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- PDB-8s50: Cryo-EM structure of the C terminal region of PTX3 with a section... -

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Basic information

Entry
Database: PDB / ID: 8s50
TitleCryo-EM structure of the C terminal region of PTX3 with a section of coiled-coil
ComponentsPentraxin-related protein PTX3
KeywordsIMMUNE SYSTEM / PTX3 / Pentraxin / pattern / recognition / coiled-coil / coil / innate / immunity / extracellular / octamer / Pentraxin-related / cryo-EM
Function / homology
Function and homology information


(1->3)-beta-D-glucan binding / negative regulation by host of viral glycoprotein metabolic process / negative regulation of glycoprotein metabolic process / ovarian cumulus expansion / negative regulation by host of viral process / opsonization / complement component C1q complex binding / response to yeast / host-mediated suppression of symbiont invasion / virion binding ...(1->3)-beta-D-glucan binding / negative regulation by host of viral glycoprotein metabolic process / negative regulation of glycoprotein metabolic process / ovarian cumulus expansion / negative regulation by host of viral process / opsonization / complement component C1q complex binding / response to yeast / host-mediated suppression of symbiont invasion / virion binding / positive regulation of phagocytosis / extracellular matrix organization / extracellular matrix / specific granule lumen / positive regulation of nitric oxide biosynthetic process / tertiary granule lumen / inflammatory response / innate immune response / Neutrophil degranulation / extracellular space / extracellular region / identical protein binding
Similarity search - Function
Pentraxin-related protein PTX3 / LamG-like jellyroll fold / LamG-like jellyroll fold domain / Pentaxin, conserved site / Pentraxin domain signature. / Pentaxin family / Pentraxin / C-reactive protein / pentaxin family / Pentraxin-related / Pentraxin (PTX) domain profile. / Concanavalin A-like lectin/glucanase domain superfamily
Similarity search - Domain/homology
Pentraxin-related protein PTX3
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.33 Å
AuthorsSnee, M. / Shah, A. / Lockhart-Cairns, M. / Collins, R. / Levy, C. / Baldock, C. / Day, A.
Funding support United Kingdom, 1items
OrganizationGrant numberCountry
Biotechnology and Biological Sciences Research Council (BBSRC) United Kingdom
Citation
Journal: Matrix Biol / Year: 2025
Title: The structural organisation of pentraxin-3 and its interactions with heavy chains of inter-α-inhibitor regulate crosslinking of the hyaluronan matrix.
Authors: Anokhi Shah / Xiaoli Zhang / Matthew Snee / Michael P Lockhart-Cairns / Colin W Levy / Thomas A Jowitt / Holly L Birchenough / Louisa Dean / Richard Collins / Rebecca J Dodd / Abigail R E ...Authors: Anokhi Shah / Xiaoli Zhang / Matthew Snee / Michael P Lockhart-Cairns / Colin W Levy / Thomas A Jowitt / Holly L Birchenough / Louisa Dean / Richard Collins / Rebecca J Dodd / Abigail R E Roberts / Jan J Enghild / Alberto Mantovani / Juan Fontana / Clair Baldock / Antonio Inforzato / Ralf P Richter / Anthony J Day /
Abstract: Pentraxin-3 (PTX3) is an octameric protein, comprised of eight identical protomers, that has diverse functions in reproductive biology, innate immunity and cancer. PTX3 interacts with the large ...Pentraxin-3 (PTX3) is an octameric protein, comprised of eight identical protomers, that has diverse functions in reproductive biology, innate immunity and cancer. PTX3 interacts with the large polysaccharide hyaluronan (HA) to which heavy chains (HCs) of the inter-α-inhibitor (IαI) family of proteoglycans are covalently attached, playing a key role in the (non-covalent) crosslinking of HC•HA complexes. These interactions stabilise the cumulus matrix, essential for ovulation and fertilisation in mammals, and are also implicated in the formation of pathogenic matrices in the context of viral lung infections. To better understand the physiological and pathological roles of PTX3 we have analysed how its quaternary structure underpins HA crosslinking via its interactions with HCs. A combination of X-ray crystallography, cryo-electron microscopy (cryo-EM) and AlphaFold predictive modelling revealed that the C-terminal pentraxin domains of the PTX3 octamer are arranged in a central cube, with two long extensions on either side, each formed from four protomers assembled into tetrameric coiled-coil regions, essentially as described by (Noone et al., 2022; doi:10.1073/pnas.2208144119). From crystallography and cryo-EM data, we identified a network of inter-protomer salt bridges that facilitate the assembly of the octamer. Small angle X-ray scattering (SAXS) validated our model for the octameric protein, including the analysis of two PTX3 constructs: a tetrameric 'Half-PTX3' and a construct missing the 24 N-terminal residues (Δ1-24_PTX3). SAXS determined a length of ∼520 Å for PTX3 and, combined with 3D variability analysis of cryo-EM data, defined the flexibility of the N-terminal extensions. Biophysical analyses revealed that the prototypical heavy chain HC1 does not interact with PTX3 at pH 7.4, consistent with our previous studies showing that, at this pH, PTX3 only associates with HC•HA complexes if they are formed in its presence. However, PTX3 binds to HC1 at acidic pH, and can also be incorporated into pre-formed HC•HA complexes under these conditions. This provides a novel mechanism for the regulation of PTX3-mediated HA crosslinking (e.g., during inflammation), likely mediated by a pH-dependent conformational change in HC1. The PTX3 octamer was found to associate simultaneously with up to eight HC1 molecules and, thus, has the potential to form a major crosslinking node within HC•HA matrices, i.e., where the physical and biochemical properties of resulting matrices could be tuned by the HC/PTX3 composition.
#1: Journal: Acta Crystallogr., Sect. D: Biol. Crystallogr. / Year: 2018
Title: Real-space refinement in PHENIX for cryo-EM and crystallography
Authors: Afonine, P. / Poon, B. / Read, R. / Sobolev, O. / Terwilliger, T. / Urzhumtsev, A. / Adams, P.
History
DepositionFeb 22, 2024Deposition site: PDBE / Processing site: PDBE
Revision 1.0Jan 22, 2025Provider: repository / Type: Initial release
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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
B: Pentraxin-related protein PTX3
A: Pentraxin-related protein PTX3
C: Pentraxin-related protein PTX3
D: Pentraxin-related protein PTX3
F: Pentraxin-related protein PTX3
G: Pentraxin-related protein PTX3
J: Pentraxin-related protein PTX3
K: Pentraxin-related protein PTX3
hetero molecules


Theoretical massNumber of molelcules
Total (without water)323,41916
Polymers321,6498
Non-polymers1,7708
Water3,855214
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

#1: Protein
Pentraxin-related protein PTX3 / Pentaxin-related protein PTX3 / Tumor necrosis factor alpha-induced protein 5 / TNF alpha-induced ...Pentaxin-related protein PTX3 / Tumor necrosis factor alpha-induced protein 5 / TNF alpha-induced protein 5 / Tumor necrosis factor-inducible gene 14 protein / TSG-14


Mass: 40206.141 Da / Num. of mol.: 8
Source method: isolated from a genetically manipulated source
Details: Full length pentraxin 3 is cleaved by the expressing cells during secretion
Source: (gene. exp.) Homo sapiens (human) / Gene: PTX3, TNFAIP5, TSG14 / Cell line (production host): CHO / Production host: Cricetulus griseus (Chinese hamster) / References: UniProt: P26022
#2: Sugar
ChemComp-NAG / 2-acetamido-2-deoxy-beta-D-glucopyranose / N-acetyl-beta-D-glucosamine / 2-acetamido-2-deoxy-beta-D-glucose / 2-acetamido-2-deoxy-D-glucose / 2-acetamido-2-deoxy-glucose / N-ACETYL-D-GLUCOSAMINE


Type: D-saccharide, beta linking / Mass: 221.208 Da / Num. of mol.: 8 / Source method: obtained synthetically / Formula: C8H15NO6
IdentifierTypeProgram
DGlcpNAcbCONDENSED IUPAC CARBOHYDRATE SYMBOLGMML 1.0
N-acetyl-b-D-glucopyranosamineCOMMON NAMEGMML 1.0
b-D-GlcpNAcIUPAC CARBOHYDRATE SYMBOLPDB-CARE 1.0
GlcNAcSNFG CARBOHYDRATE SYMBOLGMML 1.0
#3: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 214 / Source method: isolated from a natural source / Formula: H2O
Has ligand of interestN
Has protein modificationY

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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: Pentraxin 3 / Type: COMPLEX
Details: Pentraxin 3 is a pattern recognition protein which forms a homo-octameric complex
Entity ID: #1 / Source: RECOMBINANT
Molecular weightValue: 0.321 MDa / Experimental value: NO
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Cricetulus griseus (Chinese hamster)
Buffer solutionpH: 7.4 / Details: PBS pH 7.4
SpecimenConc.: 0.25 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GOLD / Grid mesh size: 200 divisions/in. / Grid type: Quantifoil R2/2
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277.15 K

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 165000 X / Nominal defocus max: 2000 nm / Nominal defocus min: 1000 nm / Cs: 2.7 mm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 4.4 sec. / Electron dose: 40 e/Å2 / Film or detector model: FEI FALCON IV (4k x 4k)

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Processing

EM software
IDNameVersionCategory
2EPUimage acquisition
4cryoSPARC4.3.1CTF correction
7UCSF Chimera1.13.1model fitting
9cryoSPARC4.3.1initial Euler assignment
10cryoSPARC4.3.1final Euler assignment
13PHENIX1.19model refinement
CTF correctionType: NONE
SymmetryPoint symmetry: D4 (2x4 fold dihedral)
3D reconstructionResolution: 3.33 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 23457 / Algorithm: FOURIER SPACE / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT / Space: REAL / Target criteria: RSC
Details: Initial fitting was done using chimera followed by real-space refinement in phenix and rebuilding in COOT
Atomic model buildingPDB-ID: 8PVQ

8pvq


Accession code: 8PVQ / Source name: PDB / Type: experimental model
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00314768
ELECTRON MICROSCOPYf_angle_d0.520072
ELECTRON MICROSCOPYf_dihedral_angle_d13.6835256
ELECTRON MICROSCOPYf_chiral_restr0.0452232
ELECTRON MICROSCOPYf_plane_restr0.0032568

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