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- EMDB-20049: CryoEM focus classification map of the hyperactive ClpB mutant K4... -

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Basic information

Entry
Database: EMDB / ID: EMD-20049
TitleCryoEM focus classification map of the hyperactive ClpB mutant K476C, bound to casein, pre-state
Map dataEM focus classification map of the hyperactive ClpB mutant K476C, bound to casein, pre-state
Sample
  • Complex: hyperactive disaggregase ClpB K476C, bound to casein
    • Protein or peptide: Hyperactive disaggregase ClpB
  • Ligand: ADENOSINE-5'-DIPHOSPHATE
  • Ligand: PHOSPHOTHIOPHOSPHORIC ACID-ADENYLATE ESTER
Keywordsdisaggregase / CLPB / AAA+ / CHAPERONE
Function / homology
Function and homology information


cellular response to heat / response to heat / protein refolding / ATP hydrolysis activity / ATP binding / identical protein binding / membrane / cytosol / 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 ...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 / 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
Chaperone protein ClpB
Similarity search - Component
Biological speciesEscherichia coli K-12 (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.3 Å
AuthorsRizo AR / Lin J-B
CitationJournal: Nat Commun / Year: 2019
Title: Structural basis for substrate gripping and translocation by the ClpB AAA+ disaggregase.
Authors: Alexandrea N Rizo / JiaBei Lin / Stephanie N Gates / Eric Tse / Stephen M Bart / Laura M Castellano / Frank DiMaio / James Shorter / Daniel R Southworth /
Abstract: Bacterial ClpB and yeast Hsp104 are homologous Hsp100 protein disaggregases that serve critical functions in proteostasis by solubilizing protein aggregates. Two AAA+ nucleotide binding domains (NBDs) ...Bacterial ClpB and yeast Hsp104 are homologous Hsp100 protein disaggregases that serve critical functions in proteostasis by solubilizing protein aggregates. Two AAA+ nucleotide binding domains (NBDs) power polypeptide translocation through a central channel comprised of a hexameric spiral of protomers that contact substrate via conserved pore-loop interactions. Here we report cryo-EM structures of a hyperactive ClpB variant bound to the model substrate, casein in the presence of slowly hydrolysable ATPγS, which reveal the translocation mechanism. Distinct substrate-gripping interactions are identified for NBD1 and NBD2 pore loops. A trimer of N-terminal domains define a channel entrance that binds the polypeptide substrate adjacent to the topmost NBD1 contact. NBD conformations at the seam interface reveal how ATP hydrolysis-driven substrate disengagement and re-binding are precisely tuned to drive a directional, stepwise translocation cycle.
History
DepositionApr 1, 2019-
Header (metadata) releaseMay 1, 2019-
Map releaseJun 12, 2019-
UpdateMar 20, 2024-
Current statusMar 20, 2024Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.0464
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 0.0464
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6og1
  • Surface level: 0.0464
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_20049.png

Downloads & links

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Map

FileDownload / File: emd_20049.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationEM focus classification map of the hyperactive ClpB mutant K476C, bound to casein, pre-state
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.03 Å/pix.
x 256 pix.
= 264.192 Å		1.03 Å/pix.
x 256 pix.
= 264.192 Å		1.03 Å/pix.
x 256 pix.
= 264.192 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.032 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.0464 / Movie #1: 0.0464
Minimum - Maximum-0.34293023 - 0.46456644
Average (Standard dev.)0.00029417133 (±0.008100012)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 264.192 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.0321.0321.032
M x/y/z256256256
origin x/y/z0.0000.0000.000
length x/y/z264.192264.192264.192
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS256256256
D min/max/mean-0.3430.4650.000

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Supplemental data

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Sample components

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Entire : hyperactive disaggregase ClpB K476C, bound to casein

EntireName: hyperactive disaggregase ClpB K476C, bound to casein
Components
  • Complex: hyperactive disaggregase ClpB K476C, bound to casein
    • Protein or peptide: Hyperactive disaggregase ClpB
  • Ligand: ADENOSINE-5'-DIPHOSPHATE
  • Ligand: PHOSPHOTHIOPHOSPHORIC ACID-ADENYLATE ESTER

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Supramolecule #1: hyperactive disaggregase ClpB K476C, bound to casein

SupramoleculeName: hyperactive disaggregase ClpB K476C, bound to casein / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: Escherichia coli K-12 (bacteria)

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Macromolecule #1: Hyperactive disaggregase ClpB

MacromoleculeName: Hyperactive disaggregase ClpB / type: protein_or_peptide / ID: 1 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Escherichia coli K-12 (bacteria)
Molecular weightTheoretical: 97.018469 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MRLDRLTNKF QLALADAQSL ALGHDNQFIE PLHLMSALLN QEGGSVSPLL TSAGINAGQL RTDINQALNR LPQVEGTGGD VQPSQDLVR VLNLCDNVAQ KRGDNFISSE LFVLAALESR GTVADILKAA GATTANITQA IEQMRGGESV NDQGAEDQRQ A LKKYTIDL ...String:
MRLDRLTNKF QLALADAQSL ALGHDNQFIE PLHLMSALLN QEGGSVSPLL TSAGINAGQL RTDINQALNR LPQVEGTGGD VQPSQDLVR VLNLCDNVAQ KRGDNFISSE LFVLAALESR GTVADILKAA GATTANITQA IEQMRGGESV NDQGAEDQRQ A LKKYTIDL TERAEQGKLD PVIGRDEEIR RTIQVLQRRT KNNPVLIGEP GVGKTAIVEG LAQRIINGEV PEGLKGRRVL AL DMGALVA GAKYRGEFEE RLKGVLNDLA KQEGNVILFI DELHTMVGAG KADGAMDAGN MLKPALARGE LHCVGATTLD EYR QYIEKD AALERRFQKV FVAEPSVEDT IAILRGLKER YELHHHVQIT DPAIVAAATL SHRYIADRQL PDKAIDLIDE AASS IRMQI DSKPEELDRL DRRIIQLKLE QQALMKESDE ASKKRLDMLN EELSDKERQY SELEEEWKAE KASLSGTQTI KCELE QAKI AIEQARRVGD LARMSELQYG KIPELEKQLE AATQLEGKTM RLLRNKVTDA EIAEVLARWT GIPVSRMMES EREKLL RME QELHHRVIGQ NEAVDAVSNA IRRSRAGLAD PNRPIGSFLF LGPTGVGKTE LCKALANFMF DSDEAMVRID MSEFMEK HS VSRLVGAPPG YVGYEEGGYL TEAVRRRPYS VILLDEVEKA HPDVFNILLQ VLDDGRLTDG QGRTVDFRNT VVIMTSNL G SDLIQERFGE LDYAHMKELV LGVVSHNFRP EFINRIDEVV VFHPLGEQHI ASIAQIQLKR LYKRLEERGY EIHISDEAL KLLSENGYDP VYGARPLKRA IQQQIENPLA QQILSGELVP GKVIRLEVNE DRIVAVQRSR SHHHHHH

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Macromolecule #2: ADENOSINE-5'-DIPHOSPHATE

MacromoleculeName: ADENOSINE-5'-DIPHOSPHATE / type: ligand / ID: 2 / Number of copies: 3 / Formula: ADP
Molecular weightTheoretical: 427.201 Da
Chemical component information

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

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Macromolecule #3: PHOSPHOTHIOPHOSPHORIC ACID-ADENYLATE ESTER

MacromoleculeName: PHOSPHOTHIOPHOSPHORIC ACID-ADENYLATE ESTER / type: ligand / ID: 3 / Number of copies: 1 / 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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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

BufferpH: 7.4
GridDetails: unspecified
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Average electron dose: 56.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 2.6 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: INSILICO MODEL
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 3.3 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 221083
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD

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