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- EMDB-34004: Spiral hexamer of the substrate-free Lon protease with an S678A m... -

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Basic information

Entry
Database: EMDB / ID: EMD-34004
TitleSpiral hexamer of the substrate-free Lon protease with an S678A mutation
Map dataSubstrate-free hexamer of MtaLon-S678A
Sample
  • Complex: Substrate-free hexamer of MtaLon-S678A in the presence of ADP
    • Protein or peptide: Substrate-free hexamer of Lon protease with S678A mutation
Keywordshydrolysis / AAA proteins / HYDROLASE
Biological speciesMeiothermus taiwanensis (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 4.7 Å
AuthorsLi S / Hsieh KY / Kou CI / Lee SH / Ho MR / Wang CH / Zhang K / Chang CI
Funding support Taiwan, 1 items
OrganizationGrant numberCountry
Ministry of Science and Technology (MoST, Taiwan)108-2320-B-001-011-MY3 Taiwan
CitationJournal: Nat Commun / Year: 2023
Title: A 5+1 assemble-to-activate mechanism of the Lon proteolytic machine.
Authors: Shanshan Li / Kan-Yen Hsieh / Chiao-I Kuo / Tzu-Chi Lin / Szu-Hui Lee / Yi-Ru Chen / Chun-Hsiung Wang / Meng-Ru Ho / See-Yeun Ting / Kaiming Zhang / Chung-I Chang /
Abstract: Many AAA+ (ATPases associated with diverse cellular activities) proteins function as protein or DNA remodelers by threading the substrate through the central pore of their hexameric assemblies. In ...Many AAA+ (ATPases associated with diverse cellular activities) proteins function as protein or DNA remodelers by threading the substrate through the central pore of their hexameric assemblies. In this ATP-dependent translocating state, the substrate is gripped by the pore loops of the ATPase domains arranged in a universal right-handed spiral staircase organization. However, the process by which a AAA+ protein is activated to adopt this substrate-pore-loop arrangement remains unknown. We show here, using cryo-electron microscopy (cryo-EM), that the activation process of the Lon AAA+ protease may involve a pentameric assembly and a substrate-dependent incorporation of the sixth protomer to form the substrate-pore-loop contacts seen in the translocating state. Based on the structural results, we design truncated monomeric mutants that inhibit Lon activity by binding to the native pentamer and demonstrated that expressing these monomeric mutants in Escherichia coli cells containing functional Lon elicits specific phenotypes associated with lon deficiency, including the inhibition of persister cell formation. These findings uncover a substrate-dependent assembly process for the activation of a AAA+ protein and demonstrate a targeted approach to selectively inhibit its function within cells.
History
DepositionAug 3, 2022-
Header (metadata) releaseOct 25, 2023-
Map releaseOct 25, 2023-
UpdateNov 29, 2023-
Current statusNov 29, 2023Processing site: PDBj / Status: Released

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Structure visualization

Supplemental images

emd_34004.png

Downloads & links

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Map

FileDownload / File: emd_34004.map.gz / Format: CCP4 / Size: 125 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationSubstrate-free hexamer of MtaLon-S678A
Voxel sizeX=Y=Z: 1.061 Å
Density
Contour LevelBy AUTHOR: 0.0713
Minimum - Maximum-0.11005958 - 0.38717023
Average (Standard dev.)0.00040768314 (±0.014477006)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions320320320
Spacing320320320
CellA=B=C: 339.52 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: Half map B

Fileemd_34004_half_map_1.map
AnnotationHalf map B
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map A

Fileemd_34004_half_map_2.map
AnnotationHalf map A
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Substrate-free hexamer of MtaLon-S678A in the presence of ADP

EntireName: Substrate-free hexamer of MtaLon-S678A in the presence of ADP
Components
  • Complex: Substrate-free hexamer of MtaLon-S678A in the presence of ADP
    • Protein or peptide: Substrate-free hexamer of Lon protease with S678A mutation

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Supramolecule #1: Substrate-free hexamer of MtaLon-S678A in the presence of ADP

SupramoleculeName: Substrate-free hexamer of MtaLon-S678A in the presence of ADP
type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Meiothermus taiwanensis (bacteria)
Molecular weightTheoretical: 530 KDa

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Macromolecule #1: Substrate-free hexamer of Lon protease with S678A mutation

MacromoleculeName: Substrate-free hexamer of Lon protease with S678A mutation
type: protein_or_peptide / ID: 1 / Enantiomer: LEVO
Source (natural)Organism: Meiothermus taiwanensis (bacteria)
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MRLELPVIPL RNTVILPHTT TPVDVGRAKS KRAVEEAMGA DRLIFLVAQR DPEVDDPAPD DLYTWGVQA VVKQAMRLPD GTLQVMVEAR ARAQVTDYIP GPYLRARGEV FSEIFPIDEA V VRVLVEEL KEAFEKYVAN HKSLRLDRYQ LEAVKGTSDP AMLADTIAYH ...String:
MRLELPVIPL RNTVILPHTT TPVDVGRAKS KRAVEEAMGA DRLIFLVAQR DPEVDDPAPD DLYTWGVQA VVKQAMRLPD GTLQVMVEAR ARAQVTDYIP GPYLRARGEV FSEIFPIDEA V VRVLVEEL KEAFEKYVAN HKSLRLDRYQ LEAVKGTSDP AMLADTIAYH ATWTVAEKQE IL ELTDLEA RLKKVLGLLS RDLERFELDK RVAQRVKEQM DTNQREYYLR EQMKAIQKEL GGE DGLSDL EALRKKIEEV GMPEAVKTKA LKELDRLERM QQGSPEATVA RTYLDWLTEV PWSK ADPEV LDINHTRQVL DEDHYGLKDV KERILEYLAV RQLTQGLDVR NKAPILVLVG PPGVG KTSL GRSIARSMNR KFHRISLGGV RDEAEIRGHR RTYIGAMPGK LIHAMKQVGV INPVIL LDE IDKMSSDWRG DPASAMLEVL DPEQNNTFTD HYLDVPYDLS KVFFITTANT LQTIPRP LL DRMEVIEIPG YTNMEKQAIA RQYLWPKQVR ESGMEGRIEV TDAAILRVIS EYTREAGV R GLERELGKIA RKGAKFWLEG AWEGLRTIDA SDIPTYLGIP RYRPDKAETE PQVGTAQGL AWTPVGGTLL TIEVAAVPGS GKLSLTGQLG EVMKESAQAA LTYLRAHTQD YGLPEDFYNK VDLHVHVPD GATPKDGPaA GITMATAIAS ALSRRPARMD IAMTGEVSLR GKVMPIGGVK E KLLAAHQA GIHKIVLPKD NEAQLEELPK EVLEGLEIKL VEDVGEVLEY LLLPEPTMPP VV QPSDNRQ QPGAGA

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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

Concentration0.5 mg/mL
BufferpH: 8
Component:
ConcentrationFormulaName
20.0 mM(HOCH2)3CNH2tris(hydroxymethyl)aminomethane
150.0 mMNaClSodium chloridesodium chloride
10.0 mMMgCl2magnesium chloride
1.0 mMC4H10O2S2dithiothreitol
1.0 mMC10H15N5O10P2Adenosine diphosphate
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 200 / Support film - Material: CARBON / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 60 sec.
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K
DetailsLon protease was incubated with alpha-S1-casein

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 2.2 µm / Nominal defocus min: 1.4000000000000001 µm / Nominal magnification: 81000
Sample stageCooling holder cryogen: NITROGEN
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Number grids imaged: 2 / Number real images: 12546 / Average exposure time: 2.52 sec. / Average electron dose: 59.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 1756270
Startup modelType of model: OTHER
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 3.2)
Final angle assignmentType: PROJECTION MATCHING / Software - Name: cryoSPARC (ver. 3.2)
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 4.7 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 3.2) / Number images used: 100882

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