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- EMDB-45430: Human Mitochondrial LONP1 Degrading Casein, ATP-bound closed form -

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Basic information

Entry
Database: EMDB / ID: EMD-45430
TitleHuman Mitochondrial LONP1 Degrading Casein, ATP-bound closed form
Map dataSharpened map of LONP1 enzymatic assembly
Sample
  • Complex: LONP1 degrading casein
    • Protein or peptide: Lon protease homolog, mitochondrial
    • Protein or peptide: Bound substrate segment undergoing translocation and subsequent degradation
  • Ligand: ADENOSINE-5'-TRIPHOSPHATE
  • Ligand: MAGNESIUM ION
  • Ligand: ADENOSINE-5'-DIPHOSPHATE
KeywordsATPase / 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
Lon protease AAA+ ATPase lid domain / Lon protease homologue, chloroplastic/mitochondrial / 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 protease AAA+ ATPase lid domain / Lon protease homologue, chloroplastic/mitochondrial / 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
Lon protease homolog, mitochondrial
Similarity search - Component
Biological speciesHomo sapiens (human) / Bos taurus (cattle)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.31 Å
AuthorsMindrebo JT / Lander GC
Funding support United States, 2 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)F32GM145143 United States
National Institutes of Health/National Institute of Neurological Disorders and Stroke (NIH/NINDS)NS095892 United States
CitationJournal: 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
DepositionJun 20, 2024-
Header (metadata) releaseAug 7, 2024-
Map releaseAug 7, 2024-
UpdateAug 7, 2024-
Current statusAug 7, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_45430.png

Downloads & links

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Map

FileDownload / File: emd_45430.map.gz / Format: CCP4 / Size: 91.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationSharpened map of LONP1 enzymatic assembly
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.15 Å/pix.
x 288 pix.
= 331.2 Å		1.15 Å/pix.
x 288 pix.
= 331.2 Å		1.15 Å/pix.
x 288 pix.
= 331.2 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.15 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.35
Minimum - Maximum-0.7422462 - 1.4338692
Average (Standard dev.)0.0012781628 (±0.045678146)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions288288288
Spacing288288288
CellA=B=C: 331.19998 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_45430_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: Unsharpened map of LONP1 enzymatic assembly

Fileemd_45430_additional_1.map
AnnotationUnsharpened map of LONP1 enzymatic assembly
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: LONP1 enzymatic assembly half map B

Fileemd_45430_half_map_1.map
AnnotationLONP1 enzymatic assembly half map B
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: LONP1 enzymatic assembly half map A

Fileemd_45430_half_map_2.map
AnnotationLONP1 enzymatic assembly half 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 : LONP1 degrading casein

EntireName: LONP1 degrading casein
Components
  • Complex: LONP1 degrading casein
    • Protein or peptide: Lon protease homolog, mitochondrial
    • Protein or peptide: Bound substrate segment undergoing translocation and subsequent degradation
  • Ligand: ADENOSINE-5'-TRIPHOSPHATE
  • Ligand: MAGNESIUM ION
  • Ligand: ADENOSINE-5'-DIPHOSPHATE

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Supramolecule #1: LONP1 degrading casein

SupramoleculeName: LONP1 degrading casein / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#2
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 583.95 KDa

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Macromolecule #1: Lon protease homolog, mitochondrial

MacromoleculeName: Lon protease homolog, mitochondrial / type: protein_or_peptide / ID: 1 / Number of copies: 6 / Enantiomer: LEVO / EC number: endopeptidase La
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 97.468156 KDa
Recombinant expressionOrganism: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)
SequenceString: 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

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Macromolecule #2: Bound substrate segment undergoing translocation and subsequent d...

MacromoleculeName: Bound substrate segment undergoing translocation and subsequent degradation
type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Bos taurus (cattle)
Molecular weightTheoretical: 1.039273 KDa
SequenceString:
(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK) (UNK)(UNK)

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Macromolecule #3: ADENOSINE-5'-TRIPHOSPHATE

MacromoleculeName: ADENOSINE-5'-TRIPHOSPHATE / type: ligand / ID: 3 / Number of copies: 4 / Formula: ATP
Molecular weightTheoretical: 507.181 Da
Chemical component information

ChemComp-ATP:
ADENOSINE-5'-TRIPHOSPHATE / ATP, energy-carrying molecule*YM

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Macromolecule #4: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 4 / Number of copies: 4 / Formula: MG
Molecular weightTheoretical: 24.305 Da

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Macromolecule #5: ADENOSINE-5'-DIPHOSPHATE

MacromoleculeName: ADENOSINE-5'-DIPHOSPHATE / type: ligand / ID: 5 / Number of copies: 2 / Formula: ADP
Molecular weightTheoretical: 427.201 Da
Chemical component information

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

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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

Concentration3 mg/mL
BufferpH: 8
Component:
ConcentrationFormulaName
75.0 mMKClpotassium chloride
50.0 mMC4H11NO3Tris
GridModel: 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
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 298 K / Instrument: FEI VITROBOT MARK IV
DetailsSample was monodisperse with ~300 particles per image.

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Electron microscopy

MicroscopeFEI TALOS ARCTICA
Image recordingFilm 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: 1938 / Average exposure time: 9.8 sec. / Average electron dose: 50.0 e/Å2
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 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 stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 740316 / Details: Template Picker
Startup modelType of model: OTHER / Details: Ab initio model generated in cryosparc
Final reconstructionNumber classes used: 1 / Resolution.type: BY AUTHOR / Resolution: 3.31 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 4.3) / Number images used: 53025
Initial angle assignmentType: PROJECTION MATCHING / Software - Name: cryoSPARC (ver. 4.3)
Final angle assignmentType: PROJECTION MATCHING / Software - Name: cryoSPARC (ver. 4.3)
Final 3D classificationNumber classes: 6 / Software - Name: cryoSPARC (ver. 4.3)
FSC plot (resolution estimation)

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