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- EMDB-1981: Asymmetric cryo-EM reconstruction of E. coli DegQ 12-mer in compl... -

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Basic information

Entry
Database: EMDB / ID: EMD-1981
TitleAsymmetric cryo-EM reconstruction of E. coli DegQ 12-mer in complex with lysozyme
Map dataAsymmetric cryo-EM map of E. coli DegQ 12-mer in complex with lysozyme
Sample
  • Sample: Escherichia coli DegQ Gallus gallus lysozyme
  • Protein or peptide: DegQ
  • Protein or peptide: Lysozyme
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 / Lactose synthesis / Antimicrobial peptides / Neutrophil degranulation / beta-N-acetylglucosaminidase activity / cell wall macromolecule catabolic process / lysozyme / lysozyme activity ...peptidase Do / protein quality control for misfolded or incompletely synthesized proteins / proteolysis involved in protein catabolic process / Lactose synthesis / Antimicrobial peptides / Neutrophil degranulation / beta-N-acetylglucosaminidase activity / cell wall macromolecule catabolic process / lysozyme / lysozyme activity / peptidase activity / defense response to Gram-negative bacterium / killing of cells of another organism / periplasmic space / defense response to Gram-positive bacterium / defense response to bacterium / serine-type endopeptidase activity / endoplasmic reticulum / proteolysis / extracellular space / identical protein binding / cytoplasm
Similarity search - Function
Glycoside hydrolase, family 23 / Peptidase S1C, Do / Peptidase S1C / Trypsin-like peptidase domain / PDZ domain / PDZ domain profile. / Domain present in PSD-95, Dlg, and ZO-1/2. / Glycoside hydrolase, family 22, lysozyme / PDZ domain / Glycoside hydrolase family 22 domain ...Glycoside hydrolase, family 23 / Peptidase S1C, Do / Peptidase S1C / Trypsin-like peptidase domain / PDZ domain / PDZ domain profile. / Domain present in PSD-95, Dlg, and ZO-1/2. / Glycoside hydrolase, family 22, lysozyme / PDZ domain / Glycoside hydrolase family 22 domain / Glycosyl hydrolases family 22 (GH22) domain signature. / Glycoside hydrolase, family 22 / C-type lysozyme/alpha-lactalbumin family / Glycosyl hydrolases family 22 (GH22) domain profile. / Alpha-lactalbumin / lysozyme C / PDZ superfamily / Lysozyme-like domain superfamily / Peptidase S1, PA clan
Similarity search - Domain/homology
Lysozyme C / Periplasmic pH-dependent serine endoprotease DegQ
Similarity search - Component
Biological speciesEscherichia coli (E. coli) / Gallus gallus (chicken)
Methodsingle particle reconstruction / cryo EM / Resolution: 14.2 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 6, 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: 0.2
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 0.2
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-4a8a
  • Surface level: 0.2
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_1981.tif

Downloads & links

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Map

FileDownload / File: emd_1981.map.gz / Format: CCP4 / Size: 7.8 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationAsymmetric cryo-EM map of E. coli DegQ 12-mer in complex with lysozyme
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
2.8 Å/pix.
x 128 pix.
= 358.4 Å		2.8 Å/pix.
x 128 pix.
= 358.4 Å		2.8 Å/pix.
x 128 pix.
= 358.4 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 2.8 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.2 / Movie #1: 0.2
Minimum - Maximum-2.79762 - 40.962499999999999
Average (Standard dev.)-0.00000476495 (±1.00186)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-64-64-64
Dimensions128128128
Spacing128128128
CellA=B=C: 358.4 Å
α=β=γ: 90 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z2.82.82.8
M x/y/z128128128
origin x/y/z0.0000.0000.000
length x/y/z358.400358.400358.400
α/β/γ90.00090.00090.000
start NX/NY/NZ-56-56-55
NX/NY/NZ112112112
MAP C/R/S123
start NC/NR/NS-64-64-64
NC/NR/NS128128128
D min/max/mean-2.79840.963-0.000

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Supplemental data

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Sample components

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Entire : Escherichia coli DegQ Gallus gallus lysozyme

EntireName: Escherichia coli DegQ Gallus gallus lysozyme
Components
  • Sample: Escherichia coli DegQ Gallus gallus lysozyme
  • Protein or peptide: DegQ
  • Protein or peptide: Lysozyme

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Supramolecule #1000: Escherichia coli DegQ Gallus gallus lysozyme

SupramoleculeName: Escherichia coli DegQ Gallus gallus lysozyme / type: sample / ID: 1000
Oligomeric state: Six lysozyme monomers bind to one DegQ 12-mer
Number unique components: 2
Molecular weightExperimental: 625 KDa / Theoretical: 625 KDa
Method: Native mass spectrometry Size elution chromatography

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Macromolecule #1: DegQ

MacromoleculeName: DegQ / type: protein_or_peptide / ID: 1 / Name.synonym: DegQ / Number of copies: 12 / Oligomeric state: Dodecamer / Recombinant expression: Yes
Source (natural)Organism: Escherichia coli (E. coli) / Strain: K-12 / Cell: Escherichia coli / 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: Lysozyme

MacromoleculeName: Lysozyme / type: protein_or_peptide / ID: 2 / Name.synonym: Lysozyme / Number of copies: 6 / Oligomeric state: Monomer / Recombinant expression: No
Source (natural)Organism: Gallus gallus (chicken) / synonym: Chicken / Tissue: Egg white
Molecular weightExperimental: 14.3 KDa / Theoretical: 14.3 KDa
SequenceGO: lysozyme activity / InterPro: Glycoside hydrolase, family 23

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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
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
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 50000 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / 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
Experimental equipment
Model: Tecnai F20 / Image courtesy: FEI Company

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Image processing

CTF correctionDetails: Phase flipping
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 14.2 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: IMAGIC-5, SPIDER / Number images used: 13432

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Atomic model buiding 1

Initial modelPDB ID:

3stj


Chain - #0 - Chain ID: A / Chain - #1 - Chain ID: B / Chain - #2 - Chain ID: C
SoftwareName: Chimera, Flex-EM
DetailsPDBEntryID_givenInChain. Protocol: Rigid body and flexible fitting. Protease and PDZ1 trimers extracted from 3STJ pdb entry. PDZ2 domain modelled with MODELLER from E. coli DegP pdb entry 3CS0.
RefinementSpace: REAL / Protocol: FLEXIBLE FIT / Target criteria: Cross-correlation, energy
Output model

PDB-4a8a:
Asymmetric cryo-EM reconstruction of E. coli DegQ 12-mer in complex with lysozyme

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Atomic model buiding 2

Initial modelPDB ID:

1dpx


Chain - Chain ID: A
SoftwareName: Flex-EM
DetailsPDBEntryID_givenInChain. Protocol: Rigid body
RefinementSpace: REAL / Protocol: RIGID BODY FIT / Target criteria: Cross-correlation, energy
Output model

PDB-4a8a:
Asymmetric cryo-EM reconstruction of E. coli DegQ 12-mer in complex with lysozyme

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