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- EMDB-5607: Structural dynamics and inter-ring communication of the MecA-ClpC... -

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Basic information

Entry
Database: EMDB / ID: EMD-5607
TitleStructural dynamics and inter-ring communication of the MecA-ClpC complex during active substrate unfolding and translocation revealed by cryo-EM
Map dataReconstruction of MecA-ClpC(E280A) with ATP, MW-ATP
Sample
  • Sample: MecA-ClpC(E280A)
  • Protein or peptide: MecA
  • Protein or peptide: ClpC
Keywordsunfolding / AAA+ ATPase / MecA / ClpC
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)
Methodsingle particle reconstruction / cryo EM / Resolution: 9.0 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
DepositionMar 20, 2013-
Header (metadata) releaseMay 8, 2013-
Map releaseMay 15, 2013-
UpdateAug 28, 2013-
Current statusAug 28, 2013Processing site: PDBj / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 1.5
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 1.5
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-3j3t
  • Surface level: 1.5
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_5607_1.jpg

Downloads & links

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Map

FileDownload / File: emd_5607.map.gz / Format: CCP4 / Size: 12.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationReconstruction of MecA-ClpC(E280A) with ATP, MW-ATP
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.5 Å/pix.
x 150 pix.
= 225. Å		1.5 Å/pix.
x 150 pix.
= 225. Å		1.5 Å/pix.
x 150 pix.
= 225. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.5 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 1.5 / Movie #1: 1.5
Minimum - Maximum-4.42900324 - 8.11595058
Average (Standard dev.)0.0016236 (±0.99658817)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-74-74-74
Dimensions150150150
Spacing150150150
CellA=B=C: 225.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.51.51.5
M x/y/z150150150
origin x/y/z0.0000.0000.000
length x/y/z225.000225.000225.000
α/β/γ90.00090.00090.000
start NX/NY/NZ-132-122-147
NX/NY/NZ250274261
MAP C/R/S123
start NC/NR/NS-74-74-74
NC/NR/NS150150150
D min/max/mean-4.4298.1160.002

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Supplemental data

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Sample components

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Entire : MecA-ClpC(E280A)

EntireName: MecA-ClpC(E280A)
Components
  • Sample: MecA-ClpC(E280A)
  • Protein or peptide: MecA
  • Protein or peptide: ClpC

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Supramolecule #1000: MecA-ClpC(E280A)

SupramoleculeName: MecA-ClpC(E280A) / type: sample / ID: 1000
Details: Mutant was generated by introducing Walker B mutations: E280A. The mutant ClpC can bind ATP but not be able to hydrolyze ATP.The protein complex was diluted with buffer, followed by an ...Details: Mutant was generated by introducing Walker B mutations: E280A. The mutant ClpC can bind ATP but not be able to hydrolyze ATP.The protein complex was diluted with buffer, followed by an incubation in water bath at 300K for 40 minutes.
Oligomeric state: Hexamer of ClpC with 6 bound MecA / Number unique components: 2
Molecular weightExperimental: 600 KDa / Theoretical: 600 KDa

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Macromolecule #1: MecA

MacromoleculeName: MecA / type: protein_or_peptide / ID: 1 / Number of copies: 6 / Recombinant expression: Yes
Source (natural)Organism: Bacillus subtilis (bacteria) / Strain: 168
Recombinant expressionOrganism: Escherichia coli (E. coli) / Recombinant strain: BL21 / Recombinant plasmid: PET-27A

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Macromolecule #2: ClpC

MacromoleculeName: ClpC / type: protein_or_peptide / ID: 2 / Number of copies: 6 / Oligomeric state: Hexamer / Recombinant expression: Yes
Source (natural)Organism: Bacillus subtilis (bacteria) / Strain: 168
Recombinant expressionOrganism: Escherichia coli (E. coli) / Recombinant strain: BL21 / Recombinant plasmid: PET-27A

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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

Concentration0.03 mg/mL
BufferpH: 7.5 / Details: 50mM kCl, 10mM Tris-HCL,2mM MgCl2, 2mM ATP
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 90 K / Instrument: FEI VITROBOT MARK IV / Method: Blot for 2 seconds before plunging

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Electron microscopy

MicroscopeFEI TITAN KRIOS
DateMar 9, 2011
Image recordingCategory: CCD / Film or detector model: FEI EAGLE (4k x 4k) / Number real images: 994 / Average electron dose: 20 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 3.0 µm / Nominal defocus min: 1.5 µm / Nominal magnification: 59000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

CTF correctionDetails: each defocus group on 3D level
Final reconstructionAlgorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 9.0 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: SPIDER / Number images used: 45514

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Atomic model buiding 1

Initial modelPDB ID:

3pxi

SoftwareName: MDFF
DetailsProtocol: 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.
RefinementSpace: REAL / Protocol: FLEXIBLE FIT / Target criteria: Cross-correlation
Output model

PDB-3j3t:
Structural dynamics of the MecA-ClpC complex revealed by cryo-EM

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