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- EMDB-18830: C1 complex binding to a tetrameric C-reactive protein platform -

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Basic information

Entry
Database: EMDB / ID: EMD-18830
TitleC1 complex binding to a tetrameric C-reactive protein platform
Map dataMap low pass filtered to 16 A.
Sample
  • Complex: C1 complex bound to a membrane bound tetrameric CRP platform
KeywordsC-reactive protein / C1 / Complex / Complement / Classical pathway / CRP / activation / IMMUNE SYSTEM
Biological speciesHomo sapiens (human)
Methodsubtomogram averaging / cryo EM / Resolution: 24.0 Å
AuthorsSharp TH / Noone DP
Funding supportEuropean Union, 1 items
OrganizationGrant numberCountry
European Research Council (ERC)759517European Union
CitationJournal: Proc Natl Acad Sci U S A / Year: 2024
Title: Structural basis for surface activation of the classical complement cascade by the short pentraxin C-reactive protein.
Authors: Dylan P Noone / Marjolein M E Isendoorn / Sebastiaan M W R Hamers / Mariska E Keizer / Jip Wulffelé / Tijn T van der Velden / Douwe J Dijkstra / Leendert A Trouw / Dmitri V Filippov / Thomas H Sharp /
Abstract: Human C-reactive protein (CRP) is a pentameric complex involved in immune defense and regulation of autoimmunity. CRP is also a therapeutic target, with both administration and depletion of serum CRP ...Human C-reactive protein (CRP) is a pentameric complex involved in immune defense and regulation of autoimmunity. CRP is also a therapeutic target, with both administration and depletion of serum CRP being pursued as a possible treatment for autoimmune and cardiovascular diseases, among others. CRP binds to phosphocholine (PC) moieties on membranes to activate the complement system via the C1 complex, but it is unknown how CRP, or any pentraxin, binds to C1. Here, we present a cryoelectron tomography (cryoET)-derived structure of CRP bound to PC ligands and the C1 complex. To gain control of CRP binding, a synthetic mimotope of PC was synthesized and used to decorate cell-mimetic liposome surfaces. Structure-guided mutagenesis of CRP yielded a fully active complex able to bind PC-coated liposomes that was ideal for cryoET and subtomogram averaging. In contrast to antibodies, which form Fc-mediated hexameric platforms to bind and activate the C1 complex, CRP formed rectangular platforms assembled from four laterally associated CRP pentamers that bind only four of the six available globular C1 head groups. Potential residues mediating lateral association of CRP were identified from interactions between unit cells in existing crystal structures, which rationalized previously unexplained mutagenesis data regarding CRP-mediated complement activation. The structure also enabled interpretation of existing biochemical data regarding interactions mediating C1 binding and identified additional residues for further mutagenesis studies. These structural data therefore provide a possible mechanism for regulation of complement by CRP, which limits complement progression and has consequences for how the innate immune system influences autoimmunity.
History
DepositionNov 2, 2023-
Header (metadata) releaseSep 4, 2024-
Map releaseSep 4, 2024-
UpdateSep 18, 2024-
Current statusSep 18, 2024Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_18830.png

Downloads & links

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Map

FileDownload / File: emd_18830.map.gz / Format: CCP4 / Size: 18.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationMap low pass filtered to 16 A.
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
3.48 Å/pix.
x 168 pix.
= 584.64 Å		3.48 Å/pix.
x 168 pix.
= 584.64 Å		3.48 Å/pix.
x 168 pix.
= 584.64 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 3.48 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.9
Minimum - Maximum-3.771058 - 6.4991913
Average (Standard dev.)0.044856805 (±0.32271105)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-84-84-84
Dimensions168168168
Spacing168168168
CellA=B=C: 584.64 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_18830_msk_1.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 : C1 complex bound to a membrane bound tetrameric CRP platform

EntireName: C1 complex bound to a membrane bound tetrameric CRP platform
Components
  • Complex: C1 complex bound to a membrane bound tetrameric CRP platform

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Supramolecule #1: C1 complex bound to a membrane bound tetrameric CRP platform

SupramoleculeName: C1 complex bound to a membrane bound tetrameric CRP platform
type: complex / ID: 1 / Parent: 0
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 1226 kDa/nm

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Experimental details

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Structure determination

Methodcryo EM
Processingsubtomogram averaging
Aggregation stateparticle

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Sample preparation

BufferpH: 7.4
Component:
ConcentrationNameFormula
50.0 mMTris
150.0 mMNaCl
5.0 mMCaCl2
GridModel: EMS Lacey Carbon / Material: COPPER / Mesh: 200 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 30 sec. / Details: 25 mA
VitrificationCryogen name: ETHANE / Chamber humidity: 65 % / Chamber temperature: 277.15 K / Instrument: LEICA EM GP
DetailsCRP was an R188A mutant expressed in human cells. C1 was purchased from complement technology (Texas, USA) and purified from human serum.

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Electron microscopy

MicroscopeFEI TALOS ARCTICA
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Average electron dose: 1.54 e/Å2
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 50.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 6.0 µm / Nominal defocus min: 4.0 µm
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company

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Image processing

Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Algorithm: BACK PROJECTION / Resolution.type: BY AUTHOR / Resolution: 24.0 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: Dynamo / Number subtomograms used: 2518
ExtractionNumber tomograms: 133 / Number images used: 3428 / Method: Manual picking / Software - Name: EMAN2
Final angle assignmentType: PROJECTION MATCHING / Software - Name: Dynamo
FSC plot (resolution estimation)

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