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- EMDB-4625: E. coli ClpB (DWB and K476C mutant) bound to casein - state KC-2 -

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Basic information

Entry
Database: EMDB / ID: EMD-4625
TitleE. coli ClpB (DWB and K476C mutant) bound to casein - state KC-2
Map dataClpB-DWB-K476C bound to casein in presence of ATPgammaS - state KC-2
Sample
  • Complex: ClpB-DWB-K476C bound to casein in presence of ATPgammaS
    • Protein or peptide: ClpB
    • Protein or peptide: casein
Biological speciesEscherichia coli (E. coli) / Bos taurus (cattle)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.4 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
DepositionFeb 20, 2019-
Header (metadata) releaseJul 3, 2019-
Map releaseJul 3, 2019-
UpdateNov 25, 2020-
Current statusNov 25, 2020Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.05
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.05
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_4625.png

Downloads & links

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Map

FileDownload / File: emd_4625.map.gz / Format: CCP4 / Size: 91.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationClpB-DWB-K476C bound to casein in presence of ATPgammaS - state KC-2
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.05 Å/pix.
x 288 pix.
= 302.4 Å		1.05 Å/pix.
x 288 pix.
= 302.4 Å		1.05 Å/pix.
x 288 pix.
= 302.4 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.05 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.05 / Movie #1: 0.05
Minimum - Maximum-0.18100557 - 0.34442452
Average (Standard dev.)0.00037129913 (±0.011882041)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions288288288
Spacing288288288
CellA=B=C: 302.4 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.051.051.05
M x/y/z288288288
origin x/y/z0.0000.0000.000
length x/y/z302.400302.400302.400
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ320320320
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS288288288
D min/max/mean-0.1810.3440.000

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Supplemental data

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Mask #1

Fileemd_4625_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map 1

Fileemd_4625_half_map_1.map
AnnotationHalf map 1
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map 2

Fileemd_4625_half_map_2.map
AnnotationHalf map 2
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-K476C bound to casein in presence of ATPgammaS

EntireName: ClpB-DWB-K476C bound to casein in presence of ATPgammaS
Components
  • Complex: ClpB-DWB-K476C bound to casein in presence of ATPgammaS
    • Protein or peptide: ClpB
    • Protein or peptide: casein

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Supramolecule #1: ClpB-DWB-K476C bound to casein in presence of ATPgammaS

SupramoleculeName: ClpB-DWB-K476C bound to casein in presence of ATPgammaS
type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Escherichia coli (E. coli)
Recombinant expressionOrganism: Escherichia coli (E. coli)
Molecular weightTheoretical: 570 KDa

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Macromolecule #1: ClpB

MacromoleculeName: ClpB / type: protein_or_peptide / ID: 1 / Enantiomer: LEVO
Source (natural)Organism: Escherichia coli (E. coli)
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MRLDRLTNKF QLALADAQSL ALGHDNQFIE PLHLMSALLN QEGGSVSPLL TSAGINAGQL RTDINQALN RLPQVEGTGG DVQPSQDLVR VLNLCDKLAQ KRGDNFISSE LFVLAALESR G TLADILKA AGATTANITQ AIEQMRGGES VNDQGAEDQR QALKKYTIDL ...String:
MRLDRLTNKF QLALADAQSL ALGHDNQFIE PLHLMSALLN QEGGSVSPLL TSAGINAGQL RTDINQALN RLPQVEGTGG DVQPSQDLVR VLNLCDKLAQ KRGDNFISSE LFVLAALESR G TLADILKA AGATTANITQ AIEQMRGGES VNDQGAEDQR QALKKYTIDL TERAEQGKLD PV IGRDEEI RRTIQVLQRR TKNNPVLIGE PGVGKTAIVE GLAQRIINGE VPEGLKGRRV LAL DMGALV AGAKYRGEFE ERLKGVLNDL AKQEGNVILF IDALHTMVGA GKADGAMDAG NMLK PALAR GELHCVGATT LDEYRQYIEK DAALERRFQK VFVAEPSVED TIAILRGLKE RYELH HHVQ ITDPAIVAAA TLSHRYIADR QLPDKAIDLI DEAASSIRMQ IDSKPEELDR LDRRII QLK LEQQALMKES DEASKKRLDM LNEELSDKER QYSELEEEWK AEKASLSGTQ TICAELE QA KIAIEQARRV GDLARMSELQ YGKIPELEKQ LEAATQLEGK TMRLLRNKVT DAEIAEVL A RWTGIPVSRM MESEREKLLR MEQELHHRVI GQNEAVDAVS NAIRRSRAGL ADPNRPIGS FLFLGPTGVG KTELCKALAN FMFDSDEAMV RIDMSEFMEK HSVSRLVGAP PGYVGYEEGG YLTEAVRRR PYSVILLDAV EKAHPDVFNI LLQVLDDGRL TDGQGRTVDF RNTVVIMTSN L GSDLIQER FGELDYAHMK ELVLGVVSHN FRPEFINRID EVVVFHPLGE QHIASIAQIQ LK RLYKRLE ERGYEIHISD EALKLLSENG YDPVYGARPL KRAIQQQIEN PLAQQILSGE LVP GKVIRL EVNEDRIVAV Q

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Macromolecule #2: casein

MacromoleculeName: casein / type: protein_or_peptide / ID: 2 / Enantiomer: LEVO
Source (natural)Organism: Bos taurus (cattle)
SequenceString: MKLLILTCLV AVALARPKHP IKHQGLPQEV LNENLLRFFV APFPEVFGKE KVNELSKDIG SESTEDQAM EDIKQMEAES ISSSEEIVPN SVEQKHIQKE DVPSERYLGY LEQLLRLKKY K VPQLEIVP NSAEERLHSM KEGIHAQQKE PMIGVNQELA YFYPELFRQF ...String:
MKLLILTCLV AVALARPKHP IKHQGLPQEV LNENLLRFFV APFPEVFGKE KVNELSKDIG SESTEDQAM EDIKQMEAES ISSSEEIVPN SVEQKHIQKE DVPSERYLGY LEQLLRLKKY K VPQLEIVP NSAEERLHSM KEGIHAQQKE PMIGVNQELA YFYPELFRQF YQLDAYPSGA WY YVPLGTQ YTDAPSFSDI PNPIGSENSE KTTMPLW

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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-HClTris-HCl
25.0 mMKClpotassium chloride
10.0 mMMgCl2magnesium chloride
2.0 mMATPgammaSadenosine5o3thiotriphosphate
1.0 mMDTTdithiothreitol
GridModel: Quantifoil, UltrAuFoil / Material: GOLD / Mesh: 300 / Support film - Material: GRAPHENE OXIDE / Support film - topology: CONTINUOUS / 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
Specialist opticsEnergy filter - Name: GIF Bioquantum
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Number grids imaged: 1 / Number real images: 6060 / Average electron dose: 1.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 70.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 3.5 µm / Nominal defocus min: 1.5 µm
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 875861
CTF correctionSoftware - Name: CTFFIND (ver. 4)
Startup modelType of model: OTHER
Details: The initial model was generated using a stochastic gradient descent algorithm
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 3.4 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 2.1) / Number images used: 152240
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 1)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 2.1)
Final 3D classificationNumber classes: 10 / Software - Name: RELION (ver. 2.1)
FSC plot (resolution estimation)

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