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- EMDB-19717: Cryo-EM structure of the C terminal region of PTX3 with a section... -

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Basic information

Entry
Database: EMDB / ID: EMD-19717
TitleCryo-EM structure of the C terminal region of PTX3 with a section of coiled-coil
Map data
Sample
  • Complex: Pentraxin 3
    • Protein or peptide: Pentraxin-related protein PTX3
  • Ligand: 2-acetamido-2-deoxy-beta-D-glucopyranose
  • Ligand: water
KeywordsPTX3 / Pentraxin / pattern / recognition / coiled-coil / coil / innate / immunity / extracellular / octamer / Pentraxin-related / cryo-EM / IMMUNE SYSTEM
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)
Methodsingle 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, 1 items
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, 2024-
Header (metadata) releaseJan 22, 2025-
Map releaseJan 22, 2025-
UpdateMar 12, 2025-
Current statusMar 12, 2025Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_19717.png

Downloads & links

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Map

FileDownload / File: emd_19717.map.gz / Format: CCP4 / Size: 244.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
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AxesZ (Sec.)Y (Row.)X (Col.)
0.92 Å/pix.
x 400 pix.
= 368.64 Å		0.92 Å/pix.
x 400 pix.
= 368.64 Å		0.92 Å/pix.
x 400 pix.
= 368.64 Å

Surface

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Images are generated by Spider.

Voxel sizeX=Y=Z: 0.9216 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.75
Minimum - Maximum-3.1164248 - 5.371554
Average (Standard dev.)0.0018122424 (±0.102109715)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions400400400
Spacing400400400
CellA=B=C: 368.64 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #1

Fileemd_19717_half_map_1.map
Projections & Slices
AxesZYX

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Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #2

Fileemd_19717_half_map_2.map
Projections & Slices
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Sample components

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Entire : Pentraxin 3

EntireName: Pentraxin 3
Components
  • Complex: Pentraxin 3
    • Protein or peptide: Pentraxin-related protein PTX3
  • Ligand: 2-acetamido-2-deoxy-beta-D-glucopyranose
  • Ligand: water

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Supramolecule #1: Pentraxin 3

SupramoleculeName: Pentraxin 3 / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1
Details: Pentraxin 3 is a pattern recognition protein which forms a homo-octameric complex
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 321 KDa

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Macromolecule #1: Pentraxin-related protein PTX3

MacromoleculeName: Pentraxin-related protein PTX3 / type: protein_or_peptide / ID: 1
Details: Full length pentraxin 3 is cleaved by the expressing cells during secretion
Number of copies: 8 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 40.206141 KDa
Recombinant expressionOrganism: Cricetulus griseus (Chinese hamster)
SequenceString: ENSDDYDLMY VNLDNEIDNG LHPTEDPTPC DCGQEHSEWD KLFIMLENSQ MRERMLLQAT DDVLRGELQR LREELGRLAE SLARPCAPG APAEARLTSA LDELLQATRD AGRRLARMEG AEAQRPEEAG RALAAVLEEL RQTRADLHAV QGWAARSWLP A GCETAILF ...String:
ENSDDYDLMY VNLDNEIDNG LHPTEDPTPC DCGQEHSEWD KLFIMLENSQ MRERMLLQAT DDVLRGELQR LREELGRLAE SLARPCAPG APAEARLTSA LDELLQATRD AGRRLARMEG AEAQRPEEAG RALAAVLEEL RQTRADLHAV QGWAARSWLP A GCETAILF PMRSKKIFGS VHPVRPMRLE SFSACIWVKA TDVLNKTILF SYGTKRNPYE IQLYLSYQSI VFVVGGEENK LV AEAMVSL GRWTHLCGTW NSEEGLTSLW VNGELAATTV EMATGHIVPE GGILQIGQEK NGCCVGGGFD ETLAFSGRLT GFN IWDSVL SNEEIRETGG AESCHIRGNI VGWGVTEIQP HGGAQYVS

UniProtKB: Pentraxin-related protein PTX3

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Macromolecule #2: 2-acetamido-2-deoxy-beta-D-glucopyranose

MacromoleculeName: 2-acetamido-2-deoxy-beta-D-glucopyranose / type: ligand / ID: 2 / Number of copies: 8 / Formula: NAG
Molecular weightTheoretical: 221.208 Da
Chemical component information

ChemComp-NAG:
2-acetamido-2-deoxy-beta-D-glucopyranose

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Macromolecule #3: water

MacromoleculeName: water / type: ligand / ID: 3 / Number of copies: 214 / Formula: HOH
Molecular weightTheoretical: 18.015 Da
Chemical component information

ChemComp-HOH:
WATER

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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

Concentration0.25 mg/mL
BufferpH: 7.4 / Details: PBS pH 7.4
GridModel: Quantifoil R2/2 / Material: GOLD / Mesh: 200
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277.15 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: FEI FALCON IV (4k x 4k) / Average exposure time: 4.4 sec. / Average electron dose: 40.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 2.0 µm / Nominal defocus min: 1.0 µm / Nominal magnification: 165000
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

Startup modelType of model: INSILICO MODEL / In silico model: cryosparc ab initio
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: D4 (2x4 fold dihedral) / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 3.33 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 23457
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 4.3.1) / Details: Cryosparc non uniform refinement
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 4.3.1)
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

8pvq


Chain - Source name: PDB / Chain - Initial model type: experimental model
DetailsInitial fitting was done using chimera followed by real-space refinement in phenix and rebuilding in COOT
RefinementSpace: REAL / Protocol: FLEXIBLE FIT / Target criteria: RSC
Output model

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

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