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

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Basic information

Entry
Database: PDB / ID: 6lsy
TitleAAA+ ATPase, ClpL from Streptococcus pneumoniae - ATP bound
ComponentsATP-dependent Clp protease, ATP-binding subunit
KeywordsCHAPERONE / AAA+ ATPase / 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)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 6.33 Å
AuthorsKim, G. / Lee, S.G. / Han, S. / Jung, J. / Jeong, H.S. / Hyun, J.K. / Rhee, D.K. / Kim, H.M. / Lee, S.
Funding support Korea, Republic Of, 4items
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, 2020Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Jan 27, 2021Provider: repository / Type: Initial release
Revision 1.1Mar 27, 2024Group: Data collection / Database references / Category: chem_comp_atom / chem_comp_bond / database_2
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession

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Structure visualization

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Assembly

Deposited unit
A: ATP-dependent Clp protease, ATP-binding subunit
B: ATP-dependent Clp protease, ATP-binding subunit
C: ATP-dependent Clp protease, ATP-binding subunit
D: ATP-dependent Clp protease, ATP-binding subunit
E: ATP-dependent Clp protease, ATP-binding subunit
F: ATP-dependent Clp protease, ATP-binding subunit
G: ATP-dependent Clp protease, ATP-binding subunit
H: ATP-dependent Clp protease, ATP-binding subunit
I: ATP-dependent Clp protease, ATP-binding subunit
J: ATP-dependent Clp protease, ATP-binding subunit
K: ATP-dependent Clp protease, ATP-binding subunit
L: ATP-dependent Clp protease, ATP-binding subunit
M: ATP-dependent Clp protease, ATP-binding subunit
N: ATP-dependent Clp protease, ATP-binding subunit


Theoretical massNumber of molelcules
Total (without water)1,086,38714
Polymers1,086,38714
Non-polymers00
Water0
1


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

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Components

#1: Protein
ATP-dependent Clp protease, ATP-binding subunit / ClpL Trap


Mass: 77599.094 Da / Num. of mol.: 14 / Mutation: E193A/E526A
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Streptococcus pneumoniae (bacteria) / Gene: clpL, SPRM200_0307 / Production host: Escherichia coli (E. coli) / References: UniProt: A0A2U3RY34

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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: ClpL Trap(E193A/E526A):ATP-bound / Type: ORGANELLE OR CELLULAR COMPONENT / Entity ID: all / Source: RECOMBINANT
Molecular weightValue: 1.1 MDa / Experimental value: YES
Source (natural)Organism: Streptococcus pneumoniae (bacteria)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 7.5
Buffer component
IDConc.NameFormulaBuffer-ID
150 mMHydroxyethyl piperazine Ethane SulfonicacidC8H18N2O4S1
2100 mMPotassium chlorideKCl1
320 mMMagnesium chlorideMgCl21
46 mMAdenosine triphosphateC10H16N5O13P31
SpecimenConc.: 0.5 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 K

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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: DARK FIELD
Image recordingElectron dose: 60 e/Å2 / Film or detector model: FEI FALCON III (4k x 4k)

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Processing

Software
NameVersionClassification
phenix.real_space_refine1.13_2998refinement
PHENIX1.13_2998refinement
EM software
IDNameVersionCategory
4CTFFIND4CTF correction
11RELION2.1classification
CTF correctionType: NONE
Particle selectionNum. of particles selected: 104148
SymmetryPoint symmetry: D7 (2x7 fold dihedral)
3D reconstructionResolution: 6.33 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 52689 / Symmetry type: POINT
Atomic model buildingProtocol: AB INITIO MODEL
RefinementCross valid method: NONE / Stereochemistry target values: GeoStd + Monomer Library
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0046136024
ELECTRON MICROSCOPYf_angle_d0.9428246554
ELECTRON MICROSCOPYf_chiral_restr0.052210794
ELECTRON MICROSCOPYf_plane_restr0.003420734
ELECTRON MICROSCOPYf_dihedral_angle_d4.073853788

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