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- EMDB-25502: Yeast Lon (PIM1) with endogenous substrate -

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Basic information

Entry
Database: EMDB / ID: EMD-25502
TitleYeast Lon (PIM1) with endogenous substrate
Map data
Sample
  • Complex: Cryo-EM structure of yeast Lon (PIM1) in complex with endogenous substrate
    • Protein or peptide: Lon protease homolog, mitochondrial
    • Protein or peptide: endogenous substrate
  • Ligand: ADENOSINE-5'-TRIPHOSPHATE
  • Ligand: MAGNESIUM ION
  • Ligand: ADENOSINE-5'-DIPHOSPHATE
KeywordsProtease / AAA+ ATPase / HYDROLASE
Function / homology
Function and homology information


regulation of mitochondrial DNA metabolic process / mitochondrial respiratory chain complex assembly / oxidation-dependent protein catabolic process / endopeptidase La / mitochondrial DNA metabolic process / Mitochondrial protein degradation / ATP-dependent peptidase activity / protein quality control for misfolded or incompletely synthesized proteins / chaperone-mediated protein complex assembly / mitochondrion organization ...regulation of mitochondrial DNA metabolic process / mitochondrial respiratory chain complex assembly / oxidation-dependent protein catabolic process / endopeptidase La / mitochondrial DNA metabolic process / Mitochondrial protein degradation / ATP-dependent peptidase activity / protein quality control for misfolded or incompletely synthesized proteins / chaperone-mediated protein complex assembly / mitochondrion organization / protein folding / single-stranded DNA binding / cellular response to oxidative stress / response to heat / sequence-specific DNA binding / mitochondrial matrix / serine-type endopeptidase activity / ATP hydrolysis activity / mitochondrion / ATP binding
Similarity search - Function
Lon protease homologue, chloroplastic/mitochondrial / Lon protease, bacterial/eukaryotic-type / Lon protease AAA+ ATPase lid domain / 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 N-terminal domain profile. ...Lon protease homologue, chloroplastic/mitochondrial / Lon protease, bacterial/eukaryotic-type / Lon protease AAA+ ATPase lid domain / 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 N-terminal domain profile. / Lon protease, N-terminal domain / Lon protease, N-terminal domain superfamily / 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 speciesSaccharomyces cerevisiae (brewer's yeast) / Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast) / Escherichia coli (E. coli)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.3 Å
AuthorsYang J / Song AS
Funding support United States, 1 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of Neurological Disorders and Stroke (NIH/NINDS) United States
CitationJournal: J Biol Chem / Year: 2022
Title: Cryo-EM structure of hexameric yeast Lon protease (PIM1) highlights the importance of conserved structural elements.
Authors: Jie Yang / Albert S Song / R Luke Wiseman / Gabriel C Lander /
Abstract: Lon protease is a conserved ATP-dependent serine protease composed of an AAA+ domain that mechanically unfolds substrates and a serine protease domain that degrades these unfolded substrates. In ...Lon protease is a conserved ATP-dependent serine protease composed of an AAA+ domain that mechanically unfolds substrates and a serine protease domain that degrades these unfolded substrates. In yeast, dysregulation of Lon protease (PIM1) attenuates lifespan and leads to gross mitochondrial morphological perturbations. Although structures of the bacterial and human Lon protease reveal a hexameric assembly, yeast PIM1 was speculated to form a heptameric assembly and is uniquely characterized by a ∼50-residue insertion between the ATPase and protease domains. To further understand the yeast-specific properties of PIM1, we determined a high-resolution cryo-electron microscopy structure of PIM1 in a substrate-translocating state. Here, we reveal that PIM1 forms a hexamer, conserved with that of bacterial and human Lon proteases, wherein the ATPase domains form a canonical closed spiral that enables pore loop residues to translocate substrates to the protease chamber. In the substrate-translocating state, PIM1 protease domains form a planar protease chamber in an active conformation and are uniquely characterized by a ∼15-residue C-terminal extension. These additional C-terminal residues form an α-helix located along the base of the protease domain. Finally, we did not observe density for the yeast-specific insertion between the ATPase and protease domains, likely due to high conformational flexibility. Biochemical studies to investigate the insertion using constructs that truncated or replaced the insertion with a glycine-serine linker suggest that the yeast-specific insertion is dispensable for PIM1's enzymatic function. Altogether, our structural and biochemical studies highlight unique components of PIM1 machinery and demonstrate evolutionary conservation of Lon protease function.
History
DepositionNov 24, 2021-
Header (metadata) releaseJan 12, 2022-
Map releaseJan 12, 2022-
UpdateOct 16, 2024-
Current statusOct 16, 2024Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.224
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 0.224
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-7sxo
  • Surface level: 0.224
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_25502.png

Downloads & links

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Map

FileDownload / File: emd_25502.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.15 Å/pix.
x 256 pix.
= 294.4 Å		1.15 Å/pix.
x 256 pix.
= 294.4 Å		1.15 Å/pix.
x 256 pix.
= 294.4 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: 0.224 / Movie #1: 0.224
Minimum - Maximum-0.84322894 - 1.7539219
Average (Standard dev.)0.0013748923 (±0.051334772)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 294.4 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.151.151.15
M x/y/z256256256
origin x/y/z0.0000.0000.000
length x/y/z294.400294.400294.400
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ720720720
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS256256256
D min/max/mean-0.8431.7540.001

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Supplemental data

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Sample components

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Entire : Cryo-EM structure of yeast Lon (PIM1) in complex with endogenous ...

EntireName: Cryo-EM structure of yeast Lon (PIM1) in complex with endogenous substrate
Components
  • Complex: Cryo-EM structure of yeast Lon (PIM1) in complex with endogenous substrate
    • Protein or peptide: Lon protease homolog, mitochondrial
    • Protein or peptide: endogenous substrate
  • Ligand: ADENOSINE-5'-TRIPHOSPHATE
  • Ligand: MAGNESIUM ION
  • Ligand: ADENOSINE-5'-DIPHOSPHATE

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Supramolecule #1: Cryo-EM structure of yeast Lon (PIM1) in complex with endogenous ...

SupramoleculeName: Cryo-EM structure of yeast Lon (PIM1) in complex with endogenous substrate
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#2
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)

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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: Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Strain: ATCC 204508 / S288c
Molecular weightTheoretical: 108.839602 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MNHKVSSHHH HHHSAGMLAL PIARRPLFPG FYKAVVISDE RVMKAIKEML DRQQPYIGAF MLKNSEEDTD VITDKNDVYD VGVLAQITS AFPSKDEKTG TETMTALLYP HRRIKIDELF PPNEEKEKSK EQAKDTDTET TVVEDANNPE DQESTSPATP K LEDIVVER ...String:
MNHKVSSHHH HHHSAGMLAL PIARRPLFPG FYKAVVISDE RVMKAIKEML DRQQPYIGAF MLKNSEEDTD VITDKNDVYD VGVLAQITS AFPSKDEKTG TETMTALLYP HRRIKIDELF PPNEEKEKSK EQAKDTDTET TVVEDANNPE DQESTSPATP K LEDIVVER IPDSELQHHK RVEATEEESE ELDDIQEGED INPTEFLKNY NVSLVNVLNL EDEPFDRKSP VINALTSEIL KV FKEISQL NTMFREQIAT FSASIQSATT NIFEEPARLA DFAAAVSAGE EDELQDILSS LNIEHRLEKS LLVLKKELMN AEL QNKISK DVETKIQKRQ REYYLMEQLK GIKRELGIDD GRDKLIDTYK ERIKSLKLPD SVQKIFDDEI TKLSTLETSM SEFG VIRNY LDWLTSIPWG KHSKEQYSIP RAKKILDEDH YGMVDVKDRI LEFIAVGKLL GKVDGKIICF VGPPGVGKTS IGKSI ARAL NRKFFRFSVG GMTDVAEIKG HRRTYIGALP GRVVQALKKC QTQNPLILID EIDKIGHGGI HGDPSAALLE VLDPEQ NNS FLDNYLDIPI DLSKVLFVCT ANSLETIPRP LLDRMEVIEL TGYVAEDKVK IAEQYLVPSA KKSAGLENSH VDMTEDA IT ALMKYYCRES GVRNLKKHIE KIYRKAALQV VKKLSIEDSP TSSADSKPKE SVSSEEKAEN NAKSSSEKTK DNNSEKTS D DIEALKTSEK INVSISQKNL KDYVGPPVYT TDRLYETTPP GVVMGLAWTN MGGCSLYVES VLEQPLHNCK HPTFERTGQ LGDVMKESSR LAYSFAKMYL AQKFPENRFF EKASIHLHCP EGATPKDGPS AGVTMATSFL SLALNKSIDP TVAMTGELTL TGKVLRIGG LREKAVAAKR SGAKTIIFPK DNLNDWEELP DNVKEGLEPL AADWYNDIFQ KLFKDVNTKE GNSVWKAEFE I LDAKKEKD

UniProtKB: Lon protease homolog, mitochondrial

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Macromolecule #2: endogenous substrate

MacromoleculeName: endogenous substrate / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Escherichia coli (E. coli)
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
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI TALOS ARCTICA
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Average electron dose: 50.0 e/Å2
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD
Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company

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Image processing

Startup modelType of model: PDB ENTRY
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.3 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 81945
Initial angle assignmentType: ANGULAR RECONSTITUTION
Final angle assignmentType: ANGULAR RECONSTITUTION

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