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- EMDB-21009: Lon Protease from Yersinia pestis -

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Basic information

Entry
Database: EMDB / ID: EMD-21009
TitleLon Protease from Yersinia pestis
Map dataFinal reconstruction used for model building and refinement
Sample
  • Complex: Lon Protease from Yersinia pestis
    • Protein or peptide: Lon protease
  • Ligand: ADENOSINE-5'-DIPHOSPHATE
KeywordsAAA+ ATPase / Quality Control / Protease / HYDROLASE
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 / 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, 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
Lon protease / Lon protease
Similarity search - Component
Biological speciesYersinia pestis (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.8 Å
AuthorsShin M / Puchades C
Funding support United States, 5 items
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, 2019-
Header (metadata) releaseJan 22, 2020-
Map releaseJan 22, 2020-
UpdateMar 6, 2024-
Current statusMar 6, 2024Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.232
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 0.232
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6v11
  • Surface level: 0.232
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_21009.png

Downloads & links

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Map

FileDownload / File: emd_21009.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationFinal reconstruction used for model building and refinement
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.15 Å/pix.
x 256 pix.
= 294.4 Å		1.15 Å/pix.
x 256 pix.
= 294.4 Å		1.15 Å/pix.
x 256 pix.
= 294.4 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.15 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.232 / Movie #1: 0.232
Minimum - Maximum-0.5951247 - 1.1104119
Average (Standard dev.)0.00036906925 (±0.038043562)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 294.4 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.151.151.15
M x/y/z256256256
origin x/y/z0.0000.0000.000
length x/y/z294.400294.400294.400
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS256256256
D min/max/mean-0.5951.1100.000

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Supplemental data

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Half map: Half-map B from final reconstruction used to calculate FSC

Fileemd_21009_half_map_1.map
AnnotationHalf-map B from final reconstruction used to calculate FSC
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half-map A from final reconstruction used to calculate FSC

Fileemd_21009_half_map_2.map
AnnotationHalf-map A from final reconstruction used to calculate FSC
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Lon Protease from Yersinia pestis

EntireName: Lon Protease from Yersinia pestis
Components
  • Complex: Lon Protease from Yersinia pestis
    • Protein or peptide: Lon protease
  • Ligand: ADENOSINE-5'-DIPHOSPHATE

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Supramolecule #1: Lon Protease from Yersinia pestis

SupramoleculeName: Lon Protease from Yersinia pestis / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1
Details: Complexes consisting of homohexameric Lon protease from Yersinia pestis were isolated using size-exclusion chromatography
Source (natural)Organism: Yersinia pestis (bacteria) / Location in cell: Cytoplasm

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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: Yersinia pestis (bacteria)
Molecular weightTheoretical: 57.780863 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: ALKRKIEAAK MPKDAREKTE AELQKLKMMS PMSAEATVVR GYIDWMLQVP WNSRSKVKKD LVKAQEVLDT DHYGLERVKD RILEYLAVQ SRVSKIKGPI LCLVGPPGVG KTSLGQSIAK ATGRQYVRMA LGGVRDEAEI RGHRRTYIGS MPGKLIQKMA K VGVKNPLF ...String:
ALKRKIEAAK MPKDAREKTE AELQKLKMMS PMSAEATVVR GYIDWMLQVP WNSRSKVKKD LVKAQEVLDT DHYGLERVKD RILEYLAVQ SRVSKIKGPI LCLVGPPGVG KTSLGQSIAK ATGRQYVRMA LGGVRDEAEI RGHRRTYIGS MPGKLIQKMA K VGVKNPLF LLDEIDKMAS DMRGDPASAL LEVLDPEQNV AFNDHYLEVD YDLSDVMFVA TSNSMNIPAP LLDRMEVIRL SG YTEDEKL NIAKQHLLPK QFERNAIKKG ELTIDDSAIM SIIRYYTREA GVRSLEREIS KLCRKAVKNL LMDKTVKHIE ING DNLKDF LGVQKVDYGR ADTENRVGQV TGLAWTEVGG DLLTIETACV PGKGKLTYTG SLGEVMQESI QAALTVVRAR ADKL GINPD FYEKRDIHVH VPEGATPKDG PSAGIAMCTA LVSCLTGNPV RADVAMTGEI TLRGLVLPIG GLKEKLLAAH RGGIK VVLI PDDNKRDLEE IPDNVIADLE IHPVKRIDDV LAIALEHPA

UniProtKB: Lon protease

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Macromolecule #2: ADENOSINE-5'-DIPHOSPHATE

MacromoleculeName: ADENOSINE-5'-DIPHOSPHATE / type: ligand / ID: 2 / Number of copies: 5 / 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

Concentration17 mg/mL
BufferpH: 8
Component:
ConcentrationFormulaName
50.0 mMTrisTris Base
75.0 mMKClPotassium Chloride
10.0 mMMgCl2Magnesium Chloride
1.0 mMTCEPTCEP
1.0 mMATPAdenosine Triphosphate

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.
GridModel: UltrAuFoil / Material: GOLD / Mesh: 400
VitrificationCryogen name: ETHANE / Chamber humidity: 95 % / Chamber temperature: 277 K / Instrument: HOMEMADE PLUNGER
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..
DetailsThis sample was monodisperse

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Electron microscopy

MicroscopeFEI TALOS ARCTICA
TemperatureMin: 80.0 K / Max: 90.0 K
Alignment procedureComa free - Residual tilt: 0.14 mrad
DetailsComa-free alignment procedure from Herzik & Wu, Nature Methods (2017). Preliminary grid screening was performed manually prior to data collection.
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Digitization - Dimensions - Width: 3710 pixel / Digitization - Dimensions - Height: 3838 pixel / Digitization - Frames/image: 0-57 / Number grids imaged: 1 / Number real images: 1864 / Average exposure time: 11.6 sec. / Average electron dose: 50.0 e/Å2
Details: Images were collected in counting mode at 5 frames per second
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 70.0 µm / Calibrated defocus max: 1.5 µm / Calibrated defocus min: 0.5 µm / Calibrated magnification: 43478 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 1.2 µm / Nominal defocus min: 0.8 µm / Nominal magnification: 36000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 412719
Details: Particles were selected from a subset of micrographs using BoxNet automated particle picker
Startup modelType of model: INSILICO MODEL
In silico model: In silico model was created using ab initio 3D reconstruction using a subset of the data in cryoSPARC
Final reconstructionNumber classes used: 2 / Resolution.type: BY AUTHOR / Resolution: 3.8 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 2.11.0)
Software - details: cryoSPARC was used to perform final reconstruction
Number images used: 140506
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 2.11.0)
Software - details: cryoSPARC was used to assign initial angles
Details: cryoSPARC was used to assign initial angles
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 2.11.0)
Software - details: cryoSPARC was used to assign final euler angles
Details: cryoSPARC was used to assign final angles
Final 3D classificationNumber classes: 5 / Avg.num./class: 57572 / Software - Name: cryoSPARC (ver. 2.11.0)
Software - details: cryoSPARC was used to perform final classification
Details: The final 3D classification had a somewhat asymmetric distribution owing to conformational heterogeneity
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

6on2


Chain - Chain ID: A / Chain - Residue range: 253-775 / Chain - Source name: PDB / Chain - Initial model type: experimental model
DetailsInitial rigid body docking was done using UCSF Chimera
RefinementSpace: REAL / Protocol: RIGID BODY FIT / Overall B value: 115 / Target criteria: Correlation coefficient
Output model

PDB-6v11:
Lon Protease from Yersinia pestis

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