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- PDB-8wv4: C-reactive protein, pentamer -

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Basic information

Entry
Database: PDB / ID: 8wv4
TitleC-reactive protein, pentamer
ComponentsC-reactive protein(1-205)
KeywordsIMMUNE SYSTEM / Pentraxin / Acute-phase reactant
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
AuthorsYadav, S. / Vinothkumar, K.R.
Funding support India, 2items
OrganizationGrant numberCountry
Department of Biotechnology (DBT, India)DBT/PR12422/MED/31/287/2014 India
Other governmentRTI4006
CitationJournal: Acta Crystallogr D Struct Biol / Year: 2024
Title: Factors affecting macromolecule orientations in thin films formed in cryo-EM.
Authors: Swati Yadav / Kutti R Vinothkumar /
Abstract: The formation of a vitrified thin film embedded with randomly oriented macromolecules is an essential prerequisite for cryogenic sample electron microscopy. Most commonly, this is achieved using the ...The formation of a vitrified thin film embedded with randomly oriented macromolecules is an essential prerequisite for cryogenic sample electron microscopy. Most commonly, this is achieved using the plunge-freeze method first described nearly 40 years ago. Although this is a robust method, the behaviour of different macromolecules shows great variation upon freezing and often needs to be optimized to obtain an isotropic, high-resolution reconstruction. For a macromolecule in such a film, the probability of encountering the air-water interface in the time between blotting and freezing and adopting preferred orientations is very high. 3D reconstruction using preferentially oriented particles often leads to anisotropic and uninterpretable maps. Currently, there are no general solutions to this prevalent issue, but several approaches largely focusing on sample preparation with the use of additives and novel grid modifications have been attempted. In this study, the effect of physical and chemical factors on the orientations of macromolecules was investigated through an analysis of selected well studied macromolecules, and important parameters that determine the behaviour of proteins on cryo-EM grids were revealed. These insights highlight the nature of the interactions that cause preferred orientations and can be utilized to systematically address orientation bias for any given macromolecule and to provide a framework to design small-molecule additives to enhance sample stability and behaviour.
History
DepositionOct 23, 2023Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Jul 17, 2024Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: C-reactive protein(1-205)
B: C-reactive protein(1-205)
C: C-reactive protein(1-205)
D: C-reactive protein(1-205)
E: C-reactive protein(1-205)
hetero molecules


Theoretical massNumber of molelcules
Total (without water)125,70815
Polymers125,3075
Non-polymers40110
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein
C-reactive protein(1-205)


Mass: 25061.477 Da / Num. of mol.: 5 / Source method: isolated from a natural source / Source: (natural) Homo sapiens (human) / 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
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 purified from human serum with calcium
Type: COMPLEX / Entity ID: #1 / Source: NATURAL
Molecular weightValue: 0.125 MDa / Experimental value: NO
Source (natural)Organism: Homo sapiens (human)
Buffer solutionpH: 8
Buffer component
IDConc.NameBuffer-ID
120 mMTris1
2280 mMNaCl1
35 mMCaCl21
40.002 %CTAB1
SpecimenConc.: 2.4 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R0.6/1
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 293 K / Details: Blot force, 0

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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: 75000 X / Calibrated magnification: 130841 X / Nominal defocus max: 3000 nm / Nominal defocus min: 1800 nm / Cs: 2.7 mm / C2 aperture diameter: 50 µm / Alignment procedure: BASIC
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 60 sec. / Electron dose: 25 e/Å2 / Detector mode: COUNTING / Film or detector model: FEI FALCON III (4k x 4k) / Num. of grids imaged: 1
Details: Images were collected in movie mode (@40 fps) and total of 25 frames were saved.
Image scansSampling size: 14 µm / Width: 4096 / Height: 4096

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Processing

EM software
IDNameVersionCategory
1RELION3.1particle selection
2EPUimage acquisition
4Gctf1.06CTF correction
5RELION3.1CTF correction
8UCSF Chimera1.15model fitting
9Coot0.9.5model fitting
14RELION3.13D reconstruction
21PHENIX1.15model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C5 (5 fold cyclic)
3D reconstructionResolution: 3.3 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 36353 / Algorithm: BACK PROJECTION / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingB value: 70.67 / Protocol: OTHER / Space: REAL
Atomic model buildingPDB-ID: 1b09

1b09


Accession code: 1b09 / Source name: PDB / Type: experimental model
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0078390
ELECTRON MICROSCOPYf_angle_d0.53411405
ELECTRON MICROSCOPYf_dihedral_angle_d5.4734880
ELECTRON MICROSCOPYf_chiral_restr0.0471235
ELECTRON MICROSCOPYf_plane_restr0.0041450

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