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- PDB-6on2: Lon Protease from Yersinia pestis with Y2853 substrate -

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Basic information

Entry
Database: PDB / ID: 6on2
TitleLon Protease from Yersinia pestis with Y2853 substrate
Components
  • ATP-dependent protease LaEndopeptidase La
  • Bound Y2853 Substrate
KeywordsHYDROLASE / 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
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 / ADENOSINE-5'-TRIPHOSPHATE / Lon protease / Lon protease
Similarity search - Component
Biological speciesYersinia pestis (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3 Å
AuthorsShin, M. / Asmita, A. / Puchades, C. / 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
DepositionApr 19, 2019Deposition site: RCSB / Processing site: RCSB
Revision 1.0May 1, 2019Provider: repository / Type: Initial release
Revision 1.1Dec 11, 2019Group: Author supporting evidence / Category: pdbx_audit_support / Item: _pdbx_audit_support.funding_organization
Revision 1.2Dec 2, 2020Group: Database references / Derived calculations / Category: citation / citation_author / struct_conn
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 / _struct_conn.pdbx_dist_value / _struct_conn.ptnr1_auth_asym_id / _struct_conn.ptnr1_auth_comp_id / _struct_conn.ptnr1_auth_seq_id / _struct_conn.ptnr1_label_asym_id / _struct_conn.ptnr1_label_atom_id / _struct_conn.ptnr1_label_comp_id / _struct_conn.ptnr1_label_seq_id / _struct_conn.ptnr2_auth_asym_id / _struct_conn.ptnr2_label_asym_id
Revision 1.3Mar 20, 2024Group: Data collection / Database references / Category: chem_comp_atom / chem_comp_bond / database_2
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession

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Structure visualization

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  • Deposited structure unit
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Structure viewerMolecule:
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Assembly

Deposited unit
A: ATP-dependent protease La
B: ATP-dependent protease La
C: ATP-dependent protease La
D: ATP-dependent protease La
E: ATP-dependent protease La
F: ATP-dependent protease La
G: Bound Y2853 Substrate
hetero molecules


Theoretical massNumber of molelcules
Total (without water)351,08318
Polymers348,0787
Non-polymers3,00511
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: assay for oligomerization, Full-length Y. pestis Lon bearing the slowly ATP hydrolyzing Walker B mutation (E424Q) was incubated with an excess of Y2853, an 18 kDa substrate. Substrate-bound ...Evidence: assay for oligomerization, Full-length Y. pestis Lon bearing the slowly ATP hydrolyzing Walker B mutation (E424Q) was incubated with an excess of Y2853, an 18 kDa substrate. Substrate-bound complexes were separated from unbound substrate using size-exclusion chromatography. Identity of elution components was confirmed using SDS-PAGE/IB
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein
ATP-dependent protease La / Endopeptidase La / DNA-binding ATP-dependent protease La / Endopeptidase La


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 Bound Y2853 Substrate


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 / ADENOSINE-5'-TRIPHOSPHATE / Adenosine triphosphate


Mass: 507.181 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: C10H16N5O13P3 / Comment: ATP, energy-carrying molecule*YM
#4: Chemical
ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 5 / Source method: obtained synthetically / Formula: Mg
#5: Chemical ChemComp-ADP / ADENOSINE-5'-DIPHOSPHATE / Adenosine diphosphate


Mass: 427.201 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C10H15N5O10P2 / Comment: ADP, energy-carrying molecule*YM

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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 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
Molecular weightUnits: MEGADALTONS / Experimental value: YES
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 TriphosphateATPAdenosine triphosphate1
SpecimenConc.: 0.95 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES / Details: This sample was monodisperse
Specimen supportDetails: 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
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 FIELDBright-field microscopy / 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 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 scansSampling size: 5 µm / Width: 3710 / Height: 3838 / Movie frames/image: 44 / Used frames/image: 0-43

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Processing

SoftwareName: PHENIX / Version: 1.11.1_2580: / Classification: refinement
EM software
IDNameVersionCategoryDetails
1FindEM1particle selectionFindEM Template Picking was used to automatically select particle images based on 2D templates
2Leginon3image acquisitionLeginon software was used for automated data collection
4RELION2.0bCTF correctionCTF correction during refinement
7Coot0.8.8model fittingCoot was used for de novo atomic model building
9RELION2.0binitial Euler assignmentRELION 2.0b was used to assign initial euler angles
10RELION2.0bfinal Euler assignmentRELION 2.0b was used to assign final euler angles
11RELION2.0bclassificationRELION 2.0b was used to perform final classification
12RELION2.0b3D reconstructionRELION 2.0b 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 RELION / Type: PHASE FLIPPING ONLY
Particle selectionNum. of particles selected: 1176206 / Details: template-based cross correlation with FindEM
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 118143 / Algorithm: BACK PROJECTION
Details: Focused classification of final reconstruction was performed on E and F "step" subunits, resulting in a reconstruction with an overall resolution of 3.5 A by FSC 0.143. The two maps were ...Details: Focused classification of final reconstruction was performed on E and F "step" subunits, resulting in a reconstruction with an overall resolution of 3.5 A by FSC 0.143. The two maps were stitched together using vop max in UCSF Chimera. All three maps (two original and final composite) are deposited in this entry.
Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingB value: 52 / Protocol: AB INITIO MODEL / Space: REAL / Target criteria: Correlation coefficient
Details: Initial homology model was built using SWISS-MODEL and initial rigid body docking was done using UCSF Chimera
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00724706
ELECTRON MICROSCOPYf_angle_d0.9233381
ELECTRON MICROSCOPYf_dihedral_angle_d6.14415370
ELECTRON MICROSCOPYf_chiral_restr0.0593863
ELECTRON MICROSCOPYf_plane_restr0.0084277

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