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- EMDB-36867: The "5+1" heteromeric structure of Lon protease consisting of a s... -

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Basic information

Entry
Database: EMDB / ID: EMD-36867
TitleThe "5+1" heteromeric structure of Lon protease consisting of a spiral pentamer with Y224S mutation and an N-terminal-truncated monomeric E613K mutant
Map dataheteromeric 5 1 complex
Sample
  • Complex: Spiral pentamer of Lon protease with a Y224S mutation in complex with the N-terminal-truncated monomeric E613K mutant of Lon.
    • Protein or peptide: Lon proteaseLon protease family
    • Protein or peptide: Lon proteaseLon protease family
  • Ligand: ADENOSINE-5'-DIPHOSPHATE
KeywordsAAA+ protein / ATPase / HYDROLASE
Function / homology
Function and homology information


endopeptidase La / ATP-dependent peptidase activity / protein quality control for misfolded or incompletely synthesized proteins / cellular response to heat / sequence-specific DNA binding / serine-type endopeptidase activity / ATP hydrolysis activity / ATP binding / identical protein binding / cytoplasm
Similarity search - Function
Lon protease, bacterial / Lon protease, bacterial/eukaryotic-type / Peptidase S16, active site / ATP-dependent serine proteases, lon family, serine active site. / Lon proteolytic domain profile. / Peptidase S16, Lon proteolytic domain / Lon protease / Lon protease (S16) C-terminal proteolytic domain / Lon protease, N-terminal domain superfamily / Lon N-terminal domain profile. ...Lon protease, bacterial / Lon protease, bacterial/eukaryotic-type / Peptidase S16, active site / ATP-dependent serine proteases, lon family, serine active site. / Lon proteolytic domain profile. / Peptidase S16, Lon proteolytic domain / Lon protease / Lon protease (S16) C-terminal proteolytic domain / Lon protease, N-terminal domain superfamily / Lon N-terminal domain profile. / Lon protease, N-terminal domain / ATP-dependent protease La (LON) substrate-binding domain / Found in ATP-dependent protease La (LON) / PUA-like superfamily / ATPase family associated with various cellular activities (AAA) / ATPase, AAA-type, core / Ribosomal protein S5 domain 2-type fold, subgroup / Ribosomal protein S5 domain 2-type fold / ATPases associated with a variety of cellular activities / AAA+ ATPase domain / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
Biological speciesMeiothermus taiwanensis (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 4.42 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_36867.png

Downloads & links

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Map

FileDownload / File: emd_36867.map.gz / Format: CCP4 / Size: 125 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationheteromeric 5 1 complex
Voxel sizeX=Y=Z: 1.061 Å
Density
Contour LevelBy AUTHOR: 0.1
Minimum - Maximum-0.18623747 - 0.7626187
Average (Standard dev.)0.0022331765 (±0.031756114)
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_36867_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_36867_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 Lon protease with a Y224S mutation in complex ...

EntireName: Spiral pentamer of Lon protease with a Y224S mutation in complex with the N-terminal-truncated monomeric E613K mutant of Lon.
Components
  • Complex: Spiral pentamer of Lon protease with a Y224S mutation in complex with the N-terminal-truncated monomeric E613K mutant of Lon.
    • Protein or peptide: Lon proteaseLon protease family
    • Protein or peptide: Lon proteaseLon protease family
  • Ligand: ADENOSINE-5'-DIPHOSPHATE

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Supramolecule #1: Spiral pentamer of Lon protease with a Y224S mutation in complex ...

SupramoleculeName: Spiral pentamer of Lon protease with a Y224S mutation in complex with the N-terminal-truncated monomeric E613K mutant of Lon.
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#2
Source (natural)Organism: Meiothermus taiwanensis (bacteria)

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Macromolecule #1: Lon protease

MacromoleculeName: Lon protease / type: protein_or_peptide / ID: 1 / Number of copies: 5 / Enantiomer: LEVO / EC number: endopeptidase La
Source (natural)Organism: Meiothermus taiwanensis (bacteria)
Molecular weightTheoretical: 89.307383 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: MRLELPVIPL RNTVILPHTT TPVDVGRAKS KRAVEEAMGA DRLIFLVAQR DPEVDDPAPD DLYTWGVQAV VKQAMRLPDG TLQVMVEAR ARAQVTDYIP GPYLRARGEV FSEIFPIDEA VVRVLVEELK EAFEKYVANH KSLRLDRYQL EAVKGTSDPA M LADTIAYH ...String:
MRLELPVIPL RNTVILPHTT TPVDVGRAKS KRAVEEAMGA DRLIFLVAQR DPEVDDPAPD DLYTWGVQAV VKQAMRLPDG TLQVMVEAR ARAQVTDYIP GPYLRARGEV FSEIFPIDEA VVRVLVEELK EAFEKYVANH KSLRLDRYQL EAVKGTSDPA M LADTIAYH ATWTVAEKQE ILELTDLEAR LKKVLGLLSR DLERFELDKR VAQRVKEQMD TNQRESYLRE QMKAIQKELG GE DGLSDLE ALRKKIEEVG MPEAVKTKAL KELDRLERMQ QGSPEATVAR TYLDWLTEVP WSKADPEVLD INHTRQVLDE DHY GLKDVK ERILEYLAVR QLTQGLDVRN KAPILVLVGP PGVGKTSLGR SIARSMNRKF HRISLGGVRD EAEIRGHRRT YIGA MPGKL IHAMKQVGVI NPVILLDEID KMSSDWRGDP ASAMLEVLDP EQNNTFTDHY LDVPYDLSKV FFITTANTLQ TIPRP LLDR MEVIEIPGYT NMEKQAIARQ YLWPKQVRES GMEGRIEVTD AAILRVISEY TREAGVRGLE RELGKIARKG AKFWLE GAW EGLRTIDASD IPTYLGIPRY RPDKAETEPQ VGTAQGLAWT PVGGTLLTIE VAAVPGSGKL SLTGQLGEVM KESAQAA LT YLRAHTQDYG LPEDFYNKVD LHVHVPDGAT PKDGPSAGIT MATAIASALS RRPARMDIAM TGEVSLRGKV MPIGGVKE K LLAAHQAGIH KIVLPKDNEA QLEELPKEVL EGLEIKLVED VGEVLEYLLL PEPTMPPVVQ PSDNRQQPGA GAHHHHHH

UniProtKB: Lon protease

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Macromolecule #2: Lon protease

MacromoleculeName: Lon protease / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO / EC number: endopeptidase La
Source (natural)Organism: Meiothermus taiwanensis (bacteria)
Molecular weightTheoretical: 63.185266 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: HHHHHHSSGE NLYFQGHMGL SDLEALRKKI EEVGMPEAVK TKALKELDRL ERMQQGSPEA TVARTYLDWL TEVPWSKADP EVLDINHTR QVLDEDHYGL KDVKERILEY LAVRQLTQGL DVRNKAPILV LVGPPGVGKT SLGRSIARSM NRKFHRISLG G VRDEAEIR ...String:
HHHHHHSSGE NLYFQGHMGL SDLEALRKKI EEVGMPEAVK TKALKELDRL ERMQQGSPEA TVARTYLDWL TEVPWSKADP EVLDINHTR QVLDEDHYGL KDVKERILEY LAVRQLTQGL DVRNKAPILV LVGPPGVGKT SLGRSIARSM NRKFHRISLG G VRDEAEIR GHRRTYIGAM PGKLIHAMKQ VGVINPVILL DEIDKMSSDW RGDPASAMLE VLDPEQNNTF TDHYLDVPYD LS KVFFITT ANTLQTIPRP LLDRMEVIEI PGYTNMEKQA IARQYLWPKQ VRESGMEGRI EVTDAAILRV ISEYTREAGV RGL ERELGK IARKGAKFWL EGAWEGLRTI DASDIPTYLG IPRYRPDKAE TEPQVGTAQG LAWTPVGGTL LTIKVAAVPG SGKL SLTGQ LGEVMKESAQ AALTYLRAHT QDYGLPEDFY NKVDLHVHVP DGATPKDGPS AGITMATAIA SALSRRPARM DIAMT GEVS LRGKVMPIGG VKEKLLAAHQ AGIHKIVLPK DNEAQLEELP KEVLEGLEIK LVEDVGEVLE YLLLPEPTMP PVVQPS DNR QQPGAGA

UniProtKB: Lon protease

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Macromolecule #3: ADENOSINE-5'-DIPHOSPHATE

MacromoleculeName: ADENOSINE-5'-DIPHOSPHATE / type: ligand / ID: 3 / Number of copies: 4 / Formula: ADP
Molecular weightTheoretical: 427.201 Da
Chemical component information

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

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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
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: 4.42 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 70828
FSC plot (resolution estimation)

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