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- EMDB-3274: Proteolytically inactive Human mitochondrial Lon protease incubat... -

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Basic information

Entry
Database: EMDB / ID: EMD-3274
TitleProteolytically inactive Human mitochondrial Lon protease incubated with ADP
Map dataProteolytically inactive Human Lon protease incubated with ADP
Sample
  • Sample: Proteolytically inactive Human mitochondrial Lon protease (S855A) incubated with ADP
  • Protein or peptide: Human mitochondrial Lon protease S855A
KeywordsAAA / Lon protease / Human
Function / homology
Function and homology information


oxidation-dependent protein catabolic process / PH domain binding / mitochondrial protein catabolic process / G-quadruplex DNA binding / endopeptidase La / mitochondrial DNA metabolic process / mitochondrial genome maintenance / ATP-dependent peptidase activity / protein quality control for misfolded or incompletely synthesized proteins / mitochondrial nucleoid ...oxidation-dependent protein catabolic process / PH domain binding / mitochondrial protein catabolic process / G-quadruplex DNA binding / endopeptidase La / mitochondrial DNA metabolic process / mitochondrial genome maintenance / ATP-dependent peptidase activity / protein quality control for misfolded or incompletely synthesized proteins / mitochondrial nucleoid / insulin receptor substrate binding / chaperone-mediated protein complex assembly / DNA polymerase binding / regulation of peptidyl-tyrosine phosphorylation / negative regulation of insulin receptor signaling pathway / Mitochondrial protein degradation / proteolysis involved in protein catabolic process / mitochondrion organization / ADP binding / protein catabolic process / single-stranded DNA binding / cellular response to oxidative stress / sequence-specific DNA binding / single-stranded RNA binding / response to hypoxia / mitochondrial matrix / serine-type endopeptidase activity / ATP hydrolysis activity / mitochondrion / nucleoplasm / ATP binding / identical protein binding / membrane / cytosol
Similarity search - Function
Lon protease homologue, chloroplastic/mitochondrial / 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 homologue, chloroplastic/mitochondrial / 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 homolog, mitochondrial
Similarity search - Component
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 21.0 Å
AuthorsKereiche S / Kovacik L / Bednar J / Pevala V / Kunova N / Ondrovicova G / Bauer J / Ambro L / Bellova J / Kutejova E / Raska I
CitationJournal: Sci Rep / Year: 2016
Title: The N-terminal domain plays a crucial role in the structure of a full-length human mitochondrial Lon protease.
Authors: Sami Kereïche / Lubomír Kováčik / Jan Bednár / Vladimír Pevala / Nina Kunová / Gabriela Ondrovičová / Jacob Bauer / Ľuboš Ambro / Jana Bellová / Eva Kutejová / Ivan Raška /
Abstract: Lon is an essential, multitasking AAA(+) protease regulating many cellular processes in species across all kingdoms of life. Altered expression levels of the human mitochondrial Lon protease (hLon) ...Lon is an essential, multitasking AAA(+) protease regulating many cellular processes in species across all kingdoms of life. Altered expression levels of the human mitochondrial Lon protease (hLon) are linked to serious diseases including myopathies, paraplegia, and cancer. Here, we present the first 3D structure of full-length hLon using cryo-electron microscopy. hLon has a unique three-dimensional structure, in which the proteolytic and ATP-binding domains (AP-domain) form a hexameric chamber, while the N-terminal domain is arranged as a trimer of dimers. These two domains are linked by a narrow trimeric channel composed likely of coiled-coil helices. In the presence of AMP-PNP, the AP-domain has a closed-ring conformation and its N-terminal entry gate appears closed, but in ADP binding, it switches to a lock-washer conformation and its N-terminal gate opens, which is accompanied by a rearrangement of the N-terminal domain. We have also found that both the enzymatic activities and the 3D structure of a hLon mutant lacking the first 156 amino acids are severely disturbed, showing that hLon's N-terminal domains are crucial for the overall structure of the hLon, maintaining a conformation allowing its proper functioning.
History
DepositionDec 9, 2015-
Header (metadata) releaseDec 16, 2015-
Map releaseOct 5, 2016-
UpdateOct 5, 2016-
Current statusOct 5, 2016Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.05
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 0.05
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_3274.png

Downloads & links

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Map

FileDownload / File: emd_3274.map.gz / Format: CCP4 / Size: 41.9 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationProteolytically inactive Human Lon protease incubated with ADP
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.8 Å/pix.
x 224 pix.
= 403.536 Å		1.8 Å/pix.
x 224 pix.
= 403.536 Å		1.8 Å/pix.
x 224 pix.
= 403.536 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.8015 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.05 / Movie #1: 0.05
Minimum - Maximum-0.02438934 - 0.13706979
Average (Standard dev.)0.00144868 (±0.01156716)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions224224224
Spacing224224224
CellA=B=C: 403.53598 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.80151.80151.8015
M x/y/z224224224
origin x/y/z0.0000.0000.000
length x/y/z403.536403.536403.536
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS224224224
D min/max/mean-0.0240.1370.001

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Supplemental data

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Segmentation: Proteolytically inactive Human Lon protease incubated with ADP

AnnotationProteolytically inactive Human Lon protease incubated with ADP
Fileemd_3274_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Proteolytically inactive Human mitochondrial Lon protease (S855A)...

EntireName: Proteolytically inactive Human mitochondrial Lon protease (S855A) incubated with ADP
Components
  • Sample: Proteolytically inactive Human mitochondrial Lon protease (S855A) incubated with ADP
  • Protein or peptide: Human mitochondrial Lon protease S855A

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Supramolecule #1000: Proteolytically inactive Human mitochondrial Lon protease (S855A)...

SupramoleculeName: Proteolytically inactive Human mitochondrial Lon protease (S855A) incubated with ADP
type: sample / ID: 1000 / Oligomeric state: hexameric / Number unique components: 1
Molecular weightExperimental: 650 KDa / Theoretical: 650 KDa

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Macromolecule #1: Human mitochondrial Lon protease S855A

MacromoleculeName: Human mitochondrial Lon protease S855A / type: protein_or_peptide / ID: 1 / Name.synonym: LonP1 / Number of copies: 6 / Oligomeric state: hexameric / Recombinant expression: Yes
Source (natural)Organism: Homo sapiens (human) / synonym: human / Organelle: mitochondria / Location in cell: mitochondria
Molecular weightExperimental: 650 KDa / Theoretical: 650 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli) / Recombinant strain: DE3
SequenceUniProtKB: Lon protease homolog, mitochondrial

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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

Concentration1 mg/mL
BufferpH: 8 / Details: 20 mM HEPES, 150 mM NaCl, 5 mM MgCl2
GridDetails: 200 mesh quanti-foil R2/2 with thin carbon support, glow discharged in standard vaccum atmosphere
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 90 K / Instrument: FEI VITROBOT MARK IV / Method: blot 2 seconds before plunging

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Electron microscopy

MicroscopeFEI POLARA 300
DateApr 4, 2014
Image recordingCategory: CCD / Film or detector model: FEI FALCON I (4k x 4k) / Digitization - Sampling interval: 14 µm / Number real images: 498 / Average electron dose: 10 e/Å2 / Bits/pixel: 16
Electron beamAcceleration voltage: 100 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 77712 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.0 mm / Nominal defocus max: 2.6 µm / Nominal defocus min: 1.2 µm / Nominal magnification: 59000
Sample stageSpecimen holder model: OTHER
Experimental equipment
Model: Tecnai Polara / Image courtesy: FEI Company

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Image processing

CTF correctionDetails: ctffind3
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 21.0 Å / Resolution method: OTHER / Software - Name: RELION / Number images used: 38100
Final two d classificationNumber classes: 20

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Atomic model buiding 1

Initial modelPDB ID:

3m6a


Chain - Chain ID: A
SoftwareName: sculptor, situs, Chimera
DetailsThe domains were separately fitted by interactive docking in the situs programme.
RefinementSpace: REAL / Protocol: RIGID BODY FIT

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Atomic model buiding 2

Initial modelPDB ID:

3jlc


Chain - Chain ID: A
SoftwareName: sculptor, situs, Chimera
DetailsThe N-terminal domains were fitted with 6 shortened copy of 3jlc from E.Coli in chimera
RefinementSpace: REAL / Protocol: RIGID BODY FIT

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