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- PDB-4a9g: Symmetrized cryo-EM reconstruction of E. coli DegQ 24-mer in comp... -

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Basic information

Entry
Database: PDB / ID: 4a9g
TitleSymmetrized cryo-EM reconstruction of E. coli DegQ 24-mer in complex with beta-casein
ComponentsPERIPLASMIC PH-DEPENDENT SERINE ENDOPROTEASE DEGQ
KeywordsHYDROLASE / CHAPERONE
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
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)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 7.5 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, S, T, U, V, W, Y
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 26, 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_image_scans / 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

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Structure visualization

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  • Deposited structure unit
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  • Superimposition on EM map
  • EMDB-1984
  • Imaged by UCSF Chimera
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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: PERIPLASMIC PH-DEPENDENT SERINE ENDOPROTEASE DEGQ
N: PERIPLASMIC PH-DEPENDENT SERINE ENDOPROTEASE DEGQ
O: PERIPLASMIC PH-DEPENDENT SERINE ENDOPROTEASE DEGQ
P: PERIPLASMIC PH-DEPENDENT SERINE ENDOPROTEASE DEGQ
Q: PERIPLASMIC PH-DEPENDENT SERINE ENDOPROTEASE DEGQ
R: PERIPLASMIC PH-DEPENDENT SERINE ENDOPROTEASE DEGQ
S: PERIPLASMIC PH-DEPENDENT SERINE ENDOPROTEASE DEGQ
T: PERIPLASMIC PH-DEPENDENT SERINE ENDOPROTEASE DEGQ
U: PERIPLASMIC PH-DEPENDENT SERINE ENDOPROTEASE DEGQ
V: PERIPLASMIC PH-DEPENDENT SERINE ENDOPROTEASE DEGQ
W: PERIPLASMIC PH-DEPENDENT SERINE ENDOPROTEASE DEGQ
Y: PERIPLASMIC PH-DEPENDENT SERINE ENDOPROTEASE DEGQ


Theoretical massNumber of molelcules
Total (without water)1,093,02424
Polymers1,093,02424
Non-polymers00
Water0
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.: 24 / Mutation: YES
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) ESCHERICHIA COLI (E. coli) / Strain: K-12 / Production host: ESCHERICHIA COLI (E. coli) / References: UniProt: P39099, peptidase Do
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 ENGINEERED RESIDUE IN CHAIN M, SER 214 TO ALA ENGINEERED RESIDUE IN CHAIN N, SER 214 TO ALA ENGINEERED RESIDUE IN CHAIN O, SER 214 TO ALA ENGINEERED RESIDUE IN CHAIN P, SER 214 TO ALA ENGINEERED RESIDUE IN CHAIN Q, SER 214 TO ALA ENGINEERED RESIDUE IN CHAIN R, SER 214 TO ALA ENGINEERED RESIDUE IN CHAIN S, SER 214 TO ALA ENGINEERED RESIDUE IN CHAIN T, SER 214 TO ALA ENGINEERED RESIDUE IN CHAIN U, SER 214 TO ALA ENGINEERED RESIDUE IN CHAIN V, SER 214 TO ALA ENGINEERED RESIDUE IN CHAIN W, SER 214 TO ALA ENGINEERED RESIDUE IN CHAIN Y, 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: DEGQ 24-MER BOUND TO BETA- CASEIN / Type: COMPLEX
Buffer solutionName: 150MM NACL, 20MM HEPES/NAOH / pH: 7.5 / Details: 150MM NACL, 20MM HEPES/NAOH
SpecimenConc.: 0.2 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: OTHER
VitrificationInstrument: HOMEMADE PLUNGER / Cryogen name: ETHANE / Details: LIQUID ETHANE

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Electron microscopy imaging

Experimental equipment
Model: Tecnai F20 / Image courtesy: FEI Company
MicroscopyModel: FEI TECNAI F20
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / 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: -0.5 °
Image recordingElectron dose: 15 e/Å2 / Film or detector model: KODAK SO-163 FILM
Radiation wavelengthRelative weight: 1

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Processing

EM software
IDNameCategory
1Flex-EMmodel fitting
2MODELLERmodel fitting
3UCSF Chimeramodel fitting
4IMAGIC3D reconstruction
5SPIDER3D reconstruction
CTF correctionDetails: FULL CTF CORRECTION
SymmetryPoint symmetry: O (octahedral)
3D reconstructionMethod: CROSS-COMMON LINE, PROJECTION MATCHING / Resolution: 7.5 Å / Num. of particles: 9848 / Nominal pixel size: 1.4 Å / Actual pixel size: 1.4 Å
Details: BETA-CASEIN WERE NOT FITTED AS BETA-CASEIN IS AN INTRISICALLY DISORDERED PROTEIN. HOWEVER BETA-CASEIN IS PRESENT IN THE DEGQ 24-MER COMPLEX. SUBMISSION BASED ON EXPERIMENTAL DATA FROM EMDB ...Details: BETA-CASEIN WERE NOT FITTED AS BETA-CASEIN IS AN INTRISICALLY DISORDERED PROTEIN. HOWEVER BETA-CASEIN IS PRESENT IN THE DEGQ 24-MER COMPLEX. SUBMISSION BASED ON EXPERIMENTAL DATA FROM EMDB EMD-1984. (DEPOSITION ID: 10375).
Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT / Space: REAL / Target criteria: Cross-correlation coefficient
Details: METHOD--FLEXIBLE FITTING REFINEMENT PROTOCOL--X-RAY
Atomic model buildingPDB-ID: 3STJ

3stj

RefinementHighest resolution: 7.5 Å
Refinement stepCycle: LAST / Highest resolution: 7.5 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms9360 0 0 0 9360

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