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- PDB-7pkg: C-reactive protein pentamer at pH 5 -

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Basic information

Entry
Database: PDB / ID: 7pkg
TitleC-reactive protein pentamer at pH 5
ComponentsC-reactive protein
KeywordsIMMUNE SYSTEM / C-reactive protein / CRP / innate immunity
Function / homology
Function and homology information


regulation of interleukin-8 production / opsonization / complement component C1q complex binding / low-density lipoprotein particle binding / negative regulation of mononuclear cell proliferation / vasoconstriction / choline binding / Classical antibody-mediated complement activation / low-density lipoprotein particle receptor binding / negative regulation of macrophage derived foam cell differentiation ...regulation of interleukin-8 production / opsonization / complement component C1q complex binding / low-density lipoprotein particle binding / negative regulation of mononuclear cell proliferation / vasoconstriction / choline binding / Classical antibody-mediated complement activation / low-density lipoprotein particle receptor binding / negative regulation of macrophage derived foam cell differentiation / negative regulation of lipid storage / positive regulation of superoxide anion generation / acute-phase response / defense response to Gram-positive bacterium / inflammatory response / innate immune response / calcium ion binding / positive regulation of gene expression / extracellular space / extracellular region / identical protein binding
Similarity search - Function
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
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.3 Å
AuthorsNoone, D.P. / Sharp, T.H.
Funding supportEuropean Union, 1items
OrganizationGrant numberCountry
European Research Council (ERC)759517European Union
CitationJournal: Front Immunol / Year: 2021
Title: Cryo-Electron Microscopy and Biochemical Analysis Offer Insights Into the Effects of Acidic pH, Such as Occur During Acidosis, on the Complement Binding Properties of C-Reactive Protein.
Authors: Dylan P Noone / Tijn T van der Velden / Thomas H Sharp /
Abstract: The pentraxin family of proteins includes C-reactive protein (CRP), a canonical marker for the acute phase inflammatory response. As compared to normal physiological conditions in human serum, under ...The pentraxin family of proteins includes C-reactive protein (CRP), a canonical marker for the acute phase inflammatory response. As compared to normal physiological conditions in human serum, under conditions associated with damage and inflammation, such as acidosis and the oxidative burst, CRP exhibits modulated biochemical properties that may have a structural basis. Here, we explore how pH and ligand binding affect the structure and biochemical properties of CRP. Cryo-electron microscopy was used to solve structures of CRP at pH 7.5 or pH 5 and in the presence or absence of the ligand phosphocholine (PCh), which yielded 7 new high-resolution structures of CRP, including pentameric and decameric complexes. Structures previously derived from crystallography were imperfect pentagons, as shown by the variable angles between each subunit, whereas pentameric CRP derived from cryoEM was found to have C5 symmetry, with subunits forming a regular pentagon with equal angles. This discrepancy indicates flexibility at the interfaces of monomers that may relate to activation of the complement system by the C1 complex. CRP also appears to readily decamerise in solution into dimers of pentamers, which obscures the postulated binding sites for C1. Subtle structural rearrangements were observed between the conditions tested, including a putative change in histidine protonation that may prime the disulphide bridges for reduction and enhanced ability to activate the immune system. Enzyme-linked immunosorbent assays showed that CRP had markedly increased association to the C1 complex and immunoglobulins under conditions associated with acidosis, whilst a reduction in the Ca concentration lowered this pH-sensitivity for C1q, but not immunoglobulins, suggesting different modes of binding. These data suggest a model whereby a change in the ionic nature of CRP and immunological proteins can make it more adhesive to potential ligands without large structural rearrangements.
History
DepositionAug 25, 2021Deposition site: PDBE / Processing site: PDBE
Revision 1.0Dec 22, 2021Provider: repository / Type: Initial release
Revision 1.1Jan 12, 2022Group: Database references / Category: citation / citation_author
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_PubMed / _citation.title / _citation_author.identifier_ORCID / _citation_author.name

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Assembly

Deposited unit
E: C-reactive protein
D: C-reactive protein
C: C-reactive protein
B: C-reactive protein
A: C-reactive protein
hetero molecules


Theoretical massNumber of molelcules
Total (without water)115,74115
Polymers115,3405
Non-polymers40110
Water905
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area7580 Å2
ΔGint-144 kcal/mol
Surface area39860 Å2

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Components

#1: Protein
C-reactive protein


Mass: 23068.039 Da / Num. of mol.: 5
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: CRP, PTX1 / Production host: Escherichia coli (E. coli) / References: UniProt: P02741
#2: Chemical
ChemComp-CA / CALCIUM ION


Mass: 40.078 Da / Num. of mol.: 10 / Source method: obtained synthetically / Formula: Ca / Feature type: SUBJECT OF INVESTIGATION
#3: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 5 / Source method: isolated from a natural source / Formula: H2O
Has ligand of interestY

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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: C-reactive protein pentamer / Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT
Molecular weightValue: 0.115 MDa / Experimental value: NO
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 5
Buffer component
IDConc.NameFormulaBuffer-ID
1140 mMSodium acetate1
240 mMAcetic acid1
3140 mMNaCl1
42 mMCaCl21
SpecimenConc.: 6 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 400 divisions/in. / Grid type: C-flat-1.2/1.3
VitrificationInstrument: LEICA EM GP / Cryogen name: ETHANE / Humidity: 65 % / 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: 105000 X / Nominal defocus max: 2000 nm / Nominal defocus min: 800 nm / Cs: 2.7 mm / C2 aperture diameter: 50 µm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 2.25 sec. / Electron dose: 50 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 3131
EM imaging opticsEnergyfilter name: GIF Bioquantum / Details: Gatan BioQuantum K3 / Energyfilter slit width: 20 eV

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Processing

SoftwareName: PHENIX / Version: 1.18.2_3874: / Classification: refinement
EM software
IDNameVersionCategoryDetails
1EMAN22.9particle selectionNeural Network
2EPUimage acquisition
4RELION3.1CTF correction
7ISOLDEmodel fitting
9PHENIXmodel refinement
10RELION3.1initial Euler assignment
11RELION3.1final Euler assignment
12RELION3.1classification
13RELION3.13D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 740919
3D reconstructionResolution: 3.3 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 171942 / Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT
Atomic model buildingPDB-ID: 1B09

1b09

Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0058390
ELECTRON MICROSCOPYf_angle_d0.85411405
ELECTRON MICROSCOPYf_dihedral_angle_d8.8811100
ELECTRON MICROSCOPYf_chiral_restr0.1661235
ELECTRON MICROSCOPYf_plane_restr0.0051450

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