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- EMDB-31590: Processive cleavage of substrate at individual proteolytic active... -

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Basic information

Entry
Database: EMDB / ID: EMD-31590
TitleProcessive cleavage of substrate at individual proteolytic active sites of the Lon protease complex (conformation 2)
Map data
Sample
  • Complex: Lon bound to endogenous substrate
    • Protein or peptide: Lon proteaseLon protease family
    • Protein or peptide: Unknown endogenous substrate
  • Ligand: PHOSPHOTHIOPHOSPHORIC ACID-ADENYLATE ESTER
  • Ligand: ADENOSINE-5'-DIPHOSPHATE
Function / homology
Function and homology information


endopeptidase La / ATP-dependent peptidase activity / protein quality control for misfolded or incompletely synthesized proteins / cellular response to heat / sequence-specific DNA binding / serine-type endopeptidase activity / ATP hydrolysis activity / ATP binding / identical protein binding / cytoplasm
Similarity search - Function
Lon protease, bacterial / 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, bacterial / 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
Biological speciesMeiothermus taiwanensis WR-220 (bacteria) / Meiothermus taiwanensis (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.36 Å
AuthorsLi S / Hsieh K / Kuo C / Su S / Huang K / Zhang K / Chang CI
Funding support Taiwan, 1 items
OrganizationGrant numberCountry
Ministry of Science and Technology (MoST, Taiwan)108-2320-B-001-011-MY3 Taiwan
CitationJournal: Sci Adv / Year: 2021
Title: Processive cleavage of substrate at individual proteolytic active sites of the Lon protease complex.
Authors: Shanshan Li / Kan-Yen Hsieh / Chiao-I Kuo / Shih-Chieh Su / Kai-Fa Huang / Kaiming Zhang / Chung-I Chang /
Abstract: The Lon protease is the prototype of a family of proteolytic machines with adenosine triphosphatase modules built into a substrate degradation chamber. Lon is known to degrade protein substrates in a ...The Lon protease is the prototype of a family of proteolytic machines with adenosine triphosphatase modules built into a substrate degradation chamber. Lon is known to degrade protein substrates in a processive fashion, cutting a protein chain processively into small peptides before commencing cleavages of another protein chain. Here, we present structural and biochemical evidence demonstrating that processive substrate degradation occurs at each of the six proteolytic active sites of Lon, which forms a deep groove that partially encloses the substrate polypeptide chain by accommodating only the unprimed residues and permits processive cleavage in the C-to-N direction. We identify a universally conserved acidic residue at the exit side of the binding groove indispensable for the proteolytic activity. This noncatalytic residue likely promotes processive proteolysis by carboxyl-carboxylate interactions with cleaved intermediates. Together, these results uncover a previously unrecognized mechanism for processive substrate degradation by the Lon protease.
History
DepositionJul 31, 2021-
Header (metadata) releaseNov 24, 2021-
Map releaseNov 24, 2021-
UpdateNov 24, 2021-
Current statusNov 24, 2021Processing site: PDBj / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.204
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.204
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-7fie
  • Surface level: 0.204
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
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Supplemental images

Downloads & links

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Map

FileDownload / File: emd_31590.map.gz / Format: CCP4 / Size: 144.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 0.82 Å
Density
Contour LevelBy AUTHOR: 0.204 / Movie #1: 0.204
Minimum - Maximum-1.0436804 - 4.0523105
Average (Standard dev.)0.0004395443 (±0.1067115)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions336336336
Spacing336336336
CellA=B=C: 275.52 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z0.820.820.82
M x/y/z336336336
origin x/y/z0.0000.0000.000
length x/y/z275.520275.520275.520
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ200200200
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS336336336
D min/max/mean-1.0444.0520.000

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Supplemental data

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Sample components

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Entire : Lon bound to endogenous substrate

EntireName: Lon bound to endogenous substrate
Components
  • Complex: Lon bound to endogenous substrate
    • Protein or peptide: Lon proteaseLon protease family
    • Protein or peptide: Unknown endogenous substrate
  • Ligand: PHOSPHOTHIOPHOSPHORIC ACID-ADENYLATE ESTER
  • Ligand: ADENOSINE-5'-DIPHOSPHATE

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Supramolecule #1: Lon bound to endogenous substrate

SupramoleculeName: Lon bound to endogenous substrate / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#2
Source (natural)Organism: Meiothermus taiwanensis WR-220 (bacteria)
Recombinant expressionOrganism: Escherichia coli (E. coli)
Molecular weightExperimental: 600 KDa

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Macromolecule #1: Lon protease

MacromoleculeName: Lon protease / type: protein_or_peptide / ID: 1 / Number of copies: 6 / Enantiomer: LEVO / EC number: endopeptidase La
Source (natural)Organism: Meiothermus taiwanensis (bacteria)
Molecular weightTheoretical: 90.081336 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MRLELPVIPL RNTVILPHTT TPVDVGRAKS KRAVEEAMGA DRLIFLVAQR DPEVDDPAPD DLYTWGVQAV VKQAMRLPDG TLQVMVEAR ARAQVTDYIP GPYLRARGEV FSEIFPIDEA VVRVLVEELK EAFEKYVANH KSLRLDRYQL EAVKGTSDPA M LADTIAYH ...String:
MRLELPVIPL RNTVILPHTT TPVDVGRAKS KRAVEEAMGA DRLIFLVAQR DPEVDDPAPD DLYTWGVQAV VKQAMRLPDG TLQVMVEAR ARAQVTDYIP GPYLRARGEV FSEIFPIDEA VVRVLVEELK EAFEKYVANH KSLRLDRYQL EAVKGTSDPA M LADTIAYH ATWTVAEKQE ILELTDLEAR LKKVLGLLSR DLERFELDKR VAQRVKEQMD TNQREYYLRE QMKAIQKELG GE DGLSDLE ALRKKIEEVG MPEAVKTKAL KELDRLERMQ QGSPEATVAR TYLDWLTEVP WSKADPEVLD INHTRQVLDE DHY GLKDVK ERILEYLAVR QLTQGLDVRN KAPILVLVGP PGVGKTSLGR SIARSMNRKF HRISLGGVRD EAEIRGHRRT YIGA MPGKL IHAMKQVGVI NPVILLDEID KMSSDWRGDP ASAMLEVLDP EQNNTFTDHY LDVPYDLSKV FFITTANTLQ TIPRP LLDR MEVIEIPGYT NMEKQAIARQ YLWPKQVRES GMEGRIEVTD AAILRVISEY TREAGVRGLE RELGKIARKG AKFWLE GAW EGLRTIDASD IPTYLGIPRY RPDKAETEPQ VGTAQGLAWT PVGGTLLTIE VAAVPGSGKL SLTGQLGEVM KESAQAA LT YLRAHTQDYG LPEDFYNKVD LHVHVPDGAT PKDGPSAGIT MATAIASALS RRPARMDIAM TGEVSLRGKV MPIGGVKE K LLAAHQAGIH KIVLPKDNEA QLEELPKEVL EGLEIKLVED VGEVLEYLLL PEPTMPPVVQ PSDNRQQPGA GAKLAAALE HHHHHH

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Macromolecule #2: Unknown endogenous substrate

MacromoleculeName: Unknown endogenous substrate / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Meiothermus taiwanensis WR-220 (bacteria)
Molecular weightTheoretical: 1.890321 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK) (UNK)(UNK)(UNK)(UNK)(UNK)(UNK) (UNK)(UNK)(UNK) (UNK)(UNK)(UNK)

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Macromolecule #3: PHOSPHOTHIOPHOSPHORIC ACID-ADENYLATE ESTER

MacromoleculeName: PHOSPHOTHIOPHOSPHORIC ACID-ADENYLATE ESTER / type: ligand / ID: 3 / Number of copies: 4 / Formula: AGS
Molecular weightTheoretical: 523.247 Da
Chemical component information

ChemComp-AGS:
PHOSPHOTHIOPHOSPHORIC ACID-ADENYLATE ESTER / ATP-gamma-S, energy-carrying molecule analogue*YM

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Macromolecule #4: ADENOSINE-5'-DIPHOSPHATE

MacromoleculeName: ADENOSINE-5'-DIPHOSPHATE / type: ligand / ID: 4 / Number of copies: 2 / Formula: ADP
Molecular weightTheoretical: 427.201 Da
Chemical component information

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

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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

BufferpH: 7.5
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 200 / Pretreatment - Type: GLOW DISCHARGE
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy
Image recordingFilm or detector model: FEI FALCON IV (4k x 4k) / Average electron dose: 48.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 2.36 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 3.0) / Number images used: 361692

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