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- EMDB-21870: Molecular basis for the ATPase-powered substrate translocation by... -

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Basic information

Entry
Database: EMDB / ID: EMD-21870
TitleMolecular basis for the ATPase-powered substrate translocation by the Lon AAA+ protease
Map dataMeiothermus taiwanensis LonA (MtaLonA) in a substrate-engaged state
Sample
  • Complex: Meiothermus taiwanensis LonA (MtaLonA) in a substrate-engaged state
    • Complex: Lon protease
      • Protein or peptide: Lon protease
    • Complex: Ig2 substrate
      • Protein or peptide: Ig2 substrate
  • Ligand: PHOSPHOTHIOPHOSPHORIC ACID-ADENYLATE ESTER
  • Ligand: N-[(1R)-1-(dihydroxyboranyl)-2-phenylethyl]-Nalpha-(pyrazin-2-ylcarbonyl)-L-phenylalaninamide
  • Ligand: ADENOSINE-5'-DIPHOSPHATE
KeywordsLon AAA+ / protease / cryo-EM / dual pore-loops / MOTOR PROTEIN
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 / metal ion binding / identical protein binding / cytoplasm
Similarity search - Function
Lon protease, bacterial / : / 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 protease, bacterial / : / 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
Biological speciesMeiothermus taiwanensis (bacteria) / Dictyostelium discoideum (eukaryote)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.6 Å
AuthorsZhang K / Li S
Funding support United States, 3 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)P41GM103832 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01GM079429 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)S10OD021600 United States
CitationJournal: J Biol Chem / Year: 2021
Title: Molecular basis for ATPase-powered substrate translocation by the Lon AAA+ protease.
Authors: Shanshan Li / Kan-Yen Hsieh / Shih-Chieh Su / Grigore D Pintilie / Kaiming Zhang / Chung-I Chang /
Abstract: The Lon AAA+ (adenosine triphosphatases associated with diverse cellular activities) protease (LonA) converts ATP-fuelled conformational changes into sufficient mechanical force to drive ...The Lon AAA+ (adenosine triphosphatases associated with diverse cellular activities) protease (LonA) converts ATP-fuelled conformational changes into sufficient mechanical force to drive translocation of a substrate into a hexameric proteolytic chamber. To understand the structural basis for the substrate translocation process, we determined the cryo-electron microscopy (cryo-EM) structure of Meiothermus taiwanensis LonA (MtaLonA) in a substrate-engaged state at 3.6 Å resolution. Our data indicate that substrate interactions are mediated by the dual pore loops of the ATPase domains, organized in spiral staircase arrangement from four consecutive protomers in different ATP-binding and hydrolysis states. However, a closed AAA+ ring is maintained by two disengaged ADP-bound protomers transiting between the lowest and highest position. This structure reveals a processive rotary translocation mechanism mediated by LonA-specific nucleotide-dependent allosteric coordination among the ATPase domains, which is induced by substrate binding.
History
DepositionApr 28, 2020-
Header (metadata) releaseJun 9, 2021-
Map releaseJun 9, 2021-
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: 0.32
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 0.32
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6wqh
  • Surface level: 0.32
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_21870.png

Downloads & links

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Map

FileDownload / File: emd_21870.map.gz / Format: CCP4 / Size: 144.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationMeiothermus taiwanensis LonA (MtaLonA) in a substrate-engaged state
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.65 Å/pix.
x 336 pix.
= 218.4 Å		0.65 Å/pix.
x 336 pix.
= 218.4 Å		0.65 Å/pix.
x 336 pix.
= 218.4 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.65 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.32 / Movie #1: 0.32
Minimum - Maximum-0.6366026 - 1.2187893
Average (Standard dev.)0.009393275 (±0.0757663)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions336336336
Spacing336336336
CellA=B=C: 218.4 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z0.650.650.65
M x/y/z336336336
origin x/y/z0.0000.0000.000
length x/y/z218.400218.400218.400
α/β/γ90.00090.00090.000
start NX/NY/NZ727265
NX/NY/NZ157157169
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS336336336
D min/max/mean-0.6371.2190.009

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Supplemental data

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Sample components

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Entire : Meiothermus taiwanensis LonA (MtaLonA) in a substrate-engaged state

EntireName: Meiothermus taiwanensis LonA (MtaLonA) in a substrate-engaged state
Components
  • Complex: Meiothermus taiwanensis LonA (MtaLonA) in a substrate-engaged state
    • Complex: Lon protease
      • Protein or peptide: Lon protease
    • Complex: Ig2 substrate
      • Protein or peptide: Ig2 substrate
  • Ligand: PHOSPHOTHIOPHOSPHORIC ACID-ADENYLATE ESTER
  • Ligand: N-[(1R)-1-(dihydroxyboranyl)-2-phenylethyl]-Nalpha-(pyrazin-2-ylcarbonyl)-L-phenylalaninamide
  • Ligand: ADENOSINE-5'-DIPHOSPHATE

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Supramolecule #1: Meiothermus taiwanensis LonA (MtaLonA) in a substrate-engaged state

SupramoleculeName: Meiothermus taiwanensis LonA (MtaLonA) in a substrate-engaged state
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#2
Molecular weightTheoretical: 360 KDa

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Supramolecule #2: Lon protease

SupramoleculeName: Lon protease / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1
Source (natural)Organism: Meiothermus taiwanensis (bacteria)

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Supramolecule #3: Ig2 substrate

SupramoleculeName: Ig2 substrate / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #2
Source (natural)Organism: Dictyostelium discoideum (eukaryote)

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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: 88.701766 KDa
Recombinant expressionOrganism: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)
SequenceString: MRLELPVIPL RNTVILPHTT TPVDVGRAKS KRAVEEAMGA DRLIFLVAQR DPEVDDPAPD DLYTWGVQAV VKQAMRLPDG TLQVMVEAR ARAFQVTDYI PGPYLRARGE VFSEIFPIDE AVVRVLVEEL KEAFEKYVAN HKSLRLDRYQ LEAVKGTSDP A MLADTIAY ...String:
MRLELPVIPL RNTVILPHTT TPVDVGRAKS KRAVEEAMGA DRLIFLVAQR DPEVDDPAPD DLYTWGVQAV VKQAMRLPDG TLQVMVEAR ARAFQVTDYI PGPYLRARGE VFSEIFPIDE AVVRVLVEEL KEAFEKYVAN HKSLRLDRYQ LEAVKGTSDP A MLADTIAY HATWTVAEKQ EILELTDLEA RLKKVLGLLS RDLERFELDK RVAQRVKEQM DTNQREYYLR EQMKAIQKEL GG EDGLSDL EALRKKIEEV GMPEAVKTKA LKELDRLERM QQGSPEATVA RTYLDWLTEV PWSKADPEVL DINHTRQVLD EDH YGLKDV KERILEYLAV RQLTQGLDVR NKAPILVLVG PPGVGKTSLG RSIARSMNRK FHRISLGGVR DEAEIRGHRR TYIG AMPGK LIHAMKQVGV INPVILLDEI DKMSSDWRGD PASAMLEVLD PEQNNTFTDH YLDVPYDLSK VFFITTANTL QTIPR PLLD RMEVIEIPGY TNMEKQAIAR QYLWPKQVRE SGMEGRIEVT DAAILRVISE YTREAGVRGL ERELGKIARK GAKFWL EGA WEGLRTIDAS DIPTYLGIPR YRPDKAETEP QVGTAQGLAW TPVGGTLLTI EVAAVPGSGK LSLTGQLGEV MKESAQA AL TYLRAHTQDY GLPEDFYNKV DLHVHVPDGA TPKDGPSAGI TMATAIASAL SRRPARMDIA MTGEVSLRGK VMPIGGVK E KLLAAHQAGI HKIVLPKDNE AQLEELPKEV LEGLEIKLVE DVGEVLEYLL LPEPTMPPVV QPSDNRQQPG AGA

UniProtKB: Lon protease

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Macromolecule #2: Ig2 substrate

MacromoleculeName: Ig2 substrate / type: protein_or_peptide / ID: 2 / Details: Unfolded substate / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Dictyostelium discoideum (eukaryote)
Molecular weightTheoretical: 954.168 Da
Recombinant expressionOrganism: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)
SequenceString:
(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: 3 / 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: N-[(1R)-1-(dihydroxyboranyl)-2-phenylethyl]-Nalpha-(pyrazin-2-ylc...

MacromoleculeName: N-[(1R)-1-(dihydroxyboranyl)-2-phenylethyl]-Nalpha-(pyrazin-2-ylcarbonyl)-L-phenylalaninamide
type: ligand / ID: 4 / Number of copies: 6 / Formula: 4KZ
Molecular weightTheoretical: 418.253 Da
Chemical component information

ChemComp-4KZ:
N-[(1R)-1-(dihydroxyboranyl)-2-phenylethyl]-Nalpha-(pyrazin-2-ylcarbonyl)-L-phenylalaninamide

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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

Concentration0.15 mg/mL
BufferpH: 8
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 200 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 40 sec.
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Number real images: 1800 / Average exposure time: 6.0 sec. / Average electron dose: 8.5 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 70.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: NONE
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 3.6 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.0.2) / Number images used: 23487
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD

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Atomic model buiding 1

Initial modelPDB ID:

4ypl


Chain - Source name: PDB / Chain - Initial model type: experimental model
Output model

PDB-6wqh:
Molecular basis for the ATPase-powered substrate translocation by the Lon AAA+ protease

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