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- PDB-6v11: Lon Protease from Yersinia pestis -

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Basic information

Entry
Database: PDB / ID: 6v11
TitleLon Protease from Yersinia pestis
ComponentsLon protease
KeywordsHYDROLASE / AAA+ ATPase / Quality Control / Protease
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
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
ADENOSINE-5'-DIPHOSPHATE / Lon protease / Lon protease
Similarity search - Component
Biological speciesYersinia pestis (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.8 Å
AuthorsShin, M. / Puchades, C. / Asmita, A. / Puri, N. / Adjei, E. / Wiseman, R.L. / Karzai, A.W. / Lander, G.C.
Funding support United States, 5items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of Biomedical Imaging and Bioengineering (NIH/NIBIB)DP2EB020402 United States
National Institutes of Health/National Institute of Neurological Disorders and Stroke (NIH/NINDS)NS095892 United States
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)AI-127533 United States
National Institutes of Health/National Institute on Aging (NIH/NIA)AG061697 United States
National Institutes of Health/Office of the DirectorS10OD021634 United States
CitationJournal: 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
DepositionNov 19, 2019Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jan 22, 2020Provider: repository / Type: Initial release
Revision 1.1Jan 29, 2020Group: Data processing / Category: em_3d_reconstruction / Item: _em_3d_reconstruction.resolution
Revision 1.2Jun 17, 2020Group: Database references / Category: citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year
Revision 1.3Mar 6, 2024Group: Data collection / Database references / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / em_3d_fitting_list / pdbx_initial_refinement_model
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id / _em_3d_fitting_list.source_name / _em_3d_fitting_list.type

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Structure visualization

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  • Deposited structure unit
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  • Imaged by UCSF Chimera
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Structure viewerMolecule:
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Assembly

Deposited unit
A: Lon protease
B: Lon protease
C: Lon protease
D: Lon protease
E: Lon protease
F: Lon protease
hetero molecules


Theoretical massNumber of molelcules
Total (without water)348,82111
Polymers346,6856
Non-polymers2,1365
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein
Lon protease / ATP-dependent protease La


Mass: 57780.863 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: Chemical
ChemComp-ADP / ADENOSINE-5'-DIPHOSPHATE


Mass: 427.201 Da / Num. of mol.: 5 / Source method: obtained synthetically / Formula: C10H15N5O10P2 / Comment: ADP, energy-carrying molecule*YM
Has ligand of interestN

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: Lon Protease from Yersinia pestis / Type: COMPLEX
Details: Complexes consisting of homohexameric Lon protease from Yersinia pestis were isolated using size-exclusion chromatography
Entity ID: #1 / Source: RECOMBINANT
Molecular weightUnits: MEGADALTONS / Experimental value: NO
Source (natural)Organism: Yersinia pestis (bacteria) / Cellular location: Cytoplasm
Source (recombinant)Organism: Escherichia coli (E. coli) / Plasmid: pET28b-lon
Buffer solutionpH: 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
IDConc.NameFormulaBuffer-ID
150 mMTris BaseTris1
275 mMPotassium ChlorideKCl1
310 mMMagnesium ChlorideMgCl21
41 mMTCEPTCEP1
51 mMAdenosine TriphosphateATP1
SpecimenConc.: 17 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES / Details: This sample was monodisperse
Specimen supportGrid material: GOLD / Grid mesh size: 400 divisions/in. / Grid type: UltrAuFoil
VitrificationInstrument: 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.

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Electron microscopy imaging

Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company
MicroscopyModel: 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 gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: FLOOD BEAM
Electron lensMode: 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 holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Temperature (max): 90 K / Temperature (min): 80 K / Residual tilt: 0.14 mradians
Image recordingAverage exposure time: 11.6 sec. / Electron dose: 50 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 1864
Details: Images were collected in counting mode at 5 frames per second
Image scansSampling size: 5 µm / Width: 3710 / Height: 3838 / Movie frames/image: 58 / Used frames/image: 0-57

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Processing

EM software
IDNameVersionCategoryDetails
2Leginon3image acquisitionLeginon software was used for automated data collection
4Warp1.0.7CTF correction
7Coot0.8.8model fittingCoot was used for de novo atomic model building
9cryoSPARC2.11.0initial Euler assignmentcryoSPARC was used to assign initial angles
10cryoSPARC2.11.0final Euler assignmentcryoSPARC was used to assign final euler angles
11cryoSPARC2.11.0classificationcryoSPARC was used to perform final classification
12cryoSPARC2.11.03D reconstructioncryoSPARC was used to perform final reconstruction
13PHENIX1.11.1model refinementPhenix 1.11.1 was used to perform real space refinement using the atomic model and experimentally-derived EM map
CTF correctionDetails: CTF correction in Warp / Type: PHASE FLIPPING ONLY
Particle selectionNum. of particles selected: 412719
Details: Particles were selected from a subset of micrographs using BoxNet automated particle picker
3D reconstructionResolution: 3.8 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 140506 / Num. of class averages: 2 / Symmetry type: POINT
Atomic model buildingB value: 115 / Protocol: RIGID BODY FIT / Space: REAL / Target criteria: Correlation coefficient
Details: Initial rigid body docking was done using UCSF Chimera
Atomic model buildingPDB-ID: 6ON2

6on2


Pdb chain-ID: A / Accession code: 6ON2 / Pdb chain residue range: 253-775 / Source name: PDB / Type: experimental model

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