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- PDB-4a8b: Symmetrized cryo-EM reconstruction of E. coli DegQ 12-mer in comp... -

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Basic information

Entry
Database: PDB / ID: 4a8b
TitleSymmetrized cryo-EM reconstruction of E. coli DegQ 12-mer in complex with lysozymes
Components
  • LYSOZYME C
  • PERIPLASMIC PH-DEPENDENT SERINE ENDOPROTEASE DEGQ
KeywordsHYDROLASE/HYDROLASE / HYDROLASE-HYDROLASE COMPLEX / CHAPERONE
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
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. ...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 / PDZ superfamily / C-type lysozyme/alpha-lactalbumin family / Glycosyl hydrolases family 22 (GH22) domain profile. / Alpha-lactalbumin / lysozyme C / 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)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 13 Å
Model type detailsCA ATOMS ONLY, CHAIN A, B, C, D, E, F, G, H, I, J, K, L, M, N, O, P, Q, R
AuthorsMalet, H. / Canellas, F. / Sawa, J. / Yan, J. / Thalassinos, K. / Ehrmann, M. / Clausen, T. / Saibil, H.R.
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 20, 2011Deposition site: PDBE / Processing site: PDBE
Revision 1.0Jan 11, 2012Provider: repository / Type: Initial release
Revision 1.1Feb 15, 2012Group: Other
Revision 1.2Aug 23, 2017Group: Data collection / Refinement description / Category: em_3d_fitting / em_software
Item: _em_3d_fitting.target_criteria / _em_software.fitting_id ..._em_3d_fitting.target_criteria / _em_software.fitting_id / _em_software.image_processing_id / _em_software.name
Revision 1.3May 8, 2024Group: Data collection / Database references / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / em_3d_fitting_list / pdbx_initial_refinement_model
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id / _em_3d_fitting_list.source_name / _em_3d_fitting_list.type

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Structure visualization

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Assembly

Deposited unit
A: PERIPLASMIC PH-DEPENDENT SERINE ENDOPROTEASE DEGQ
B: PERIPLASMIC PH-DEPENDENT SERINE ENDOPROTEASE DEGQ
C: PERIPLASMIC PH-DEPENDENT SERINE ENDOPROTEASE DEGQ
D: PERIPLASMIC PH-DEPENDENT SERINE ENDOPROTEASE DEGQ
E: PERIPLASMIC PH-DEPENDENT SERINE ENDOPROTEASE DEGQ
F: PERIPLASMIC PH-DEPENDENT SERINE ENDOPROTEASE DEGQ
G: PERIPLASMIC PH-DEPENDENT SERINE ENDOPROTEASE DEGQ
H: PERIPLASMIC PH-DEPENDENT SERINE ENDOPROTEASE DEGQ
I: PERIPLASMIC PH-DEPENDENT SERINE ENDOPROTEASE DEGQ
J: PERIPLASMIC PH-DEPENDENT SERINE ENDOPROTEASE DEGQ
K: PERIPLASMIC PH-DEPENDENT SERINE ENDOPROTEASE DEGQ
L: PERIPLASMIC PH-DEPENDENT SERINE ENDOPROTEASE DEGQ
M: LYSOZYME C
N: LYSOZYME C
O: LYSOZYME C
P: LYSOZYME C
Q: LYSOZYME C
R: LYSOZYME C


Theoretical massNumber of molelcules
Total (without water)632,49918
Polymers632,49918
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

#1: Protein
PERIPLASMIC PH-DEPENDENT SERINE ENDOPROTEASE DEGQ / PROTEASE DO / DEGQ / Coordinate model: Cα atoms only


Mass: 45542.664 Da / Num. of mol.: 12 / Mutation: YES
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) ESCHERICHIA COLI (E. coli) / Strain: K-12 / Tissue: PERIPLASM / Production host: ESCHERICHIA COLI (E. coli) / Strain (production host): BL21(DE3) / References: UniProt: P39099, peptidase Do
#2: Protein
LYSOZYME C / 1 / 4-BETA-N-ACETYLMURAMIDASE C / ALLERGEN GAL D IV / GAL D 4 / Coordinate model: Cα atoms only


Mass: 14331.160 Da / Num. of mol.: 6 / Source method: isolated from a natural source / Source: (natural) GALLUS GALLUS (chicken) / Tissue: EGG WHITE / References: UniProt: P00698, lysozyme
Compound detailsENGINEERED RESIDUE IN CHAIN A, SER 214 TO ALA ENGINEERED RESIDUE IN CHAIN B, SER 214 TO ALA ...ENGINEERED RESIDUE IN CHAIN A, SER 214 TO ALA ENGINEERED RESIDUE IN CHAIN B, SER 214 TO ALA ENGINEERED RESIDUE IN CHAIN C, SER 214 TO ALA ENGINEERED RESIDUE IN CHAIN D, SER 214 TO ALA ENGINEERED RESIDUE IN CHAIN E, SER 214 TO ALA ENGINEERED RESIDUE IN CHAIN F, SER 214 TO ALA ENGINEERED RESIDUE IN CHAIN G, SER 214 TO ALA ENGINEERED RESIDUE IN CHAIN H, SER 214 TO ALA ENGINEERED RESIDUE IN CHAIN I, SER 214 TO ALA ENGINEERED RESIDUE IN CHAIN J, SER 214 TO ALA ENGINEERED RESIDUE IN CHAIN K, SER 214 TO ALA ENGINEERED RESIDUE IN CHAIN L, SER 214 TO ALA

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: ESCHERICHIA COLI DEGQ 12- MER IN COMPLEX WITH LYSOZYME SUBSTRATES
Type: COMPLEX
Buffer solutionName: 20 MM HEPES/NAOH, 150 MM NACL / pH: 7.5 / Details: 20 MM HEPES/NAOH, 150 MM NACL
SpecimenConc.: 0.2 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: CARBON
VitrificationInstrument: HOMEMADE PLUNGER / Cryogen name: ETHANE
Details: VITRIFICATION 1 -- CRYOGEN- ETHANE, INSTRUMENT- MANUAL PLUNGER, METHOD- BLOT FOR 2 SECONDS BEFORE PLUNGING,

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Electron microscopy imaging

Experimental equipment
Model: Tecnai F20 / Image courtesy: FEI Company
MicroscopyModel: FEI TECNAI F20 / Details: LOW DOSE MODE
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 50000 X / Calibrated magnification: 50000 X / Nominal defocus max: 3000 nm / Nominal defocus min: 1000 nm / Cs: 2 mm
Specimen holderTemperature: 91 K / Tilt angle max: 0 ° / Tilt angle min: -0.1 °
Image recordingElectron dose: 15 e/Å2 / Film or detector model: KODAK SO-163 FILM
Image scansNum. digital images: 100
Radiation wavelengthRelative weight: 1

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Processing

EM software
IDNameVersionCategory
1Flex-EMmodel fitting
2MODELLERmodel fitting
3UCSF Chimeramodel fitting
4IMAGIC53D reconstruction
5SPIDER3D reconstruction
CTF correctionDetails: PHASE FLIPPING
SymmetryPoint symmetry: D2 (2x2 fold dihedral)
3D reconstructionMethod: COMMON-LINE, PROJECTION MATCHING / Resolution: 13 Å / Num. of particles: 13432 / Nominal pixel size: 2.8 Å / Actual pixel size: 2.8 Å
Details: SUBMISSION BASED ON EXPERIMENTAL DATA FROM EMDB EMD-1982(DEPOSITION ID: 10365).
Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT / Space: REAL / Target criteria: Cross-correlation coefficient
Details: METHOD--RIGID BODY AND FLEXIBLE FITTING REFINEMENT PROTOCOL--X-RAY
Atomic model buildingPDB-ID: 3STJ

3stj


Accession code: 3STJ / Source name: PDB / Type: experimental model
RefinementHighest resolution: 13 Å
Refinement stepCycle: LAST / Highest resolution: 13 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms5381 0 0 0 5381

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