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- EMDB-34006: Spiral hexamer of the substrate-free Lon protease with Y397A and ... -
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Open data
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Basic information
Entry | ![]() | |||||||||
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Title | Spiral hexamer of the substrate-free Lon protease with Y397A and S678A mutations | |||||||||
![]() | Hexameric state of MtaLon-Y397A-S678A in the presence of ATPgammaS and casein | |||||||||
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![]() | hydrolysis / AAA proteins / HYDROLASE | |||||||||
Biological species | ![]() | |||||||||
Method | single particle reconstruction / cryo EM / Resolution: 4.08 Å | |||||||||
![]() | Li S / Hsieh KY / Kuo CI / Lee SH / Ho MR / Wang CH / Zhang K / Chang CI | |||||||||
Funding support | ![]()
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![]() | ![]() 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 |
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Structure visualization
Supplemental images |
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Downloads & links
-EMDB archive
Map data | ![]() | 71.3 MB | ![]() | |
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Header (meta data) | ![]() ![]() | 17.8 KB 17.8 KB | Display Display | ![]() |
Images | ![]() | 93.5 KB | ||
Filedesc metadata | ![]() | 5.6 KB | ||
Others | ![]() ![]() | 134.5 MB 134.5 MB | ||
Archive directory | ![]() ![]() | HTTPS FTP |
-Validation report
Summary document | ![]() | 791.1 KB | Display | ![]() |
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Full document | ![]() | 790.6 KB | Display | |
Data in XML | ![]() | 14.5 KB | Display | |
Data in CIF | ![]() | 17.1 KB | Display | |
Arichive directory | ![]() ![]() | HTTPS FTP |
-Related structure data
Related structure data | ![]() 7yphC ![]() 7ypiC ![]() 7ypjC ![]() 7ypkC ![]() 7yuhC ![]() 7yumC ![]() 7yupC ![]() 7yutC ![]() 7yuuC ![]() 7yuvC ![]() 7yuwC ![]() 7yuxC ![]() 8k3yC C: citing same article ( |
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Links
EMDB pages | ![]() ![]() |
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Map
File | ![]() | ||||||||||||||||||||
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Annotation | Hexameric state of MtaLon-Y397A-S678A in the presence of ATPgammaS and casein | ||||||||||||||||||||
Voxel size | X=Y=Z: 1.061 Å | ||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||
Details | EMDB XML:
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-Supplemental data
-Half map: Half map A
File | emd_34006_half_map_1.map | ||||||||||||
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Annotation | Half map A | ||||||||||||
Projections & Slices |
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Density Histograms |
-Half map: Half map B
File | emd_34006_half_map_2.map | ||||||||||||
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Annotation | Half map B | ||||||||||||
Projections & Slices |
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Density Histograms |
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Sample components
-Entire : Hexameric complex of Lon protease with Y397A-S678A mutation
Entire | Name: Hexameric complex of Lon protease with Y397A-S678A mutation |
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Components |
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-Supramolecule #1: Hexameric complex of Lon protease with Y397A-S678A mutation
Supramolecule | Name: Hexameric complex of Lon protease with Y397A-S678A mutation type: complex / ID: 1 / Parent: 0 / Macromolecule list: all |
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Source (natural) | Organism: ![]() |
Molecular weight | Theoretical: 530 KDa |
-Macromolecule #1: Substrate-free hexamer of Lon protease with Y397A and S678A mutations
Macromolecule | Name: Substrate-free hexamer of Lon protease with Y397A and S678A mutations type: protein_or_peptide / ID: 1 / Enantiomer: LEVO |
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Source (natural) | Organism: ![]() |
Recombinant expression | Organism: ![]() ![]() |
Sequence | String: 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 RTaIGAMPGK 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 |
-Experimental details
-Structure determination
Method | cryo EM |
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![]() | single particle reconstruction |
Aggregation state | particle |
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Sample preparation
Concentration | 0.5 mg/mL | ||||||||||||||||||
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Buffer | pH: 8 Component:
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Grid | Model: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 200 / Support film - Material: CARBON / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 60 sec. | ||||||||||||||||||
Vitrification | Cryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K | ||||||||||||||||||
Details | Lon protease was incubated with alpha-S1-casein |
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Electron microscopy
Microscope | FEI TITAN KRIOS |
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Image recording | Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Number grids imaged: 1 / Number real images: 5554 / Average exposure time: 2.0 sec. / Average electron dose: 48.0 e/Å2 |
Electron beam | Acceleration voltage: 300 kV / Electron source: ![]() |
Electron optics | Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 2.2 µm / Nominal defocus min: 1.4000000000000001 µm / Nominal magnification: 81000 |
Sample stage | Cooling holder cryogen: NITROGEN |
Experimental equipment | ![]() Model: Titan Krios / Image courtesy: FEI Company |