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- EMDB-1984: Symmetrized cryo-EM reconstruction of E. coli DegQ 24-mer in comp... -

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Basic information

Entry
Database: EMDB / ID: EMD-1984
TitleSymmetrized cryo-EM reconstruction of E. coli DegQ 24-mer in complex with beta-casein
Map dataSymmetrized cryo-EM reconstruction of Escherichia coli DegQ 24-mer in complex with beta-casein substrates
Sample
  • Sample: Escherichia coli DegQ 24-mer in complex with beta-casein
  • Protein or peptide: DegQ
  • Protein or peptide: Beta-caseinCSN2
KeywordsChaperone / Protease
Function / homology
Function and homology information


peptidase Do / protein quality control for misfolded or incompletely synthesized proteins / proteolysis involved in protein catabolic process / peptidase activity / periplasmic space / serine-type endopeptidase activity / identical protein binding
Similarity search - Function
Casein, beta / Peptidase S1C, Do / Peptidase S1C / Trypsin-like peptidase domain / PDZ domain / PDZ domain profile. / Domain present in PSD-95, Dlg, and ZO-1/2. / PDZ domain / PDZ superfamily / Peptidase S1, PA clan
Similarity search - Domain/homology
Periplasmic pH-dependent serine endoprotease DegQ
Similarity search - Component
Biological speciesEscherichia coli (E. coli) / Bos taurus (cattle)
Methodsingle particle reconstruction / cryo EM / Resolution: 7.5 Å
AuthorsMalet H / Canellas F / Sawa J / Yan J / Thalassinos K / Ehrmann M / Clausen T / Saibil HR
CitationJournal: Nat Struct Mol Biol / Year: 2012
Title: Newly folded substrates inside the molecular cage of the HtrA chaperone DegQ.
Authors: Hélène Malet / Flavia Canellas / Justyna Sawa / Jun Yan / Konstantinos Thalassinos / Michael Ehrmann / Tim Clausen / Helen R Saibil /
Abstract: The HtrA protein family combines chaperone and protease activities and is essential for protein quality control in many organisms. Whereas the mechanisms underlying the proteolytic function of HtrA ...The HtrA protein family combines chaperone and protease activities and is essential for protein quality control in many organisms. Whereas the mechanisms underlying the proteolytic function of HtrA proteins are well characterized, their chaperone activity remains poorly understood. Here we describe cryo-EM structures of Escherichia coli DegQ in its 12- and 24-mer states in complex with model substrates, providing a structural model of HtrA chaperone action. Up to six lysozyme substrates bind inside the DegQ 12-mer cage and are visualized in a close-to-native state. An asymmetric reconstruction reveals the binding of a well-ordered lysozyme to four DegQ protomers. DegQ PDZ domains are located adjacent to substrate density and their presence is required for chaperone activity. The substrate-interacting regions appear conserved in 12- and 24-mer cages, suggesting a common mechanism of chaperone function.
History
DepositionNov 8, 2011-
Header (metadata) releaseDec 16, 2011-
Map releaseDec 21, 2011-
UpdateFeb 3, 2012-
Current statusFeb 3, 2012Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 3
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 3
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-4a9g
  • Surface level: 3
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

EMD-1984.gif

Downloads & links

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Map

FileDownload / File: emd_1984.map.gz / Format: CCP4 / Size: 100.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationSymmetrized cryo-EM reconstruction of Escherichia coli DegQ 24-mer in complex with beta-casein substrates
Voxel sizeX=Y=Z: 1.4 Å
Density
Contour LevelBy AUTHOR: 3.0 / Movie #1: 3
Minimum - Maximum-17.705500000000001 - 32.120699999999999
Average (Standard dev.)-0.0000723661 (±1.00244)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-150-150-150
Dimensions300300300
Spacing300300300
CellA=B=C: 420 Å
α=β=γ: 90 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.41.41.4
M x/y/z300300300
origin x/y/z0.0000.0000.000
length x/y/z420.000420.000420.000
α/β/γ90.00090.00090.000
start NX/NY/NZ-56-56-55
NX/NY/NZ112112112
MAP C/R/S123
start NC/NR/NS-150-150-150
NC/NR/NS300300300
D min/max/mean-17.70532.121-0.000

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Supplemental data

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Sample components

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Entire : Escherichia coli DegQ 24-mer in complex with beta-casein

EntireName: Escherichia coli DegQ 24-mer in complex with beta-casein
Components
  • Sample: Escherichia coli DegQ 24-mer in complex with beta-casein
  • Protein or peptide: DegQ
  • Protein or peptide: Beta-caseinCSN2

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Supramolecule #1000: Escherichia coli DegQ 24-mer in complex with beta-casein

SupramoleculeName: Escherichia coli DegQ 24-mer in complex with beta-casein
type: sample / ID: 1000
Details: The exact molecular weight of the complex is unknown as it is not known how many beta-casein substrates are bound
Oligomeric state: Beta-casein monomers bound to one DegQ 24-mer
Number unique components: 2
Molecular weightExperimental: 1.1 MDa / Theoretical: 1.1 MDa / Method: Size exclusion chromatography

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Macromolecule #1: DegQ

MacromoleculeName: DegQ / type: protein_or_peptide / ID: 1 / Name.synonym: DegQ / Number of copies: 24 / Oligomeric state: 24-mer / Recombinant expression: Yes
Source (natural)Organism: Escherichia coli (E. coli) / Strain: K12 / Location in cell: Periplasm
Molecular weightExperimental: 45 KDa / Theoretical: 45 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli) / Recombinant plasmid: pET26b

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Macromolecule #2: Beta-casein

MacromoleculeName: Beta-casein / type: protein_or_peptide / ID: 2 / Name.synonym: Beta-casein / Oligomeric state: Monomer / Recombinant expression: No
Source (natural)Organism: Bos taurus (cattle) / synonym: Bovine
Molecular weightExperimental: 24 KDa / Theoretical: 24 KDa
SequenceInterPro: Casein, beta

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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

Concentration0.2 mg/mL
BufferpH: 7.5 / Details: 20 mM HEPES/NaOH, 150 mM NaCl
GridDetails: C-flat grids (CF-2/2-4C-100 Protochips)
VitrificationCryogen name: ETHANE / Instrument: HOMEMADE PLUNGER / Details: Vitrification instrument: Manual plunger / Method: Blot for 2 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: 50000 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2 mm / Nominal defocus max: 3.0 µm / Nominal defocus min: 1.0 µm / Nominal magnification: 50000
Sample stageSpecimen holder: Single tilt cryo / Specimen holder model: GATAN LIQUID NITROGEN
TemperatureMin: 90 K / Max: 92 K / Average: 91 K
Alignment procedureLegacy - Astigmatism: objective lens astigmatism was corrected at 150,000 times magnification
Legacy - Electron beam tilt params: 0
DetailsLow dose mode
Image recordingCategory: FILM / Film or detector model: KODAK SO-163 FILM / Digitization - Scanner: ZEISS SCAI / Digitization - Sampling interval: 7 µm / Number real images: 100 / Average electron dose: 15 e/Å2 / Bits/pixel: 8
Tilt angle min0
Tilt angle max0
Experimental equipment
Model: Tecnai F20 / Image courtesy: FEI Company

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Image processing

CTF correctionDetails: Phase flipping, full CTF correction
Final reconstructionApplied symmetry - Point group: O (octahedral) / Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 7.5 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: IMAGIC-5, SPIDER / Number images used: 9848

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Atomic model buiding 1

Initial modelPDB ID:

3stj


Chain - Chain ID: A
SoftwareName: Chimera, Flex-EM
DetailsPDBEntryID_givenInChain. Protocol: Rigid body and flexible fitting. Protease and PDZ1 trimers extracted from pdb entry 3STJ.
RefinementSpace: REAL / Protocol: FLEXIBLE FIT / Target criteria: Cross-correlation, energy
Output model

PDB-4a9g:
Symmetrized cryo-EM reconstruction of E. coli DegQ 24-mer in complex with beta-casein

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Atomic model buiding 2

Initial modelPDB ID:

3stj


Chain - Chain ID: B
SoftwareName: Chimera, Flex-EM
DetailsPDBEntryID_givenInChain. Protocol: Rigid body and flexible fitting. Protease and PDZ1 trimers extracted from pdb entry 3STJ.
RefinementSpace: REAL / Protocol: FLEXIBLE FIT / Target criteria: Cross-correlation, energy
Output model

PDB-4a9g:
Symmetrized cryo-EM reconstruction of E. coli DegQ 24-mer in complex with beta-casein

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Atomic model buiding 3

Initial modelPDB ID:

3stj


Chain - Chain ID: C
SoftwareName: Chimera, Flex-EM
DetailsPDBEntryID_givenInChain. Protocol: Rigid body and flexible fitting. Protease and PDZ1 trimers extracted from pdb entry 3STJ.
RefinementSpace: REAL / Protocol: FLEXIBLE FIT / Target criteria: Cross-correlation, energy
Output model

PDB-4a9g:
Symmetrized cryo-EM reconstruction of E. coli DegQ 24-mer in complex with beta-casein

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Atomic model buiding 4

Initial modelPDB ID:

3cs0

SoftwareName: MODELLER, Chimera, Flex-EM
DetailsProtocol: Rigid body. PDZ2 domain modelled with MODELLER from E. coli DegP pdb entry 3CS0.
RefinementSpace: REAL / Protocol: RIGID BODY FIT / Target criteria: Cross-correlation, energy
Output model

PDB-4a9g:
Symmetrized cryo-EM reconstruction of E. coli DegQ 24-mer in complex with beta-casein

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