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Yorodumi- EMDB-34003: Close-ring hexamer of the substrate-bound Lon protease with an S6... -
+Open data
-Basic information
Entry | Database: EMDB / ID: EMD-34003 | |||||||||
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Title | Close-ring hexamer of the substrate-bound Lon protease with an S678A mutation | |||||||||
Map data | Substrate-bound Lon protease | |||||||||
Sample |
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Keywords | Lon protease / hydrolysis / AAA proteins / 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 | |||||||||
Biological species | Bos taurus (cattle) / Meiothermus taiwanensis (bacteria) | |||||||||
Method | single particle reconstruction / cryo EM / Resolution: 3.4 Å | |||||||||
Authors | Li S / Hsieh KY / Kuo CI / Lee SH / Ho MR / Wang CH / Zhang K / Chang CI | |||||||||
Funding support | Taiwan, 1 items
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Citation | Journal: 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 |
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-Structure visualization
Supplemental images |
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-Downloads & links
-EMDB archive
Map data | emd_34003.map.gz | 62.8 MB | EMDB map data format | |
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Header (meta data) | emd-34003-v30.xml emd-34003.xml | 20.1 KB 20.1 KB | Display Display | EMDB header |
Images | emd_34003.png | 131 KB | ||
Filedesc metadata | emd-34003.cif.gz | 6.9 KB | ||
Others | emd_34003_half_map_1.map.gz emd_34003_half_map_2.map.gz | 115.9 MB 115.9 MB | ||
Archive directory | http://ftp.pdbj.org/pub/emdb/structures/EMD-34003 ftp://ftp.pdbj.org/pub/emdb/structures/EMD-34003 | HTTPS FTP |
-Related structure data
Related structure data | 7ypkMC 7yphC 7ypiC 7ypjC 7yuhC 7yumC 7yupC 7yutC 7yuuC 7yuvC 7yuwC 7yuxC 8k3yC M: atomic model generated by this map C: citing same article (ref.) |
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Similar structure data | Similarity search - Function & homologyF&H Search |
-Links
EMDB pages | EMDB (EBI/PDBe) / EMDataResource |
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Related items in Molecule of the Month |
-Map
File | Download / File: emd_34003.map.gz / Format: CCP4 / Size: 125 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) | ||||||||||||||||||||
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Annotation | Substrate-bound Lon protease | ||||||||||||||||||||
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 B
File | emd_34003_half_map_1.map | ||||||||||||
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Annotation | Half map B | ||||||||||||
Projections & Slices |
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Density Histograms |
-Half map: Half map A
File | emd_34003_half_map_2.map | ||||||||||||
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Annotation | Half map A | ||||||||||||
Projections & Slices |
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Density Histograms |
-Sample components
-Entire : Close-ring hexamer of MtaLon-S678A in a substrate-engaged state
Entire | Name: Close-ring hexamer of MtaLon-S678A in a substrate-engaged state |
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Components |
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-Supramolecule #1: Close-ring hexamer of MtaLon-S678A in a substrate-engaged state
Supramolecule | Name: Close-ring hexamer of MtaLon-S678A in a substrate-engaged state type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#2 |
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Source (natural) | Organism: Bos taurus (cattle) |
Molecular weight | Theoretical: 530 KDa |
-Supramolecule #2: alpha-S1-casein
Supramolecule | Name: alpha-S1-casein / type: complex / ID: 2 / Parent: 1 |
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Source (natural) | Organism: Meiothermus taiwanensis (bacteria) |
-Supramolecule #3: Close-ring hexamer of Lon protease
Supramolecule | Name: Close-ring hexamer of Lon protease / type: complex / ID: 3 / Parent: 1 |
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-Macromolecule #1: alpha-S1-casein
Macromolecule | Name: alpha-S1-casein / type: protein_or_peptide / ID: 1 / Details: Unfolded substate / Number of copies: 1 / Enantiomer: LEVO |
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Source (natural) | Organism: Bos taurus (cattle) |
Molecular weight | Theoretical: 1.635006 KDa |
Sequence | String: (UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK) (UNK)(UNK)(UNK)(UNK)(UNK)(UNK) (UNK)(UNK)(UNK) |
-Macromolecule #2: Lon protease
Macromolecule | Name: Lon protease / type: protein_or_peptide / ID: 2 / Number of copies: 6 / Enantiomer: LEVO / EC number: endopeptidase La |
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Source (natural) | Organism: Meiothermus taiwanensis (bacteria) |
Molecular weight | Theoretical: 88.538594 KDa |
Recombinant expression | Organism: Escherichia coli (E. coli) |
Sequence | String: 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 TNQREYYLRE 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 PKDGPAAGIT MATAIASALS RRPARMDIAM TGEVSLRGKV MPIGGVKE K LLAAHQAGIH KIVLPKDNEA QLEELPKEVL EGLEIKLVED VGEVLEYLLL PEPTMPPVVQ PSDNRQQPGA GA UniProtKB: Lon protease |
-Macromolecule #3: ADENOSINE-5'-DIPHOSPHATE
Macromolecule | Name: ADENOSINE-5'-DIPHOSPHATE / type: ligand / ID: 3 / Number of copies: 6 / Formula: ADP |
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Molecular weight | Theoretical: 427.201 Da |
Chemical component information | ChemComp-ADP: |
-Experimental details
-Structure determination
Method | cryo EM |
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Processing | single particle reconstruction |
Aggregation state | particle |
-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 |
-Electron microscopy
Microscope | FEI TITAN KRIOS |
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Electron beam | Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN |
Electron optics | Illumination 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 stage | Cooling holder cryogen: NITROGEN |
Image recording | Film 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 |
-Image processing
Particle selection | Number selected: 1756270 |
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Startup model | Type of model: OTHER |
Initial angle assignment | Type: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 3.2) |
Final angle assignment | Type: PROJECTION MATCHING / Software - Name: cryoSPARC (ver. 3.2) |
Final reconstruction | Resolution.type: BY AUTHOR / Resolution: 3.4 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 3.2) / Number images used: 207760 |