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- EMDB-11003: Campylobacter jejuni serine protease HtrA -

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Basic information

Entry
Database: EMDB / ID: EMD-11003
TitleCampylobacter jejuni serine protease HtrA
Map data
Sample
  • Complex: HtrA dodecamer
Function / homology
Function and homology information


peptidase Do / periplasmic space / serine-type endopeptidase activity
Similarity search - Function
Peptidase S1C, Do / PDZ domain / Peptidase S1C / Domain present in PSD-95, Dlg, and ZO-1/2. / PDZ domain / PDZ superfamily / Peptidase S1, PA clan
Similarity search - Domain/homology
Periplasmic serine endoprotease DegP-like
Similarity search - Component
Biological speciesCampylobacter jejuni (Campylobacter)
Methodsingle particle reconstruction / cryo EM / Resolution: 5.8 Å
AuthorsGrinzato A / Kandiah E / Zanotti G
CitationJournal: Gut Microbes / Year: 2020
Title: Functional analysis and cryo-electron microscopy of serine protease HtrA.
Authors: Urszula Zarzecka / Alessandro Grinzato / Eaazhisai Kandiah / Dominik Cysewski / Paola Berto / Joanna Skorko-Glonek / Giuseppe Zanotti / Steffen Backert /
Abstract: is a predominant zoonotic pathogen causing gastroenteritis and other diseases in humans. An important bacterial virulence factor is the secreted serine protease HtrA (HtrA ), which targets tight ... is a predominant zoonotic pathogen causing gastroenteritis and other diseases in humans. An important bacterial virulence factor is the secreted serine protease HtrA (HtrA ), which targets tight and adherens junctional proteins in the gut epithelium. Here we have investigated the function and structure of HtrA using biochemical assays and cryo-electron microscopy. Mass spectrometry analysis identified differences and similarities in the cleavage site specificity for HtrA by comparison to the HtrA counterparts from and . We defined the architecture of HtrA at 5.8 Å resolution as a dodecamer, built of four trimers. The contacts between the trimers are quite loose, a fact that explains the flexibility and mobility of the dodecameric assembly. This flexibility has also been studied through molecular dynamics simulation, which revealed opening of the dodecamer to expose the proteolytically active site of the protease. Moreover, we examined the rearrangements at the level of oligomerization in the presence or absence of substrate using size exclusion chromatography, which revealed hexamers, dodecamers and larger oligomeric forms, as well as remarkable stability of higher oligomeric forms (> 12-mers) compared to previously tested homologs from other bacteria. Extremely dynamic decay of the higher oligomeric forms into lower forms was observed after full cleavage of the substrate by the proteolytically active variant of HtrA . Together, this is the first report on the in-depth functional and structural analysis of HtrA , which may allow the construction of therapeutically relevant HtrA inhibitors in the near future.
History
DepositionMay 5, 2020-
Header (metadata) releaseSep 30, 2020-
Map releaseSep 30, 2020-
UpdateMay 26, 2021-
Current statusMay 26, 2021Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.02
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 0.02
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6z05
  • Surface level: 0.02
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_11003.png

Downloads & links

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Map

FileDownload / File: emd_11003.map.gz / Format: CCP4 / Size: 76.8 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 1.207 Å
Density
Contour LevelBy AUTHOR: 0.02 / Movie #1: 0.02
Minimum - Maximum-0.026209008 - 0.066626936
Average (Standard dev.)0.00048996235 (±0.00403843)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions272272272
Spacing272272272
CellA=B=C: 328.30402 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.2071.2071.207
M x/y/z272272272
origin x/y/z0.0000.0000.000
length x/y/z328.304328.304328.304
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS272272272
D min/max/mean-0.0260.0670.000

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Supplemental data

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Sample components

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Entire : HtrA dodecamer

EntireName: HtrA dodecamer
Components
  • Complex: HtrA dodecamer

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Supramolecule #1: HtrA dodecamer

SupramoleculeName: HtrA dodecamer / type: complex / ID: 1 / Parent: 0
Source (natural)Organism: Campylobacter jejuni (Campylobacter)
Recombinant expressionOrganism: Escherichia coli (E. coli)

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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

BufferpH: 7.4
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeTFS GLACIOS
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy
Image recordingFilm or detector model: FEI FALCON III (4k x 4k) / Average electron dose: 44.0 e/Å2

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Image processing

Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
Final reconstructionApplied symmetry - Point group: T (tetrahedral) / Resolution.type: BY AUTHOR / Resolution: 5.8 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 50000

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