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- PDB-3j3u: Structural dynamics of the MecA-ClpC complex revealed by cryo-EM -

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Basic information

Entry
Database: PDB / ID: 3j3u
TitleStructural dynamics of the MecA-ClpC complex revealed by cryo-EM
Components
  • Adapter protein MecA 1
  • Negative regulator of genetic competence ClpC/MecB
KeywordsCHAPERONE / ClpC / MecA / AAA+ ATPase / PROTEIN unfolding
Function / homology
Function and homology information


negative regulation of establishment of competence for transformation / negative regulation of sporulation resulting in formation of a cellular spore / establishment of competence for transformation / sporulation resulting in formation of a cellular spore / protein-macromolecule adaptor activity / ATP hydrolysis activity / ATP binding
Similarity search - Function
MecA, C-terminal domain superfamily / Negative regulator of genetic competence, MecA / Negative regulator of genetic competence (MecA) / UVR domain / UVR domain profile. / ClpA/B, conserved site 2 / Chaperonins clpA/B signature 2. / ClpA/B, conserved site 1 / Chaperonins clpA/B signature 1. / ClpA/ClpB, AAA lid domain ...MecA, C-terminal domain superfamily / Negative regulator of genetic competence, MecA / Negative regulator of genetic competence (MecA) / UVR domain / UVR domain profile. / ClpA/B, conserved site 2 / Chaperonins clpA/B signature 2. / ClpA/B, conserved site 1 / Chaperonins clpA/B signature 1. / ClpA/ClpB, AAA lid domain / AAA lid domain / Clp amino terminal domain, pathogenicity island component / : / Clp, repeat (R) domain / Clp repeat (R) domain profile. / Clp, N-terminal domain superfamily / 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
Negative regulator of genetic competence ClpC/MecB / Adapter protein MecA 1
Similarity search - Component
Biological speciesBacillus subtilis (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 10 Å
AuthorsLiu, J. / Mei, Z. / Li, N. / Qi, Y. / Xu, Y. / Shi, Y. / Wang, F. / Lei, J. / Gao, N.
CitationJournal: J Biol Chem / Year: 2013
Title: Structural dynamics of the MecA-ClpC complex: a type II AAA+ protein unfolding machine.
Authors: Jing Liu / Ziqing Mei / Ningning Li / Yutao Qi / Yanji Xu / Yigong Shi / Feng Wang / Jianlin Lei / Ning Gao /
Abstract: The MecA-ClpC complex is a bacterial type II AAA(+) molecular machine responsible for regulated unfolding of substrates, such as transcription factors ComK and ComS, and targeting them to ClpP for ...The MecA-ClpC complex is a bacterial type II AAA(+) molecular machine responsible for regulated unfolding of substrates, such as transcription factors ComK and ComS, and targeting them to ClpP for degradation. The six subunits of the MecA-ClpC complex form a closed barrel-like structure, featured with three stacked rings and a hollow passage, where substrates are threaded and translocated through successive pores. Although the general concepts of how polypeptides are unfolded and translocated by internal pore loops of AAA(+) proteins have long been conceived, the detailed mechanistic model remains elusive. With cryoelectron microscopy, we captured four different structures of the MecA-ClpC complexes. These complexes differ in the nucleotide binding states of the two AAA(+) rings and therefore might presumably reflect distinctive, representative snapshots from a dynamic unfolding cycle of this hexameric complex. Structural analysis reveals that nucleotide binding and hydrolysis modulate the hexameric complex in a number of ways, including the opening of the N-terminal ring, the axial and radial positions of pore loops, the compactness of the C-terminal ring, as well as the relative rotation between the two nucleotide-binding domain rings. More importantly, our structural and biochemical data indicate there is an active allosteric communication between the two AAA(+) rings and suggest that concerted actions of the two AAA(+) rings are required for the efficiency of the substrate unfolding and translocation. These findings provide important mechanistic insights into the dynamic cycle of the MecA-ClpC unfoldase and especially lay a foundation toward the complete understanding of the structural dynamics of the general type II AAA(+) hexamers.
History
DepositionApr 18, 2013Deposition site: RCSB / Processing site: PDBJ
Revision 1.0May 15, 2013Provider: repository / Type: Initial release
Revision 1.1Aug 28, 2013Group: Database references
Revision 1.2Oct 22, 2014Group: Other
Revision 1.3Jan 21, 2015Group: Other
Revision 1.4Dec 18, 2019Group: Data collection / Database references / Structure summary
Category: database_2 / em_image_scans ...database_2 / em_image_scans / em_software / struct_keywords / struct_ref_seq_dif
Item: _em_software.fitting_id / _em_software.image_processing_id ..._em_software.fitting_id / _em_software.image_processing_id / _struct_keywords.pdbx_keywords / _struct_ref_seq_dif.details
Revision 1.5Mar 20, 2024Group: Data collection / Database references / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / em_3d_fitting_list / pdbx_initial_refinement_model
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id / _em_3d_fitting_list.source_name / _em_3d_fitting_list.type

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Structure visualization

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Assembly

Deposited unit
1: Adapter protein MecA 1
2: Adapter protein MecA 1
3: Adapter protein MecA 1
4: Adapter protein MecA 1
5: Adapter protein MecA 1
6: Adapter protein MecA 1
A: Negative regulator of genetic competence ClpC/MecB
B: Negative regulator of genetic competence ClpC/MecB
C: Negative regulator of genetic competence ClpC/MecB
D: Negative regulator of genetic competence ClpC/MecB
E: Negative regulator of genetic competence ClpC/MecB
F: Negative regulator of genetic competence ClpC/MecB


Theoretical massNumber of molelcules
Total (without water)695,56412
Polymers695,56412
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

#1: Protein
Adapter protein MecA 1


Mass: 25791.617 Da / Num. of mol.: 6
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Bacillus subtilis (bacteria) / Strain: 168 / Gene: BSU11520, mecA / Plasmid: pET-27a / Production host: Escherichia coli (E. coli) / Strain (production host): BL21 / References: UniProt: P37958
#2: Protein
Negative regulator of genetic competence ClpC/MecB


Mass: 90135.648 Da / Num. of mol.: 6 / Mutation: E280A, E618A
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Bacillus subtilis (bacteria) / Strain: 168 / Gene: BSU00860, clpC, mecB / Plasmid: pET-27a / Production host: Escherichia coli (E. coli) / Strain (production host): BL21 / References: UniProt: P37571

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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: MecA-ClpC (E280A,E618A) with ATP / Type: COMPLEX / Details: hexamer
Molecular weightValue: 0.6 MDa / Experimental value: NO
Buffer solutionName: 50mM kCl, 10mM Tris-HCL, 2mM MgCl2, 2mM ATP / pH: 7.5 / Details: 50mM kCl, 10mM Tris-HCL, 2mM MgCl2, 2mM ATP
SpecimenConc.: 0.03 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Temp: 90 K / Humidity: 100 % / Method: Blot for 2 seconds before plunging

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS / Date: Sep 9, 2010
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 59000 X / Nominal defocus max: 4000 nm / Nominal defocus min: 1700 nm / Camera length: 0 mm
Specimen holderSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Temperature: 90 K / Tilt angle max: 0 ° / Tilt angle min: 0 °
Image recordingElectron dose: 20 e/Å2 / Film or detector model: FEI EAGLE (4k x 4k)
Radiation wavelengthRelative weight: 1

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Processing

EM software
IDNameCategory
1MDFFmodel fitting
2SPIDER3D reconstruction
CTF correctionDetails: each defocus group on 3D level
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionMethod: Reference Projections / Resolution: 10 Å / Resolution method: FSC 0.5 CUT-OFF / Num. of particles: 36688 / Details: (Single particle--Applied symmetry: C6) / Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT / Space: REAL / Target criteria: Cross-correlation
Details: REFINEMENT PROTOCOL--flexible DETAILS--Protocol- Initial local fitting was done using Chimera and then MDFF was used for flexible fitting. ref- Trabuco, L.G., Villa, E., Mitra, K., Frank, J. ...Details: REFINEMENT PROTOCOL--flexible DETAILS--Protocol- Initial local fitting was done using Chimera and then MDFF was used for flexible fitting. ref- Trabuco, L.G., Villa, E., Mitra, K., Frank, J. and Schulten, K. (2008) Flexible fitting of atomic structures into electron microscopy maps using molecular dynamics. Structure, 16, 673-683
Atomic model buildingPDB-ID: 3PXI

3pxi


Accession code: 3PXI / Source name: PDB / Type: experimental model
Refinement stepCycle: LAST
ProteinNucleic acidLigandSolventTotal
Num. atoms41838 0 0 0 41838

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