+Open data
-Basic information
Entry | Database: EMDB / ID: EMD-45434 | |||||||||
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Title | LONP1 stall state bound to substrate and 4 ADPs | |||||||||
Map data | Sharpened map | |||||||||
Sample |
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Keywords | ATPase / protease / HYDROLASE | |||||||||
Biological species | Homo sapiens (human) | |||||||||
Method | single particle reconstruction / cryo EM / Resolution: 3.18 Å | |||||||||
Authors | Mindrebo JT / Lander GC | |||||||||
Funding support | United States, 2 items
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Citation | Journal: bioRxiv / Year: 2024 Title: Structural and mechanistic studies on human LONP1 redefine the hand-over-hand translocation mechanism. Authors: Jeffrey T Mindrebo / Gabriel C Lander / Abstract: AAA+ enzymes use energy from ATP hydrolysis to remodel diverse cellular targets. Structures of substrate-bound AAA+ complexes suggest that these enzymes employ a conserved hand-over-hand mechanism to ...AAA+ enzymes use energy from ATP hydrolysis to remodel diverse cellular targets. Structures of substrate-bound AAA+ complexes suggest that these enzymes employ a conserved hand-over-hand mechanism to thread substrates through their central pore. However, the fundamental aspects of the mechanisms governing motor function and substrate processing within specific AAA+ families remain unresolved. We used cryo-electron microscopy to structurally interrogate reaction intermediates from in vitro biochemical assays to inform the underlying regulatory mechanisms of the human mitochondrial AAA+ protease, LONP1. Our results demonstrate that substrate binding allosterically regulates proteolytic activity, and that LONP1 can adopt a configuration conducive to substrate translocation even when the ATPases are bound to ADP. These results challenge the conventional understanding of the hand-over-hand translocation mechanism, giving rise to an alternative model that aligns more closely with biochemical and biophysical data on related enzymes like ClpX, ClpA, the 26S proteasome, and Lon protease. | |||||||||
History |
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-Structure visualization
Supplemental images |
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-Downloads & links
-EMDB archive
Map data | emd_45434.map.gz | 47.2 MB | EMDB map data format | |
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Header (meta data) | emd-45434-v30.xml emd-45434.xml | 19.9 KB 19.9 KB | Display Display | EMDB header |
FSC (resolution estimation) | emd_45434_fsc.xml | 10.7 KB | Display | FSC data file |
Images | emd_45434.png | 108.4 KB | ||
Filedesc metadata | emd-45434.cif.gz | 5.8 KB | ||
Others | emd_45434_additional_1.map.gz emd_45434_half_map_1.map.gz emd_45434_half_map_2.map.gz | 46 MB 84.6 MB 84.6 MB | ||
Archive directory | http://ftp.pdbj.org/pub/emdb/structures/EMD-45434 ftp://ftp.pdbj.org/pub/emdb/structures/EMD-45434 | HTTPS FTP |
-Validation report
Summary document | emd_45434_validation.pdf.gz | 1003.8 KB | Display | EMDB validaton report |
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Full document | emd_45434_full_validation.pdf.gz | 1003.4 KB | Display | |
Data in XML | emd_45434_validation.xml.gz | 17.7 KB | Display | |
Data in CIF | emd_45434_validation.cif.gz | 22.5 KB | Display | |
Arichive directory | https://ftp.pdbj.org/pub/emdb/validation_reports/EMD-45434 ftp://ftp.pdbj.org/pub/emdb/validation_reports/EMD-45434 | HTTPS FTP |
-Related structure data
-Links
EMDB pages | EMDB (EBI/PDBe) / EMDataResource |
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-Map
File | Download / File: emd_45434.map.gz / Format: CCP4 / Size: 91.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) | ||||||||||||||||||||||||||||||||||||
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Annotation | Sharpened map | ||||||||||||||||||||||||||||||||||||
Projections & slices | Image control
Images are generated by Spider. | ||||||||||||||||||||||||||||||||||||
Voxel size | X=Y=Z: 1.15 Å | ||||||||||||||||||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||||||||||||||||||
Details | EMDB XML:
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-Supplemental data
-Additional map: Unsharpened map
File | emd_45434_additional_1.map | ||||||||||||
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Annotation | Unsharpened map | ||||||||||||
Projections & Slices |
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Density Histograms |
-Half map: Half map B
File | emd_45434_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_45434_half_map_2.map | ||||||||||||
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Annotation | Half map A | ||||||||||||
Projections & Slices |
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Density Histograms |
-Sample components
-Entire : LONP1 bound to substrate and ADP
Entire | Name: LONP1 bound to substrate and ADP |
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Components |
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-Supramolecule #1: LONP1 bound to substrate and ADP
Supramolecule | Name: LONP1 bound to substrate and ADP / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all |
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Source (natural) | Organism: Homo sapiens (human) |
Molecular weight | Theoretical: 583.95 KDa |
-Macromolecule #1: Human mitochondrial Lon Protease homolog
Macromolecule | Name: Human mitochondrial Lon Protease homolog / type: protein_or_peptide / ID: 1 / Details: homohexameric assembly / Enantiomer: LEVO / EC number: endopeptidase La |
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Source (natural) | Organism: Homo sapiens (human) |
Recombinant expression | Organism: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria) |
Sequence | String: MHHHHHHENL YFQGAHMMTI PDVFPHLPLI AITRNPVFPR FIKIIEVKNK KLVELLRRKV RLAQPYVGVF LKRDDSNESD VVESLDEIYH TGTFAQIHEM QDLGDKLRMI VMGHRRVHIS RQLEVEPEEP EAENKHKPRR KSKRGKKEAE DELSARHPAE LAMEPTPELP ...String: MHHHHHHENL YFQGAHMMTI PDVFPHLPLI AITRNPVFPR FIKIIEVKNK KLVELLRRKV RLAQPYVGVF LKRDDSNESD VVESLDEIYH TGTFAQIHEM QDLGDKLRMI VMGHRRVHIS RQLEVEPEEP EAENKHKPRR KSKRGKKEAE DELSARHPAE LAMEPTPELP AEVLMVEVEN VVHEDFQVTE EVKALTAEIV KTIRDIIALN PLYRESVLQM MQAGQRVVDN PIYLSDMGAA LTGAESHELQ DVLEETNIPK RLYKALSLLK KEFELSKLQQ RLGREVEEKI KQTHRKYLLQ EQLKIIKKEL GLEKDDKDAI EEKFRERLKE LVVPKHVMDV VDEELSKLGL LDNHSSEFNV TRNYLDWLTS IPWGKYSNEN LDLARAQAVL EEDHYGMEDV KKRILEFIAV SQLRGSTQGK ILCFYGPPGV GKTSIARSIA RALNREYFRF SVGGMTDVAE IKGHRRTYVG AMPGKIIQCL KKTKTENPLI LIDEVDKIGR GYQGDPSSAL LELLDPEQNA NFLDHYLDVP VDLSKVLFIC TANVTDTIPE PLRDRMEMIN VSGYVAQEKL AIAERYLVPQ ARALCGLDES KAKLSSDVLT LLIKQYCRES GVRNLQKQVE KVLRKSAYKI VSGEAESVEV TPENLQDFVG KPVFTVERMY DVTPPGVVMG LAWTAMGGST LFVETSLRRP QDKDAKGDKD GSLEVTGQLG EVMKESARIA YTFARAFLMQ HAPANDYLVT SHIHLHVPEG ATPKDGPSAG CTIVTALLSL AMGRPVRQNL AMTGEVSLTG KILPVGGIKE KTIAAKRAGV TCIVLPAENK KDFYDLAAFI TEGLEVHFVE HYREIFDIAF PDEQAEALAV ER |
-Experimental details
-Structure determination
Method | cryo EM |
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Processing | single particle reconstruction |
Aggregation state | particle |
-Sample preparation
Concentration | 3 mg/mL | |||||||||
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Buffer | pH: 8 Component:
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Grid | Model: Quantifoil / Material: GOLD / Mesh: 300 / Support film - Material: GOLD / Support film - topology: HOLEY ARRAY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 25 sec. / Pretreatment - Atmosphere: OTHER | |||||||||
Vitrification | Cryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 298 K / Instrument: FEI VITROBOT MARK IV | |||||||||
Details | Sample was monodisperse with ~300 particles per image. |
-Electron microscopy
Microscope | FEI TALOS ARCTICA |
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Image recording | Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Digitization - Dimensions - Width: 3840 pixel / Digitization - Dimensions - Height: 3712 pixel / Number grids imaged: 1 / Number real images: 1944 / Average exposure time: 9.8 sec. / Average electron dose: 50.0 e/Å2 |
Electron beam | Acceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN |
Electron optics | C2 aperture diameter: 100.0 µm / Calibrated magnification: 43478 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 1.6 µm / Nominal defocus min: 0.9 µm / Nominal magnification: 36000 |
Sample stage | Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN |
Experimental equipment | Model: Talos Arctica / Image courtesy: FEI Company |