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- EMDB-36865: Spiral pentameric form of the substrate-free Lon protease with a ... -

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Entry
Database: EMDB / ID: EMD-36865
TitleSpiral pentameric form of the substrate-free Lon protease with a Y224S mutation in the presence of the N-terminal-truncated monomeric mutant bearing an E613K mutation
Map dataspiral pentamer of Y224S:dN-E613K:ADP
Sample
  • Complex: Spiral pentamer of the substrate-free Lon protease with a Y224S mutation in the presence of the N-terminal-truncated monomeric mutant bearing a E613K mutation.
    • Protein or peptide: homo-pentamer of Lon protease with a Y224S mutation
KeywordsAAA+ protein / ATPase / HYDROLASE
Biological speciesMeiothermus taiwanensis (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.8 Å
AuthorsLi S / Hsieh KY / Kuo CI / Zhang K / Chang CI
Funding support Taiwan, 2 items
OrganizationGrant numberCountry
Ministry of Science and Technology (MoST, Taiwan)108-2320-B-001-011-MY3 Taiwan
Ministry of Science and Technology (MoST, Taiwan)111-2320-B-001-013-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
DepositionJul 17, 2023-
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_36865.png

Downloads & links

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Map

FileDownload / File: emd_36865.map.gz / Format: CCP4 / Size: 144.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationspiral pentamer of Y224S:dN-E613K:ADP
Voxel sizeX=Y=Z: 1.061 Å
Density
Contour LevelBy AUTHOR: 0.13
Minimum - Maximum-0.2622223 - 0.9575852
Average (Standard dev.)0.0016456145 (±0.032124706)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions336336336
Spacing336336336
CellA=B=C: 356.496 Å
α=β=γ: 90.0 °

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Supplemental data

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Additional map: sharpened map

Fileemd_36865_additional_1.map
Annotationsharpened map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: half map B

Fileemd_36865_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_36865_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 : Spiral pentamer of the substrate-free Lon protease with a Y224S m...

EntireName: Spiral pentamer of the substrate-free Lon protease with a Y224S mutation in the presence of the N-terminal-truncated monomeric mutant bearing a E613K mutation.
Components
  • Complex: Spiral pentamer of the substrate-free Lon protease with a Y224S mutation in the presence of the N-terminal-truncated monomeric mutant bearing a E613K mutation.
    • Protein or peptide: homo-pentamer of Lon protease with a Y224S mutation

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Supramolecule #1: Spiral pentamer of the substrate-free Lon protease with a Y224S m...

SupramoleculeName: Spiral pentamer of the substrate-free Lon protease with a Y224S mutation in the presence of the N-terminal-truncated monomeric mutant bearing a E613K mutation.
type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Meiothermus taiwanensis (bacteria)

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Macromolecule #1: homo-pentamer of Lon protease with a Y224S mutation

MacromoleculeName: homo-pentamer of Lon protease with a Y224S mutation / type: protein_or_peptide / ID: 1 / Enantiomer: LEVO
Source (natural)Organism: Meiothermus taiwanensis (bacteria)
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: MRLELPVIPL RNTVILPHTT TPVDVGRAKS KRAVEEAMGA DRLIFLVAQR DPEVDDPAPD DLYTWGVQAV VKQAMRLPDG TLQVMVEARA RAQVTDYIPG PYLRARGEVF SEIFPIDEAV VRVLVEELKE AFEKYVANHK SLRLDRYQLE AVKGTSDPAM LADTIAYHAT ...String:
MRLELPVIPL RNTVILPHTT TPVDVGRAKS KRAVEEAMGA DRLIFLVAQR DPEVDDPAPD DLYTWGVQAV VKQAMRLPDG TLQVMVEARA RAQVTDYIPG PYLRARGEVF SEIFPIDEAV VRVLVEELKE AFEKYVANHK SLRLDRYQLE AVKGTSDPAM LADTIAYHAT WTVAEKQEIL ELTDLEARLK KVLGLLSRDL ERFELDKRVA QRVKEQMDTN QRESYLREQM KAIQKELGGE DGLSDLEALR KKIEEVGMPE AVKTKALKEL DRLERMQQGS PEATVARTYL DWLTEVPWSK ADPEVLDINH TRQVLDEDHY GLKDVKERIL EYLAVRQLTQ GLDVRNKAPI LVLVGPPGVG KTSLGRSIAR SMNRKFHRIS LGGVRDEAEI RGHRRTYIGA MPGKLIHAMK QVGVINPVIL LDEIDKMSSD WRGDPASAML EVLDPEQNNT FTDHYLDVPY DLSKVFFITT ANTLQTIPRP LLDRMEVIEI PGYTNMEKQA IARQYLWPKQ VRESGMEGRI EVTDAAILRV ISEYTREAGV RGLERELGKI ARKGAKFWLE GAWEGLRTID ASDIPTYLGI PRYRPDKAET EPQVGTAQGL AWTPVGGTLL TIEVAAVPGS GKLSLTGQLG EVMKESAQAA LTYLRAHTQD YGLPEDFYNK VDLHVHVPDG ATPKDGPSAG ITMATAIASA LSRRPARMDI AMTGEVSLRG KVMPIGGVKE KLLAAHQAGI HKIVLPKDNE AQLEELPKEV LEGLEIKLVE DVGEVLEYLL LPEPTMPPVV QPSDNRQQPG AGAHHHHHH

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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

Concentration0.7 mg/mL
BufferpH: 8
Component:
ConcentrationFormulaName
20.0 mMC4H11NO3Tris(hydroxymethyl)aminomethane
200.0 mMNaClSodium chloridesodium chloride
10.0 mMMgCl2Magnesium chloride
1.0 mMC4H10O2S2Dithiothreitol

Details: 20 mM Tris-HCl, 200 mM NaCl, 10 mM MgCl2, 1 mM DTT
GridMaterial: COPPER / Mesh: 200 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 60 sec.
VitrificationCryogen name: ETHANE

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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
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Average electron dose: 52.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: NONE
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: PROJECTION MATCHING
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.8 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 102693
FSC plot (resolution estimation)

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