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- EMDB-20133: Lon Protease from Yersinia pestis with Y2853 substrate -

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Basic information

Entry
Database: EMDB / ID: EMD-20133
TitleLon Protease from Yersinia pestis with Y2853 substrateLon protease family
Map dataLon protease family
SampleLon protease bound to Y2853 substrate
  • ATP-dependent protease LaEndopeptidase La
  • Bound Y2853 Substrate
  • (ligand) x 3
Function / homologyendopeptidase 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 binding / cytoplasm / ATP-dependent protease La
Function and homology information
SourceYersinia pestis (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 3 Å
AuthorsShin M / Asmita A / Puchades C / Adjei E / Wiseman RL / Karzai AW / Lander GC
CitationJournal: To Be Published
Title: Distinct Structural Features of the Lon Protease Drive Conserved Hand-over-Hand Substrate Translocation
Authors: Shin M / Asmita A / Puchades C / Adjei E / Wiseman RL / Karzai AW / Lander GC
Validation ReportPDB-ID: 6on2

SummaryFull reportAbout validation report
DateDeposition: Apr 19, 2019 / Header (metadata) release: May 1, 2019 / Map release: May 1, 2019 / Update: May 1, 2019

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.0403
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 0.0403
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: : PDB-6on2
  • Surface level: 0.0403
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

Downloads & links

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Map

FileDownload / File: emd_20133.map.gz / Format: CCP4 / Size: 8.4 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.15 Å/pix.
x 130 pix.
= 149.5 Å
1.15 Å/pix.
x 130 pix.
= 149.5 Å
1.15 Å/pix.
x 130 pix.
= 149.5 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
Contour LevelBy AUTHOR: 0.0403 / Movie #1: 0.0403
Minimum - Maximum-0.021715725 - 0.20937862
Average (Standard dev.)0.0051819608 (±0.01397938)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions130130130
Spacing130130130
CellA=B=C: 149.5 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.151.151.15
M x/y/z130130130
origin x/y/z0.0000.0000.000
length x/y/z149.500149.500149.500
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS130130130
D min/max/mean-0.0220.2090.005

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Supplemental data

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Sample components

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Entire Lon protease bound to Y2853 substrate

EntireName: Lon protease bound to Y2853 substrate
Details: Complexes consisting of homohexameric Lon protease from Yersinia pestis bound to Y2853 substrate were isolated using size-exclusion chromatography
Number of components: 6

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Component #1: protein, Lon protease bound to Y2853 substrate

ProteinName: Lon protease bound to Y2853 substrate
Details: Complexes consisting of homohexameric Lon protease from Yersinia pestis bound to Y2853 substrate were isolated using size-exclusion chromatography
Recombinant expression: No
SourceSpecies: Yersinia pestis (bacteria)
Source (engineered)Expression System: Escherichia coli (E. coli) / Vector: pET28b-lon
Source (natural)Location in cell: Cytoplasm

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Component #2: protein, ATP-dependent protease La

ProteinName: ATP-dependent protease LaEndopeptidase La / Number of Copies: 6 / Recombinant expression: No
MassTheoretical: 57.927051 kDa
SourceSpecies: Yersinia pestis (bacteria)
Source (engineered)Expression System: Escherichia coli (E. coli)

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Component #3: protein, Bound Y2853 Substrate

ProteinName: Bound Y2853 Substrate
Details: Y2853 substrate was added to Lon and modeled here as a polyalanine chain
Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 0.51556 kDa
SourceSpecies: Yersinia pestis (bacteria)
Source (engineered)Expression System: Escherichia coli (E. coli)

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Component #4: ligand, ADENOSINE-5'-TRIPHOSPHATE

LigandName: ADENOSINE-5'-TRIPHOSPHATE / Number of Copies: 4 / Recombinant expression: No
MassTheoretical: 0.507181 kDa

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Component #5: ligand, MAGNESIUM ION

LigandName: MAGNESIUM ION / Number of Copies: 5 / Recombinant expression: No
MassTheoretical: 2.430505 MDa

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Component #6: ligand, ADENOSINE-5'-DIPHOSPHATE

LigandName: ADENOSINE-5'-DIPHOSPHATE / Number of Copies: 2 / Recombinant expression: No
MassTheoretical: 0.427201 kDa

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Experimental details

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Sample preparation

SpecimenSpecimen state: Particle / Method: cryo EM
Sample solutionSpecimen conc.: 0.95 mg/mL
Buffer solution: 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.
pH: 8
Support filmGrids were plasma treated for 30 seconds using a 15 mA current operating under atmospheric gases using a glow discharger (Electron Microscopy Sciences).
VitrificationInstrument: HOMEMADE PLUNGER / Cryogen name: ETHANE / Temperature: 277 K / Humidity: 95 %
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
ImagingMicroscope: 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 / Electron dose: 52 e/Å2 / Illumination mode: FLOOD BEAM
LensMagnification: 36000.0 X (nominal), 43478.0 X (calibrated) / Cs: 2.7 mm / Imaging mode: BRIGHT FIELD / Defocus: 800.0 - 1200.0 nm
Specimen HolderModel: FEI TITAN KRIOS AUTOGRID HOLDER / Temperature: (80.0 - 90.0 K)
CameraDetector: GATAN K2 SUMMIT (4k x 4k)

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Image acquisition

Image acquisitionNumber of digital images: 4071 / Sampling size: 5 µm
Details: Images were collected in counting mode at 4 frames per second

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Image processing

ProcessingMethod: single particle reconstruction / Applied symmetry: C1 (asymmetric) / Number of projections: 118143
3D reconstructionAlgorithm: BACK PROJECTION / Software: RELION / CTF correction: CTF correction in RELION / Resolution: 3 Å / Resolution method: FSC 0.143 CUT-OFF
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.
Euler angles: RELION 2.0b was used to assign initial angles
FSC plot (resolution estimation)

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Atomic model buiding

Modeling #1Target criteria: Correlation coefficient / Refinement space: REAL
Details: Initial homology model was built using SWISS-MODEL and initial rigid body docking was done using UCSF Chimera
Overall bvalue: 52
Output model

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