+Open data
-Basic information
Entry | Database: PDB / ID: 6on2 | ||||||||||||||||||
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Title | Lon Protease from Yersinia pestis with Y2853 substrate | ||||||||||||||||||
Components |
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Keywords | HYDROLASE / Lon / mitochondrial protease / AAA+ / ATPase | ||||||||||||||||||
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 / cytoplasm Similarity search - Function | ||||||||||||||||||
Biological species | Yersinia pestis (bacteria) | ||||||||||||||||||
Method | ELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3 Å | ||||||||||||||||||
Authors | Shin, M. / Asmita, A. / Puchades, C. / Adjei, E. / Wiseman, R.L. / Karzai, A.W. / Lander, G.C. | ||||||||||||||||||
Funding support | United States, 5items
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Citation | Journal: Sci Adv / Year: 2020 Title: Structural basis for distinct operational modes and protease activation in AAA+ protease Lon. Authors: Mia Shin / Cristina Puchades / Ananya Asmita / Neha Puri / Eric Adjei / R Luke Wiseman / A Wali Karzai / Gabriel C Lander / Abstract: Substrate-bound structures of AAA+ protein translocases reveal a conserved asymmetric spiral staircase architecture wherein a sequential ATP hydrolysis cycle drives hand-over-hand substrate ...Substrate-bound structures of AAA+ protein translocases reveal a conserved asymmetric spiral staircase architecture wherein a sequential ATP hydrolysis cycle drives hand-over-hand substrate translocation. However, this configuration is unlikely to represent the full conformational landscape of these enzymes, as biochemical studies suggest distinct conformational states depending on the presence or absence of substrate. Here, we used cryo-electron microscopy to determine structures of the Lon AAA+ protease in the absence and presence of substrate, uncovering the mechanistic basis for two distinct operational modes. In the absence of substrate, Lon adopts a left-handed, "open" spiral organization with autoinhibited proteolytic active sites. Upon the addition of substrate, Lon undergoes a reorganization to assemble an enzymatically active, right-handed "closed" conformer with active protease sites. These findings define the mechanistic principles underlying the operational plasticity required for processing diverse protein substrates. | ||||||||||||||||||
History |
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-Structure visualization
Movie |
Movie viewer |
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Structure viewer | Molecule: MolmilJmol/JSmol |
-Downloads & links
-Download
PDBx/mmCIF format | 6on2.cif.gz | 526.1 KB | Display | PDBx/mmCIF format |
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PDB format | pdb6on2.ent.gz | 436.2 KB | Display | PDB format |
PDBx/mmJSON format | 6on2.json.gz | Tree view | PDBx/mmJSON format | |
Others | Other downloads |
-Validation report
Summary document | 6on2_validation.pdf.gz | 1.7 MB | Display | wwPDB validaton report |
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Full document | 6on2_full_validation.pdf.gz | 1.8 MB | Display | |
Data in XML | 6on2_validation.xml.gz | 86.1 KB | Display | |
Data in CIF | 6on2_validation.cif.gz | 127.7 KB | Display | |
Arichive directory | https://data.pdbj.org/pub/pdb/validation_reports/on/6on2 ftp://data.pdbj.org/pub/pdb/validation_reports/on/6on2 | HTTPS FTP |
-Related structure data
Related structure data | 20133MC 6v11C M: map data used to model this data C: citing same article (ref.) |
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Similar structure data |
-Links
-Assembly
Deposited unit |
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1 |
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-Components
#1: Protein | Mass: 57927.051 Da / Num. of mol.: 6 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Yersinia pestis (bacteria) / Gene: lon, YP_0776, EGT45_05820, NCTC144_01047 / Production host: Escherichia coli (E. coli) References: UniProt: A0A3N4AY83, UniProt: A0A5P8YJ65*PLUS, endopeptidase La #2: Protein/peptide | | Mass: 515.560 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Details: Y2853 substrate was added to Lon and modeled here as a polyalanine chain Source: (gene. exp.) Yersinia pestis (bacteria) / Production host: Escherichia coli (E. coli) #3: Chemical | ChemComp-ATP / #4: Chemical | ChemComp-MG / #5: Chemical | |
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-Experimental details
-Experiment
Experiment | Method: ELECTRON MICROSCOPY |
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EM experiment | Aggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction |
-Sample preparation
Component | Name: Lon protease bound to Y2853 substrate / Type: COMPLEX Details: Complexes consisting of homohexameric Lon protease from Yersinia pestis bound to Y2853 substrate were isolated using size-exclusion chromatography Entity ID: #1-#2 / Source: RECOMBINANT | ||||||||||||||||||||||||||||||
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Molecular weight | Units: MEGADALTONS / Experimental value: YES | ||||||||||||||||||||||||||||||
Source (natural) | Organism: Yersinia pestis (bacteria) / Cellular location: Cytoplasm | ||||||||||||||||||||||||||||||
Source (recombinant) | Organism: Escherichia coli (E. coli) / Plasmid: pET28b-lon | ||||||||||||||||||||||||||||||
Buffer solution | pH: 8 Details: Solutions were made fresh from concentrated and filtered using a 0.1 um syringe filter to avoid microbial contamination. Buffers were stored on ice and used within 15 minutes of mixing in ...Details: Solutions were made fresh from concentrated and filtered using a 0.1 um syringe filter to avoid microbial contamination. Buffers were stored on ice and used within 15 minutes of mixing in order to avoid excess ATP hydrolysis. | ||||||||||||||||||||||||||||||
Buffer component |
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Specimen | Conc.: 0.95 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES / Details: This sample was monodisperse | ||||||||||||||||||||||||||||||
Specimen support | Details: Grids were plasma treated for 30 seconds using a 15 mA current operating under atmospheric gases using a glow discharger (Electron Microscopy Sciences). Grid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil, UltrAuFoil, R1.2/1.3 | ||||||||||||||||||||||||||||||
Vitrification | Instrument: HOMEMADE PLUNGER / Cryogen name: ETHANE / Humidity: 95 % / Chamber temperature: 277 K Details: 4 uL of sample was applied per grid and manually blotted for 4 seconds followed by immediately plunge-freezing in liquid ethane cooled by liquid nitrogen. |
-Electron microscopy imaging
Experimental equipment | Model: Talos Arctica / Image courtesy: FEI Company |
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Microscopy | Model: FEI TALOS ARCTICA Details: Coma-free alignment procedure from Herzik & Wu, Nature Methods (2017). Preliminary grid screening was performed manually prior to data collection. |
Electron gun | Electron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: FLOOD BEAM |
Electron lens | Mode: BRIGHT FIELD / Nominal magnification: 36000 X / Calibrated magnification: 43478 X / Nominal defocus max: 1200 nm / Nominal defocus min: 800 nm / Calibrated defocus min: 500 nm / Calibrated defocus max: 1500 nm / Cs: 2.7 mm / C2 aperture diameter: 70 µm / Alignment procedure: COMA FREE |
Specimen holder | Cryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Temperature (max): 90 K / Temperature (min): 80 K / Residual tilt: 0.14 mradians |
Image recording | Average exposure time: 11 sec. / Electron dose: 52 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of grids imaged: 2 / Num. of real images: 4071 Details: Images were collected in counting mode at 4 frames per second |
Image scans | Sampling size: 5 µm / Width: 3710 / Height: 3838 / Movie frames/image: 44 / Used frames/image: 0-43 |
-Processing
Software | Name: PHENIX / Version: 1.11.1_2580: / Classification: refinement | |||||||||||||||||||||||||||||||||||||||||||||||||
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EM software |
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