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- EMDB-45434: LONP1 stall state bound to substrate and 4 ADPs -

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Basic information

Entry
Database: EMDB / ID: EMD-45434
TitleLONP1 stall state bound to substrate and 4 ADPs
Map dataSharpened map
Sample
  • Complex: LONP1 bound to substrate and ADP
    • Protein or peptide: Human mitochondrial Lon Protease homolog
KeywordsATPase / protease / HYDROLASE
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.18 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_45434.png

Downloads & links

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Map

FileDownload / File: emd_45434.map.gz / Format: CCP4 / Size: 91.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationSharpened map
Projections & slices

Image control

Size
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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)

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Images are generated by Spider.

Voxel sizeX=Y=Z: 1.15 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.25
Minimum - Maximum-0.6610794 - 1.3890935
Average (Standard dev.)0.00079117 (±0.04138167)
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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Additional map: Unsharpened map

Fileemd_45434_additional_1.map
AnnotationUnsharpened map
Projections & Slices
AxesZYX

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Half map: Half map B

Fileemd_45434_half_map_1.map
AnnotationHalf map B
Projections & Slices
AxesZYX

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Slices (1/2)
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Histogram

Histogram (log scale)

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Half map: Half map A

Fileemd_45434_half_map_2.map
AnnotationHalf map A
Projections & Slices
AxesZYX

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Slices (1/2)
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Histogram

Histogram (log scale)

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Sample components

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Entire : LONP1 bound to substrate and ADP

EntireName: LONP1 bound to substrate and ADP
Components
  • Complex: LONP1 bound to substrate and ADP
    • Protein or peptide: Human mitochondrial Lon Protease homolog

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Supramolecule #1: LONP1 bound to substrate and ADP

SupramoleculeName: LONP1 bound to substrate and ADP / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 583.95 KDa

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Macromolecule #1: Human mitochondrial Lon Protease homolog

MacromoleculeName: Human mitochondrial Lon Protease homolog / type: protein_or_peptide / ID: 1 / Details: homohexameric assembly / Enantiomer: LEVO / EC number: endopeptidase La
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)
SequenceString: 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

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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: 1944 / 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 / Algorithm: BACK PROJECTION / Resolution.type: BY AUTHOR / Resolution: 3.18 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 4.3) / Number images used: 94000
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: 5 / Software - Name: cryoSPARC (ver. 4.3)
FSC plot (resolution estimation)

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