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- EMDB-14775: Unsymmetrical (C1) cryoEM map of the long pentraxin 3 -

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Basic information

Entry
Database: EMDB / ID: EMD-14775
TitleUnsymmetrical (C1) cryoEM map of the long pentraxin 3
Map dataC1 symmetrical map of the long pentraxin 3.
Sample
  • Complex: Long pentraxin 3 pentraxin domain
    • Protein or peptide: Long pentraxin 3
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.8 Å
AuthorsNoone DP / Sharp TH
Funding supportEuropean Union, 1 items
OrganizationGrant numberCountry
European Research Council (ERC)759517European Union
CitationJournal: Proc Natl Acad Sci U S A / Year: 2022
Title: PTX3 structure determination using a hybrid cryoelectron microscopy and AlphaFold approach offers insights into ligand binding and complement activation.
Authors: Dylan P Noone / Douwe J Dijkstra / Teun T van der Klugt / Peter A van Veelen / Arnoud H de Ru / Paul J Hensbergen / Leendert A Trouw / Thomas H Sharp /
Abstract: Pattern recognition molecules (PRMs) form an important part of innate immunity, where they facilitate the response to infections and damage by triggering processes such as inflammation. The pentraxin ...Pattern recognition molecules (PRMs) form an important part of innate immunity, where they facilitate the response to infections and damage by triggering processes such as inflammation. The pentraxin family of soluble PRMs comprises long and short pentraxins, with the former containing unique N-terminal regions unrelated to other proteins or each other. No complete high-resolution structural information exists about long pentraxins, unlike the short pentraxins, where there is an abundance of both X-ray and cryoelectron microscopy (cryo-EM)-derived structures. This study presents a high-resolution structure of the prototypical long pentraxin, PTX3. Cryo-EM yielded a 2.5-Å map of the C-terminal pentraxin domains that revealed a radically different quaternary structure compared to other pentraxins, comprising a glycosylated D4 symmetrical octameric complex stabilized by an extensive disulfide network. The cryo-EM map indicated α-helices that extended N terminal of the pentraxin domains that were not fully resolved. AlphaFold was used to predict the remaining N-terminal structure of the octameric PTX3 complex, revealing two long tetrameric coiled coils with two hinge regions, which was validated using classification of cryo-EM two-dimensional averages. The resulting hybrid cryo-EM/AlphaFold structure allowed mapping of ligand binding sites, such as C1q and fibroblast growth factor-2, as well as rationalization of previous biochemical data. Given the relevance of PTX3 in conditions ranging from COVID-19 prognosis, cancer progression, and female infertility, this structure could be used to inform the understanding and rational design of therapies for these disorders and processes.
History
DepositionApr 13, 2022-
Header (metadata) releaseAug 3, 2022-
Map releaseAug 3, 2022-
UpdateAug 17, 2022-
Current statusAug 17, 2022Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_14775.png

Downloads & links

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Map

FileDownload / File: emd_14775.map.gz / Format: CCP4 / Size: 178 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationC1 symmetrical map of the long pentraxin 3.
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.84 Å/pix.
x 360 pix.
= 300.96 Å		0.84 Å/pix.
x 360 pix.
= 300.96 Å		0.84 Å/pix.
x 360 pix.
= 300.96 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.836 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.015
Minimum - Maximum-0.05328496 - 0.10900947
Average (Standard dev.)8.7842716e-05 (±0.0020781525)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions360360360
Spacing360360360
CellA=B=C: 300.96002 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_14775_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: Unmasked C1 symmetrical map of the long pentraxin 3.

Fileemd_14775_additional_1.map
AnnotationUnmasked C1 symmetrical map of the long pentraxin 3.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map from the Relion Refinement used to generate the final map.

Fileemd_14775_half_map_1.map
AnnotationHalf map from the Relion Refinement used to generate the final map.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map from the Relion Refinement used to generate the final map.

Fileemd_14775_half_map_2.map
AnnotationHalf map from the Relion Refinement used to generate the final map.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Long pentraxin 3 pentraxin domain

EntireName: Long pentraxin 3 pentraxin domain
Components
  • Complex: Long pentraxin 3 pentraxin domain
    • Protein or peptide: Long pentraxin 3

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Supramolecule #1: Long pentraxin 3 pentraxin domain

SupramoleculeName: Long pentraxin 3 pentraxin domain / type: complex / Chimera: Yes / ID: 1 / Parent: 0 / Macromolecule list: all
Details: CryoEM derived map of the pentraxin domains of PTX3. At higher isosurface threshold values the start of the coiled coil N-terminal domain can be seen.
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Homo sapiens (human)
Molecular weightTheoretical: 336 KDa

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Macromolecule #1: Long pentraxin 3

MacromoleculeName: Long pentraxin 3 / type: protein_or_peptide / ID: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: QGWAARSWLP AGCETAILFP MRSKKIFGSV HPVRPMRLES FSACIWVKAT DVLNKTILFS YGTKRNPYE IQLYLSYQSI VFVVGGEENK LVAEAMVSLG RWTHLCGTWN SEEGLTSLWV N GELAATTV EMATGHIVPE GGILQIGQEK NGCCVGGGFD ETLAFSGRLT ...String:
QGWAARSWLP AGCETAILFP MRSKKIFGSV HPVRPMRLES FSACIWVKAT DVLNKTILFS YGTKRNPYE IQLYLSYQSI VFVVGGEENK LVAEAMVSLG RWTHLCGTWN SEEGLTSLWV N GELAATTV EMATGHIVPE GGILQIGQEK NGCCVGGGFD ETLAFSGRLT GFNIWDSVLS NE EIRETGG AESCHIRGNI VGWGVTEIQP HGGAQYVS

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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

Concentration23 mg/mL
BufferpH: 8
Component:
ConcentrationNameFormula
20.0 mMTris-HClTris
500.0 mMSodium chlorideNaClSodium chloride
0.05 % (w/v)Tween-20
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 400 / Support film - Material: GRAPHENE OXIDE / Support film - topology: HOLEY
VitrificationCryogen name: ETHANE / Chamber humidity: 65 % / Chamber temperature: 277.15 K / Instrument: LEICA EM GP
DetailsPTX3 at concentrations between 1-2 uM in 20 mM Tris-HCl, 500 mM NaCl, pH 8.0 was concentrated in a 50 kDa molecular weight cut-off spin filter to concentrations between 60-70 uM.

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 50.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 2.0 µm / Nominal defocus min: 0.8 µm / Nominal magnification: 105000
Specialist opticsEnergy filter - Name: GIF Bioquantum / Energy filter - Slit width: 20 eV
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
TemperatureMin: 90.15 K / Max: 103.15 K
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Number grids imaged: 1 / Number real images: 4612 / Average electron dose: 50.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 1360284
CTF correctionSoftware - Name: Warp
Startup modelType of model: INSILICO MODEL / In silico model: Generated an initial model in Relion 3.1
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.1)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.1)
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 2.8 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.1) / Number images used: 276374
FSC plot (resolution estimation)

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