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- EMDB-40852: Structure of the C-terminal protease CtpA-LbcA complex of Pseudom... -

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Basic information

Entry
Database: EMDB / ID: EMD-40852
TitleStructure of the C-terminal protease CtpA-LbcA complex of Pseudomonas aeruginosa
Map data
Sample
  • Complex: CtpA-LbcA complex of Pseudomonas aeruginosa
    • Protein or peptide: Carboxyl-terminal protease
    • Protein or peptide: TPR repeat-containing protein PA4667
    • Protein or peptide: unidentified peptide
KeywordsCtpA / LbcA / C-terminal protease / Pseudomonas aeruginosa / HYDROLASE
Function / homology
Function and homology information


C-terminal processing peptidase / serine-type peptidase activity / proteolysis
Similarity search - Function
: / Activating protease CtpB N-terminal domain / C-terminal-processing peptidase S41A / tail specific protease / Tail specific protease / Peptidase family S41 / PDZ domain 6 / PDZ domain / Tetratricopeptide repeat / Tetratricopeptide repeat ...: / Activating protease CtpB N-terminal domain / C-terminal-processing peptidase S41A / tail specific protease / Tail specific protease / Peptidase family S41 / PDZ domain 6 / PDZ domain / Tetratricopeptide repeat / Tetratricopeptide repeat / ClpP/crotonase-like domain superfamily / TPR repeat region circular profile. / TPR repeat profile. / Tetratricopeptide repeats / Tetratricopeptide repeat / PDZ domain profile. / Domain present in PSD-95, Dlg, and ZO-1/2. / PDZ domain / PDZ superfamily / Tetratricopeptide-like helical domain superfamily
Similarity search - Domain/homology
Carboxy-terminal processing protease CtpB / TPR repeat-containing protein PA4667
Similarity search - Component
Biological speciesPseudomonas aeruginosa (bacteria) / Escherichia coli BL21(DE3) (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.94 Å
AuthorsHsu H-C / Li H
Funding support United States, 1 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)AI136901 United States
CitationJournal: EMBO J / Year: 2024
Title: P. aeruginosa CtpA protease adopts a novel activation mechanism to initiate the proteolytic process.
Authors: Hao-Chi Hsu / Michelle Wang / Amanda Kovach / Andrew J Darwin / Huilin Li /
Abstract: During bacterial cell growth, hydrolases cleave peptide cross-links between strands of the peptidoglycan sacculus to allow new strand insertion. The Pseudomonas aeruginosa carboxyl-terminal ...During bacterial cell growth, hydrolases cleave peptide cross-links between strands of the peptidoglycan sacculus to allow new strand insertion. The Pseudomonas aeruginosa carboxyl-terminal processing protease (CTP) CtpA regulates some of these hydrolases by degrading them. CtpA assembles as an inactive hexamer composed of a trimer-of-dimers, but its lipoprotein binding partner LbcA activates CtpA by an unknown mechanism. Here, we report the cryo-EM structures of the CtpA-LbcA complex. LbcA has an N-terminal adaptor domain that binds to CtpA, and a C-terminal superhelical tetratricopeptide repeat domain. One LbcA molecule attaches to each of the three vertices of a CtpA hexamer. LbcA triggers relocation of the CtpA PDZ domain, remodeling of the substrate binding pocket, and realignment of the catalytic residues. Surprisingly, only one CtpA molecule in a CtpA dimer is activated upon LbcA binding. Also, a long loop from one CtpA dimer inserts into a neighboring dimer to facilitate the proteolytic activity. This work has revealed an activation mechanism for a bacterial CTP that is strikingly different from other CTPs that have been characterized structurally.
History
DepositionMay 22, 2023-
Header (metadata) releaseMar 6, 2024-
Map releaseMar 6, 2024-
UpdateApr 24, 2024-
Current statusApr 24, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_40852.png

Downloads & links

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Map

FileDownload / File: emd_40852.map.gz / Format: CCP4 / Size: 125 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.24 Å/pix.
x 320 pix.
= 397.44 Å		1.24 Å/pix.
x 320 pix.
= 397.44 Å		1.24 Å/pix.
x 320 pix.
= 397.44 Å

Surface

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Images are generated by Spider.

Voxel sizeX=Y=Z: 1.242 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.25
Minimum - Maximum-1.9341416 - 3.0479808
Average (Standard dev.)0.0008490718 (±0.04997741)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions320320320
Spacing320320320
CellA=B=C: 397.44 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_40852_msk_1.map
Projections & Slices
AxesZYX

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Histogram

Histogram (log scale)

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Half map: #1

Fileemd_40852_half_map_1.map
Projections & Slices
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Histogram

Histogram (log scale)

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Half map: #2

Fileemd_40852_half_map_2.map
Projections & Slices
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Sample components

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Entire : CtpA-LbcA complex of Pseudomonas aeruginosa

EntireName: CtpA-LbcA complex of Pseudomonas aeruginosa
Components
  • Complex: CtpA-LbcA complex of Pseudomonas aeruginosa
    • Protein or peptide: Carboxyl-terminal protease
    • Protein or peptide: TPR repeat-containing protein PA4667
    • Protein or peptide: unidentified peptide

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Supramolecule #1: CtpA-LbcA complex of Pseudomonas aeruginosa

SupramoleculeName: CtpA-LbcA complex of Pseudomonas aeruginosa / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Pseudomonas aeruginosa (bacteria)
Molecular weightTheoretical: 420 KDa

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Macromolecule #1: Carboxyl-terminal protease

MacromoleculeName: Carboxyl-terminal protease / type: protein_or_peptide / ID: 1 / Number of copies: 6 / Enantiomer: LEVO
Source (natural)Organism: Pseudomonas aeruginosa (bacteria)
Molecular weightTheoretical: 42.920438 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: GSHMSAPLPL DELRTFAEVL DRVKAAYVEP VDDKTLLENA IKGMLSNLDP HSAYLGPEDF AELQESTSGE FGGLGIEVGS EDGFIKVVS PIDDTPAARA GIQPGDLIVQ IDGKPTKGQS MTEAVDSMRG KAGSPITLTI VRDGGRPFDV ELKRAIIKVK S VKSQVLEP ...String:
GSHMSAPLPL DELRTFAEVL DRVKAAYVEP VDDKTLLENA IKGMLSNLDP HSAYLGPEDF AELQESTSGE FGGLGIEVGS EDGFIKVVS PIDDTPAARA GIQPGDLIVQ IDGKPTKGQS MTEAVDSMRG KAGSPITLTI VRDGGRPFDV ELKRAIIKVK S VKSQVLEP GYAYLRITQF QVNTGEEVVK ALNQLRKDNK GRLKGLVLDL RNNPGGVLQS AVEVADAFLT KGLIVYTKGR IA NSELRFS ADPADPSDKV PLVVLINGGS AAAAEIVAGA LQDQKRAILM GTDSFGKGSV QTVLPLNNDR ALKLTTALYY TPN GRSIQA QGIVPDIEVG RAKVTQERSS FEGFKEADLQ GHLANGNGGA DRPTGKRAAP SERPQDSDYQ LSQALSLLKG LSVT RGN

UniProtKB: Carboxy-terminal processing protease CtpB

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Macromolecule #2: TPR repeat-containing protein PA4667

MacromoleculeName: TPR repeat-containing protein PA4667 / type: protein_or_peptide / ID: 2 / Number of copies: 3 / Enantiomer: LEVO
Source (natural)Organism: Pseudomonas aeruginosa (bacteria)
Molecular weightTheoretical: 61.742066 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: MEDTAVETKA KPEKYGSFSE DSLYSLLVAE LAGQRNRFDI ALSNYVVQAQ KTRDPGVSER AFRIAEYLGA DQEALDTSLL WARSAPDNL DAQRAAAIQL ARAGRYEESM VYMEKVLNGQ GDTHFDFLAL SAAETDPDTR AGLLQSFDHL LKKYPNNGQL L FGKALLLQ ...String:
MEDTAVETKA KPEKYGSFSE DSLYSLLVAE LAGQRNRFDI ALSNYVVQAQ KTRDPGVSER AFRIAEYLGA DQEALDTSLL WARSAPDNL DAQRAAAIQL ARAGRYEESM VYMEKVLNGQ GDTHFDFLAL SAAETDPDTR AGLLQSFDHL LKKYPNNGQL L FGKALLLQ QDGRPDEALT LLEDNSASRH EVAPLLLRSR LLQSMKRSDE ALPLLKAGIK EHPDDKRVRL AYARLLVEQN RL DDAKAEF AGLVQQFPDD DDLRFSLALV CLEAQAWDEA RIYLEELVER DSHVDAAHFN LGRLAEEQKD TARALDEYAQ VGP GNDFLP AQLRQTDVLL KAGRVDEAAQ RLDKARSEQP DYAIQLYLIE AEALSNNDQQ EKAWQAIQEG LKQYPEDLNL LYTR SMLAE KRNDLAQMEK DLRFVIAREP DNAMALNALG YTLADRTTRY GEARELILKA HKLNPDDPAI LDSMGWINYR QGKLA DAER YLRQALQRYP DHEVAAHLGE VLWAQGRQGD ARAIWREYLD KQPDSDVLRR TIKRLTGAET P

UniProtKB: TPR repeat-containing protein PA4667

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Macromolecule #3: unidentified peptide

MacromoleculeName: unidentified peptide / type: protein_or_peptide / ID: 3 / Number of copies: 3 / Enantiomer: LEVO
Source (natural)Organism: Escherichia coli BL21(DE3) (bacteria)
Molecular weightTheoretical: 613.749 Da
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString:
(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)

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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

Concentration0.8 mg/mL
BufferpH: 7.5
Component:
ConcentrationNameFormula
20.0 mMTris
100.0 mMKClKCl
5.0 mMMgCl2MgCl2
GridModel: Quantifoil R1.2/1.3 / Material: GOLD / Mesh: 300 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: PLASMA CLEANING / Pretreatment - Time: 30 sec. / Pretreatment - Atmosphere: OTHER
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 279 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Specialist opticsEnergy filter - Name: GIF Bioquantum / Energy filter - Slit width: 20 eV
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Digitization - Dimensions - Width: 11520 pixel / Digitization - Dimensions - Height: 8184 pixel / Number grids imaged: 1 / Number real images: 26257 / Average exposure time: 1.5 sec. / Average electron dose: 66.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 100.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 1.8 µm / Nominal defocus min: 1.3 µm / Nominal magnification: 105000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 2090314
Startup modelType of model: PDB ENTRY
PDB model - PDB ID:

7rqh

Final reconstructionNumber classes used: 4 / Applied symmetry - Point group: C3 (3 fold cyclic) / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 3.94 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 2.8) / Number images used: 1185591
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 2.8)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 2.8)
Final 3D classificationNumber classes: 4 / Software - Name: cryoSPARC (ver. 2.8)
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial model
PDB IDChain

7rqh

source_name: PDB, initial_model_type: experimental model

7rqf

source_name: PDB, initial_model_type: experimental model
RefinementSpace: REAL / Protocol: AB INITIO MODEL / Overall B value: 196.3
Output model

PDB-8sxh:
Structure of the C-terminal protease CtpA-LbcA complex of Pseudomonas aeruginosa

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