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- EMDB-6305: The cryo-EM structure of Meiothermus taiwanensis Lon protease wit... -

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Basic information

Entry
Database: EMDB / ID: EMD-6305
TitleThe cryo-EM structure of Meiothermus taiwanensis Lon protease with ATP and Mg2+
Map dataReconstruction of Meiothermus taiwanensis LonA protease with Mg2+ and ATP
Sample
  • Sample: Meiothermus taiwanensis LonA protease
  • Protein or peptide: Meiothermus taiwanensis LonA protease
KeywordsATP-dependent proteases
Biological speciesMeiothermus taiwanensis (bacteria)
Methodsingle particle reconstruction / cryo EM / negative staining / Resolution: 11.8 Å
AuthorsSu SC / Chang YC / Chang CI
CitationJournal: Structure / Year: 2016
Title: Structural Basis for the Magnesium-Dependent Activation and Hexamerization of the Lon AAA+ Protease.
Authors: Shih-Chieh Su / Chien-Chu Lin / Hui-Chung Tai / Mu-Yueh Chang / Meng-Ru Ho / C Satheesan Babu / Jiahn-Haur Liao / Shih-Hsiung Wu / Yuan-Chih Chang / Carmay Lim / Chung-I Chang /
Abstract: The Lon AAA+ protease (LonA) plays important roles in protein homeostasis and regulation of diverse biological processes. LonA behaves as a homomeric hexamer in the presence of magnesium (Mg(2+)) and ...The Lon AAA+ protease (LonA) plays important roles in protein homeostasis and regulation of diverse biological processes. LonA behaves as a homomeric hexamer in the presence of magnesium (Mg(2+)) and performs ATP-dependent proteolysis. However, it is also found that LonA can carry out Mg(2+)-dependent degradation of unfolded protein substrate in an ATP-independent manner. Here we show that in the presence of Mg(2+) LonA forms a non-secluded hexameric barrel with prominent openings, which explains why Mg(2+)-activated LonA can operate as a diffusion-based chambered protease to degrade unstructured protein and peptide substrates efficiently in the absence of ATP. A 1.85 Å crystal structure of Mg(2+)-activated protease domain reveals Mg(2+)-dependent remodeling of a substrate-binding loop and a potential metal-binding site near the Ser-Lys catalytic dyad, supported by biophysical binding assays and molecular dynamics simulations. Together, these findings reveal the specific roles of Mg(2+) in the molecular assembly and activation of LonA.
History
DepositionMar 15, 2015-
Header (metadata) releaseMay 6, 2015-
Map releaseMar 23, 2016-
UpdateJun 29, 2016-
Current statusJun 29, 2016Processing site: PDBj / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 1.65
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 1.65
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_6305.tiff

Downloads & links

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Map

FileDownload / File: emd_6305.map.gz / Format: CCP4 / Size: 7.8 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationReconstruction of Meiothermus taiwanensis LonA protease with Mg2+ and ATP
Voxel sizeX=Y=Z: 2.5 Å
Density
Contour LevelBy AUTHOR: 0.225 / Movie #1: 1.65
Minimum - Maximum-1.42064238 - 4.19262695
Average (Standard dev.)0.14911929 (±0.58456016)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-64-64-64
Dimensions128128128
Spacing128128128
CellA=B=C: 320.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z2.52.52.5
M x/y/z128128128
origin x/y/z0.0000.0000.000
length x/y/z320.000320.000320.000
α/β/γ90.00090.00090.000
start NX/NY/NZ-147-147-146
NX/NY/NZ294294294
MAP C/R/S123
start NC/NR/NS-64-64-64
NC/NR/NS128128128
D min/max/mean-1.4214.1930.149

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Supplemental data

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Sample components

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Entire : Meiothermus taiwanensis LonA protease

EntireName: Meiothermus taiwanensis LonA protease
Components
  • Sample: Meiothermus taiwanensis LonA protease
  • Protein or peptide: Meiothermus taiwanensis LonA protease

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Supramolecule #1000: Meiothermus taiwanensis LonA protease

SupramoleculeName: Meiothermus taiwanensis LonA protease / type: sample / ID: 1000
Oligomeric state: One homotetramer of MtaLonA in closed form
Number unique components: 1
Molecular weightExperimental: 540 KDa / Theoretical: 540 KDa / Method: Sedimentation

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Macromolecule #1: Meiothermus taiwanensis LonA protease

MacromoleculeName: Meiothermus taiwanensis LonA protease / type: protein_or_peptide / ID: 1 / Name.synonym: MtaLonA / Oligomeric state: Hexamer / Recombinant expression: Yes
Source (natural)Organism: Meiothermus taiwanensis (bacteria) / synonym: Meiothermus taiwanensis LonA protease
Molecular weightExperimental: 540 KDa / Theoretical: 540 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli) / Recombinant plasmid: pET21a

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Experimental details

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Structure determination

Methodnegative staining, cryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration0.12 mg/mL
BufferpH: 8
Details: 20mM Tris-HCl, 100mM NaCl, 1mM DTT, 10mM MgCl2, 2.5mM ATP
StainingType: NEGATIVE
Details: Grids with adsorbed protein floated on 1% w/v uranyl acetate for 60 seconds.
GridDetails: 200 mesh gold grid with thin carbon support, glow discharged in amylamine atmosphere
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Instrument: FEI VITROBOT MARK IV
Method: 1. wait for 10 seconds before blot 2. Blot for 3 seconds before plunging

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Electron microscopy

MicroscopeFEI TECNAI F20
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 85600 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2 mm / Nominal defocus max: 3.7 µm / Nominal defocus min: 1.45 µm / Nominal magnification: 62000
Sample stageSpecimen holder model: GATAN LIQUID NITROGEN
Alignment procedureLegacy - Astigmatism: Objective lens astigmatism was corrected at 100,000 times magnification
DateMar 3, 2014
Image recordingCategory: CCD / Film or detector model: GATAN ULTRASCAN 4000 (4k x 4k) / Number real images: 220 / Average electron dose: 20 e/Å2
Experimental equipment
Model: Tecnai F20 / Image courtesy: FEI Company

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Image processing

CTF correctionDetails: Each particle
Final two d classificationNumber classes: 80
Final reconstructionResolution.type: BY AUTHOR / Resolution: 11.8 Å / Resolution method: OTHER / Software - Name: EMAN2 / Number images used: 30249
DetailsThe particle images were interactively selected using EMAN Boxer program

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