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- EMDB-4940: ClpB (DWB mutant) bound to casein in presence of ATPgammaS - stat... -

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Basic information

Entry
Database: EMDB / ID: EMD-4940
TitleClpB (DWB mutant) bound to casein in presence of ATPgammaS - state WT-1
Map datamain cryoEM map of ClpB-DWB casein in presence of ATPgS -state WT-1
Sample
  • Complex: ClpB-DWB bound to casein in presence of ATPgammaS
    • Complex: ClpB-DWB
      • Protein or peptide: Chaperone protein ClpB
    • Complex: casein
      • Protein or peptide: casein
  • Ligand: PHOSPHOTHIOPHOSPHORIC ACID-ADENYLATE ESTER
  • Ligand: ADENOSINE-5'-DIPHOSPHATE
Function / homology
Function and homology information


response to heat / protein refolding / ATP hydrolysis activity / ATP binding / cytoplasm
Similarity search - Function
Chaperonin ClpB / 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 profile. / Clp, repeat (R) domain ...Chaperonin ClpB / 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 profile. / Clp, repeat (R) domain / 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
Chaperone protein ClpB
Similarity search - Component
Biological speciesEscherichia coli (E. coli) / Bos taurus (cattle) / Escherichia coli HVH 50 (4-2593475) (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 6.2 Å
AuthorsDeville C / Saibil HR
Funding support United Kingdom, 3 items
OrganizationGrant numberCountry
Wellcome Trust106252 United Kingdom
Wellcome Trust079605 United Kingdom
Wellcome Trust101488 United Kingdom
CitationJournal: Cell Rep / Year: 2019
Title: Two-Step Activation Mechanism of the ClpB Disaggregase for Sequential Substrate Threading by the Main ATPase Motor.
Authors: Célia Deville / Kamila Franke / Axel Mogk / Bernd Bukau / Helen R Saibil /
Abstract: AAA+ proteins form asymmetric hexameric rings that hydrolyze ATP and thread substrate proteins through a central channel via mobile substrate-binding pore loops. Understanding how ATPase and ...AAA+ proteins form asymmetric hexameric rings that hydrolyze ATP and thread substrate proteins through a central channel via mobile substrate-binding pore loops. Understanding how ATPase and threading activities are regulated and intertwined is key to understanding the AAA+ protein mechanism. We studied the disaggregase ClpB, which contains tandem ATPase domains (AAA1, AAA2) and shifts between low and high ATPase and threading activities. Coiled-coil M-domains repress ClpB activity by encircling the AAA1 ring. Here, we determine the mechanism of ClpB activation by comparing ATPase mechanisms and cryo-EM structures of ClpB wild-type and a constitutively active ClpB M-domain mutant. We show that ClpB activation reduces ATPase cooperativity and induces a sequential mode of ATP hydrolysis in the AAA2 ring, the main ATPase motor. AAA1 and AAA2 rings do not work synchronously but in alternating cycles. This ensures high grip, enabling substrate threading via a processive, rope-climbing mechanism.
History
DepositionMay 7, 2019-
Header (metadata) releaseJul 3, 2019-
Map releaseJul 3, 2019-
UpdateDec 2, 2020-
Current statusDec 2, 2020Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.02
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.02
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  • Surface view with fitted model
  • Atomic models: PDB-6rn2
  • Surface level: 0.02
  • Imaged by UCSF Chimera
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  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-6rn2
  • Imaged by Jmol
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_4940.png

Downloads & links

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Map

FileDownload / File: emd_4940.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationmain cryoEM map of ClpB-DWB casein in presence of ATPgS -state WT-1
Voxel sizeX=Y=Z: 1.04 Å
Density
Contour LevelBy AUTHOR: 0.01 / Movie #1: 0.02
Minimum - Maximum-0.03225943 - 0.097224854
Average (Standard dev.)0.000562657 (±0.005748253)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 266.24 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.041.041.04
M x/y/z256256256
origin x/y/z0.0000.0000.000
length x/y/z266.240266.240266.240
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ480480480
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS256256256
D min/max/mean-0.0320.0970.001

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Supplemental data

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Mask #1

Fileemd_4940_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: non postprocessed cryoEM map of ClpB-DWB casein in...

Fileemd_4940_additional_1.map
Annotationnon postprocessed cryoEM map of ClpB-DWB casein in presence of ATPgS -state WT-1
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: cryoEM map filtered according to local resolution of...

Fileemd_4940_additional_2.map
AnnotationcryoEM map filtered according to local resolution of ClpB-DWB casein in presence of ATPgS -state WT-1
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: odd cryoEM half map of ClpB-DWB casein in...

Fileemd_4940_half_map_1.map
Annotationodd cryoEM half map of ClpB-DWB casein in presence of ATPgS -state WT-1
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: even cryoEM half map of ClpB-DWB casein in...

Fileemd_4940_half_map_2.map
Annotationeven cryoEM half map of ClpB-DWB casein in presence of ATPgS -state WT-1
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : ClpB-DWB bound to casein in presence of ATPgammaS

EntireName: ClpB-DWB bound to casein in presence of ATPgammaS
Components
  • Complex: ClpB-DWB bound to casein in presence of ATPgammaS
    • Complex: ClpB-DWB
      • Protein or peptide: Chaperone protein ClpB
    • Complex: casein
      • Protein or peptide: casein
  • Ligand: PHOSPHOTHIOPHOSPHORIC ACID-ADENYLATE ESTER
  • Ligand: ADENOSINE-5'-DIPHOSPHATE

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Supramolecule #1: ClpB-DWB bound to casein in presence of ATPgammaS

SupramoleculeName: ClpB-DWB bound to casein in presence of ATPgammaS / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#2
Molecular weightTheoretical: 570 KDa

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Supramolecule #2: ClpB-DWB

SupramoleculeName: ClpB-DWB / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1
Source (natural)Organism: Escherichia coli (E. coli)
Recombinant expressionOrganism: Escherichia coli (E. coli)

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Supramolecule #3: casein

SupramoleculeName: casein / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #2
Source (natural)Organism: Bos taurus (cattle)

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Macromolecule #1: Chaperone protein ClpB

MacromoleculeName: Chaperone protein ClpB / type: protein_or_peptide / ID: 1 / Number of copies: 6 / Enantiomer: LEVO
Source (natural)Organism: Escherichia coli HVH 50 (4-2593475) (bacteria) / Strain: 4-2593475
Molecular weightTheoretical: 95.734172 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MRLDRLTNKF QLALADAQSL ALGHDNQFIE PLHLMSALLN QEGGSVSPLL TSAGINAGQL RTDINQALNR LPQVEGTGGD VQPSQDLVR VLNLCDKLAQ KRGDNFISSE LFVLAALESR GTLADILKAA GATTANITQA IEQMRGGESV NDQGAEDQRQ A LKKYTIDL ...String:
MRLDRLTNKF QLALADAQSL ALGHDNQFIE PLHLMSALLN QEGGSVSPLL TSAGINAGQL RTDINQALNR LPQVEGTGGD VQPSQDLVR VLNLCDKLAQ KRGDNFISSE LFVLAALESR GTLADILKAA GATTANITQA IEQMRGGESV NDQGAEDQRQ A LKKYTIDL TERAEQGKLD PVIGRDEEIR RTIQVLQRRT KNNPVLIGEP GVGKTAIVEG LAQRIINGEV PEGLKGRRVL AL DMGALVA GAKYRGEFEE RLKGVLNDLA KQEGNVILFI DALHTMVGAG KADGAMDAGN MLKPALARGE LHCVGATTLD EYR QYIEKD AALERRFQKV FVAEPSVEDT IAILRGLKER YELHHHVQIT DPAIVAAATL SHRYIADRQL PDKAIDLIDE AASS IRMQI DSKPEELDRL DRRIIQLKLE QQALMKESDE ASKKRLDMLN EELSDKERQY SELEEEWKAE KASLSGTQTI KAELE QAKI AIEQARRVGD LARMSELQYG KIPELEKQLE AATQLEGKTM RLLRNKVTDA EIAEVLARWT GIPVSRMMES EREKLL RME QELHHRVIGQ NEAVDAVSNA IRRSRAGLAD PNRPIGSFLF LGPTGVGKTE LCKALANFMF DSDEAMVRID MSEFMEK HS VSRLVGAPPG YVGYEEGGYL TEAVRRRPYS VILLDAVEKA HPDVFNILLQ VLDDGRLTDG QGRTVDFRNT VVIMTSNL G SDLIQERFGE LDYAHMKELV LGVVSHNFRP EFINRIDEVV VFHPLGEQHI ASIAQIQLKR LYKRLEERGY EIHISDEAL KLLSENGYDP VYGARPLKRA IQQQIENPLA QQILSGELVP GKVIRLEVNE DRIVAVQH

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Macromolecule #2: casein

MacromoleculeName: casein / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Escherichia coli (E. coli)
Molecular weightTheoretical: 1.723883 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
AAAAAAAAAA AAAAAAAAAA AAAA

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Macromolecule #3: PHOSPHOTHIOPHOSPHORIC ACID-ADENYLATE ESTER

MacromoleculeName: PHOSPHOTHIOPHOSPHORIC ACID-ADENYLATE ESTER / type: ligand / ID: 3 / Number of copies: 9 / Formula: AGS
Molecular weightTheoretical: 523.247 Da
Chemical component information

ChemComp-AGS:
PHOSPHOTHIOPHOSPHORIC ACID-ADENYLATE ESTER / ATP-gamma-S, energy-carrying molecule analogue*YM

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Macromolecule #4: ADENOSINE-5'-DIPHOSPHATE

MacromoleculeName: ADENOSINE-5'-DIPHOSPHATE / type: ligand / ID: 4 / Number of copies: 2 / Formula: ADP
Molecular weightTheoretical: 427.201 Da
Chemical component information

ChemComp-ADP:
ADENOSINE-5'-DIPHOSPHATE / ADP, energy-carrying molecule*YM / Adenosine diphosphate

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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

Concentration1.6 mg/mL
BufferpH: 7.5
Component:
ConcentrationFormulaName
25.0 mMTris-HClTrisTris-HClTris
25.0 mMKClpotassium chloride
10.0 mMMgCl2magnesium chloride
2.0 mMATPgammaSadenosine5o3thiotriphosphate
1.0 mMDTTdithiothreitol
GridModel: Quantifoil, UltrAuFoil / Material: GOLD / Mesh: 300 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Atmosphere: AIR
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 295 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 opticsC2 aperture diameter: 70.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 3.5 µm / Nominal defocus min: 1.5 µm
Specialist opticsEnergy filter - Name: GIF Bioquantum
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Number grids imaged: 1 / Number real images: 1687 / Average electron dose: 1.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 290830
CTF correctionSoftware - Name: CTFFIND (ver. 4)
Startup modelType of model: OTHER
Details: The initial model was generated using a stochastic gradient descent algorithm
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 1)
Final 3D classificationNumber classes: 10 / Software - Name: RELION (ver. 2.1)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 2.1)
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C1 (asymmetric) / Algorithm: BACK PROJECTION / Resolution.type: BY AUTHOR / Resolution: 6.2 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 2.1) / Number images used: 21927
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

1ciu

RefinementSpace: REAL / Protocol: FLEXIBLE FIT
Output model

PDB-6rn2:
ClpB (DWB mutant) bound to casein in presence of ATPgammaS - state WT-1

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