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- EMDB-5609: Structural dynamics and inter-ring communication of the MecA-ClpC... -

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Basic information

Entry
Database: EMDB / ID: EMD-5609
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,E618A) complex with ATP state
Sample
  • Sample: MecA-ClpC (E280A,E618A) with ATP
  • 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 repeat (R) N-terminal domain / : / Clp, repeat (R) domain / Clp repeat (R) domain profile. / Clp, N-terminal domain superfamily / ClpA/B family / Clp ATPase, C-terminal / C-terminal, D2-small domain, of ClpB protein / C-terminal, D2-small domain, of ClpB protein / AAA domain (Cdc48 subfamily) / 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: 10.0 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-3j3u
  • Surface level: 1.5
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_5609_1.jpg

Downloads & links

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Map

FileDownload / File: emd_5609.map.gz / Format: CCP4 / Size: 12.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationReconstruction of MecA-ClpC(E280A,E618A) complex with ATP state
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-4.93566799 - 7.70951939
Average (Standard dev.)0.00010409 (±0.99860597)
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-4.9367.7100.000

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Supplemental data

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Sample components

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Entire : MecA-ClpC (E280A,E618A) with ATP

EntireName: MecA-ClpC (E280A,E618A) with ATP
Components
  • Sample: MecA-ClpC (E280A,E618A) with ATP
  • Protein or peptide: MecA
  • Protein or peptide: ClpC

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Supramolecule #1000: MecA-ClpC (E280A,E618A) with ATP

SupramoleculeName: MecA-ClpC (E280A,E618A) with ATP / type: sample / ID: 1000
Details: Mutant was generated by introducing double Walker B mutations: E280A and E618A. The mutant ClpC can bind ATP but not be able to hydrolyze ATP.
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
DateSep 9, 2010
Image recordingCategory: CCD / Film or detector model: FEI EAGLE (4k x 4k) / Number real images: 530 / 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: 4.0 µm / Nominal defocus min: 1.7 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: 10.0 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: SPIDER / Number images used: 36688

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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. Structure, 16, 673-683
RefinementSpace: REAL / Protocol: FLEXIBLE FIT / Target criteria: Cross-correlation
Output model

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

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