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- EMDB-20051: CryoEM focus classification map of the hyperactive ClpB mutant K4... -

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Basic information

Entry
Database: EMDB / ID: EMD-20051
TitleCryoEM focus classification map of the hyperactive ClpB mutant K476C, bound to casein, NTD-trimer
Map dataEM focus classification map of the hyperactive ClpB mutant K476C, bound to casein, NTD-trimer
Sample
  • Complex: hyperactive ClpB mutant K476C bound to casein
    • Complex: hyperactive ClpB mutant K476C_NTD trimer
      • Protein or peptide: Hyperactive disaggregase ClpB
    • Complex: alpha-s1-casein
      • Protein or peptide: Alpha S1-casein
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) / Bos taurus (cattle)
Methodsingle particle reconstruction / cryo EM / Resolution: 4.1 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: 0.0117
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 0.0117
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6og3
  • Surface level: 0.0117
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_20051.png

Downloads & links

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Map

FileDownload / File: emd_20051.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, NTD-trimer
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: 0.0117 / Movie #1: 0.0117
Minimum - Maximum-0.026774807 - 0.06934049
Average (Standard dev.)0.000086781474 (±0.0012235555)
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-0.0270.0690.000

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Supplemental data

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Sample components

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Entire : hyperactive ClpB mutant K476C bound to casein

EntireName: hyperactive ClpB mutant K476C bound to casein
Components
  • Complex: hyperactive ClpB mutant K476C bound to casein
    • Complex: hyperactive ClpB mutant K476C_NTD trimer
      • Protein or peptide: Hyperactive disaggregase ClpB
    • Complex: alpha-s1-casein
      • Protein or peptide: Alpha S1-casein

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Supramolecule #1: hyperactive ClpB mutant K476C bound to casein

SupramoleculeName: hyperactive ClpB mutant K476C bound to casein / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all

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Supramolecule #2: hyperactive ClpB mutant K476C_NTD trimer

SupramoleculeName: hyperactive ClpB mutant K476C_NTD trimer / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1
Source (natural)Organism: Escherichia coli K-12 (bacteria)

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Supramolecule #3: alpha-s1-casein

SupramoleculeName: alpha-s1-casein / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #2
Details: Protein was purchased from Sigma-Aldrich. Purified from bovine milk.
Source (natural)Organism: Bos taurus (cattle)

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Macromolecule #1: Hyperactive disaggregase ClpB

MacromoleculeName: Hyperactive disaggregase ClpB / type: protein_or_peptide / ID: 1 / Number of copies: 3 / Enantiomer: LEVO
Source (natural)Organism: Escherichia coli K-12 (bacteria) / Strain: K-12
Molecular weightTheoretical: 96.960367 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 CAELE 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

UniProtKB: Chaperone protein ClpB

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Macromolecule #2: Alpha S1-casein

MacromoleculeName: Alpha S1-casein / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Bos taurus (cattle)
Molecular weightTheoretical: 783.958 Da
SequenceString:
(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)

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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

Concentration1.5 mg/mL
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) / Detector mode: SUPER-RESOLUTION / Average electron dose: 56.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.6 mm
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: 4.1 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 93588
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD

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Atomic model buiding 1

Initial modelPDB ID:

1khy


Chain - Chain ID: A / Chain - Source name: PDB / Chain - Initial model type: experimental model
RefinementProtocol: RIGID BODY FIT
Output model

PDB-6og3:
Focus classification structure of the hyperactive ClpB mutant K476C, bound to casein, NTD-trimer

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