeName: ATP-dependent Clp protease, ATP-binding subunit / type: protein_or_peptide / ID: 1 / Number of copies: 14 / Enantiomer: LEVO
Source (natural)Organism: Streptococcus pneumoniae (bacteria)
Molecular weightTheoretical: 77.599094 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MNNNFNNFNN MDDLFNQLMG GMRGYSSENR RYLINGREVT PEEFAYYRAT GQLPGNAESD VQMQQQASGM KQDGVLAKLG RNLTAEARE GKLDPVIGRN KEIQEASEIL SRRTKNNPVL VGDAGVGKTA VVEGLAQAIV NGDVPAAIKN KEIVSIDISG L EAGTQYRG ...String:
MNNNFNNFNN MDDLFNQLMG GMRGYSSENR RYLINGREVT PEEFAYYRAT GQLPGNAESD VQMQQQASGM KQDGVLAKLG RNLTAEARE GKLDPVIGRN KEIQEASEIL SRRTKNNPVL VGDAGVGKTA VVEGLAQAIV NGDVPAAIKN KEIVSIDISG L EAGTQYRG SFEENVQNLV NEVKEAGNII LFFDAIHQIL GAGSTGGDSG SKGLADILKP ALSRGELTVI GATTQDEYRN TI LKNAALA RRFNEVKVNA PSAENTFKIL QGIRDLYQQH HNVILPDEVL KAAVDYSVQY IPQRSLPDKA IDLVDVTAAH LAA QHPVTD VHAVEREIET EKDKQEKAVE AEDFEAALNY KTRIAELERK IENHTEDMKV TASVNDVAES VERMTGIPVS QMGA SDIER LKDMAHRLQD KVIGQDKAVE VVARAICRNR AGFDEGNRPI GNFLFVGSTG VGKTELAKQL ALDMFGTQDA IIRLD MSEY SDRTAVSKLI GTTAGYVGYD DNSNTLTERV RRNPYSIILL DAIEKADPQV ITLLLQVLDD GRLTDGQGNT VNFKNT VII ATSNAGFGYE ANLTEDADKP ELMDRLKPFF RPEFLNRFNA VIEFSHLTKE DLSKIVDLML AEVNQTLAKK DIDLVVS QA AKDYITEEGY DEVMGVRPLR RVVEQEIRDK VTDFHLDHLD AKHLEADMED GVLVIREKV

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

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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

Concentration0.5 mg/mL
BufferpH: 7.5
Component:
ConcentrationFormulaName
50.0 mMC8H18N2O4SHydroxyethyl piperazine Ethane Sulfonicacid
100.0 mMKClPotassium chloride
20.0 mMMgCl2Magnesium chloride
6.0 mMC10H16N5O13P3Adenosine triphosphate
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 300
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 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: DARK FIELD
Image recordingFilm or detector model: FEI FALCON III (4k x 4k) / Average electron dose: 60.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 104148
Startup modelType of model: PDB ENTRY
PDB model - PDB ID:

1qvr

Initial angle assignmentType: ANGULAR RECONSTITUTION
Final 3D classificationNumber classes: 100 / Software - Name: RELION (ver. 2.1)
Final angle assignmentType: ANGULAR RECONSTITUTION
Final reconstructionApplied symmetry - Point group: D7 (2x7 fold dihedral) / Resolution.type: BY AUTHOR / Resolution: 6.33 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 52689

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Atomic model buiding 1

RefinementProtocol: AB INITIO MODEL
Output model

PDB-6lsy:
AAA+ ATPase, ClpL from Streptococcus pneumoniae - ATP bound

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