+Open data
-Basic information
Entry | Database: EMDB / ID: EMD-45433 | |||||||||
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Title | Human Mitochondrial LONP1 Stall State + casein | |||||||||
Map data | Sharpened Map | |||||||||
Sample |
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Keywords | ATPase / protease / HYDROLASE | |||||||||
Function / homology | Function and homology information oxidation-dependent protein catabolic process / PH domain binding / mitochondrial protein catabolic process / G-quadruplex DNA binding / endopeptidase La / mitochondrial DNA metabolic process / mitochondrial genome maintenance / ATP-dependent peptidase activity / protein quality control for misfolded or incompletely synthesized proteins / mitochondrial nucleoid ...oxidation-dependent protein catabolic process / PH domain binding / mitochondrial protein catabolic process / G-quadruplex DNA binding / endopeptidase La / mitochondrial DNA metabolic process / mitochondrial genome maintenance / ATP-dependent peptidase activity / protein quality control for misfolded or incompletely synthesized proteins / mitochondrial nucleoid / insulin receptor substrate binding / chaperone-mediated protein complex assembly / DNA polymerase binding / regulation of peptidyl-tyrosine phosphorylation / negative regulation of insulin receptor signaling pathway / Mitochondrial protein degradation / proteolysis involved in protein catabolic process / mitochondrion organization / protein catabolic process / ADP binding / single-stranded DNA binding / cellular response to oxidative stress / sequence-specific DNA binding / single-stranded RNA binding / response to hypoxia / mitochondrial matrix / serine-type endopeptidase activity / ATP hydrolysis activity / mitochondrion / nucleoplasm / ATP binding / identical protein binding / membrane / cytosol Similarity search - Function | |||||||||
Biological species | Homo sapiens (human) / Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria) | |||||||||
Method | single particle reconstruction / cryo EM / Resolution: 3.23 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_45433.map.gz | 33.2 MB | EMDB map data format | |
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Header (meta data) | emd-45433-v30.xml emd-45433.xml | 23.9 KB 23.9 KB | Display Display | EMDB header |
FSC (resolution estimation) | emd_45433_fsc.xml | 9.5 KB | Display | FSC data file |
Images | emd_45433.png | 92.8 KB | ||
Filedesc metadata | emd-45433.cif.gz | 7.1 KB | ||
Others | emd_45433_additional_1.map.gz emd_45433_additional_2.map.gz emd_45433_half_map_1.map.gz emd_45433_half_map_2.map.gz | 32.4 MB 57.4 MB 59.3 MB 59.3 MB | ||
Archive directory | http://ftp.pdbj.org/pub/emdb/structures/EMD-45433 ftp://ftp.pdbj.org/pub/emdb/structures/EMD-45433 | HTTPS FTP |
-Validation report
Summary document | emd_45433_validation.pdf.gz | 961.8 KB | Display | EMDB validaton report |
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Full document | emd_45433_full_validation.pdf.gz | 961.3 KB | Display | |
Data in XML | emd_45433_validation.xml.gz | 16 KB | Display | |
Data in CIF | emd_45433_validation.cif.gz | 19.9 KB | Display | |
Arichive directory | https://ftp.pdbj.org/pub/emdb/validation_reports/EMD-45433 ftp://ftp.pdbj.org/pub/emdb/validation_reports/EMD-45433 | HTTPS FTP |
-Related structure data
Related structure data | 9cc3MC 9cc0C 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_45433.map.gz / Format: CCP4 / Size: 64 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: 1.30156 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_45433_additional_1.map | ||||||||||||
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Annotation | Unsharpened map | ||||||||||||
Projections & Slices |
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Density Histograms |
-Additional map: Deep Emhanced Map
File | emd_45433_additional_2.map | ||||||||||||
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Annotation | Deep Emhanced Map | ||||||||||||
Projections & Slices |
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Density Histograms |
-Half map: Half Map A
File | emd_45433_half_map_1.map | ||||||||||||
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Annotation | Half Map A | ||||||||||||
Projections & Slices |
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Density Histograms |
-Half map: Half Map A
File | emd_45433_half_map_2.map | ||||||||||||
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Annotation | Half Map A | ||||||||||||
Projections & Slices |
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Density Histograms |
-Sample components
-Entire : LONP1 closed state in complex with casein
Entire | Name: LONP1 closed state in complex with casein |
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Components |
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-Supramolecule #1: LONP1 closed state in complex with casein
Supramolecule | Name: LONP1 closed state in complex with casein / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#2 / Details: LONP1 incubated with casein before vitrification |
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Source (natural) | Organism: Homo sapiens (human) |
Molecular weight | Theoretical: 583.95 KDa |
-Macromolecule #1: Endogenous Co-purified substrate modeled as unknown residues
Macromolecule | Name: Endogenous Co-purified substrate modeled as unknown residues type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO |
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Source (natural) | Organism: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria) |
Molecular weight | Theoretical: 2.571161 KDa |
Sequence | String: (UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK) (UNK)(UNK)(UNK)(UNK)(UNK)(UNK) (UNK)(UNK)(UNK) (UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK) (UNK) |
-Macromolecule #2: Lon protease homolog, mitochondrial
Macromolecule | Name: Lon protease homolog, mitochondrial / type: protein_or_peptide / ID: 2 / Number of copies: 6 / Enantiomer: LEVO / EC number: endopeptidase La |
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Source (natural) | Organism: Homo sapiens (human) |
Molecular weight | Theoretical: 97.468156 KDa |
Recombinant expression | Organism: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria) |
Sequence | String: MHHHHHHENL YFQGAHMMTI PDVFPHLPLI AITRNPVFPR FIKIIEVKNK KLVELLRRKV RLAQPYVGVF LKRDDSNESD VVESLDEIY HTGTFAQIHE MQDLGDKLRM IVMGHRRVHI SRQLEVEPEE PEAENKHKPR RKSKRGKKEA EDELSARHPA E LAMEPTPE ...String: MHHHHHHENL YFQGAHMMTI PDVFPHLPLI AITRNPVFPR FIKIIEVKNK KLVELLRRKV RLAQPYVGVF LKRDDSNESD VVESLDEIY HTGTFAQIHE MQDLGDKLRM IVMGHRRVHI SRQLEVEPEE PEAENKHKPR RKSKRGKKEA EDELSARHPA E LAMEPTPE LPAEVLMVEV ENVVHEDFQV TEEVKALTAE IVKTIRDIIA LNPLYRESVL QMMQAGQRVV DNPIYLSDMG AA LTGAESH ELQDVLEETN IPKRLYKALS LLKKEFELSK LQQRLGREVE EKIKQTHRKY LLQEQLKIIK KELGLEKDDK DAI EEKFRE RLKELVVPKH VMDVVDEELS KLGLLDNHSS EFNVTRNYLD WLTSIPWGKY SNENLDLARA QAVLEEDHYG MEDV KKRIL EFIAVSQLRG STQGKILCFY GPPGVGKTSI ARSIARALNR EYFRFSVGGM TDVAEIKGHR RTYVGAMPGK IIQCL KKTK TENPLILIDE VDKIGRGYQG DPSSALLELL DPEQNANFLD HYLDVPVDLS KVLFICTANV TDTIPEPLRD RMEMIN VSG YVAQEKLAIA ERYLVPQARA LCGLDESKAK LSSDVLTLLI KQYCRESGVR NLQKQVEKVL RKSAYKIVSG EAESVEV TP ENLQDFVGKP VFTVERMYDV TPPGVVMGLA WTAMGGSTLF VETSLRRPQD KDAKGDKDGS LEVTGQLGEV MKESARIA Y TFARAFLMQH APANDYLVTS HIHLHVPEGA TPKDGPSAGC TIVTALLSLA MGRPVRQNLA MTGEVSLTGK ILPVGGIKE KTIAAKRAGV TCIVLPAENK KDFYDLAAFI TEGLEVHFVE HYREIFDIAF PDEQAEALAV ER UniProtKB: Lon protease homolog, mitochondrial |
-Macromolecule #3: ADENOSINE-5'-DIPHOSPHATE
Macromolecule | Name: ADENOSINE-5'-DIPHOSPHATE / type: ligand / ID: 3 / Number of copies: 4 / Formula: ADP |
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Molecular weight | Theoretical: 427.201 Da |
Chemical component information | ChemComp-ADP: |
-Macromolecule #4: MAGNESIUM ION
Macromolecule | Name: MAGNESIUM ION / type: ligand / ID: 4 / Number of copies: 4 / Formula: MG |
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Molecular weight | Theoretical: 24.305 Da |
-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: UltrAuFoil R1.2/1.3 / 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: 5 second blot blot force 2. | |||||||||
Details | Sample was monodisperse with ~350 particles per image |
-Electron microscopy
Microscope | FEI TITAN KRIOS |
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Specialist optics | Energy filter - Name: GIF Bioquantum / Energy filter - Slit width: 20 eV |
Image recording | Film or detector model: GATAN K3 (6k x 4k) / Digitization - Dimensions - Width: 5760 pixel / Digitization - Dimensions - Height: 4092 pixel / Number grids imaged: 1 / Number real images: 2931 / Average exposure time: 1.0 sec. / Average electron dose: 54.0 e/Å2 Details: Images were collected in movie mode at 50 frames per second. |
Electron beam | Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN |
Electron optics | C2 aperture diameter: 50.0 µm / Calibrated magnification: 60024 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 3.0 µm / Nominal defocus min: 0.4 µm / Nominal magnification: 105000 |
Sample stage | Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN |
Experimental equipment | Model: Titan Krios / Image courtesy: FEI Company |