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- EMDB-1598: Cryo-negative staining electron microscopy reveals the structure ... -

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Basic information

Entry
Database: EMDB / ID: EMD-1598
TitleCryo-negative staining electron microscopy reveals the structure and oligomeric state of Limulus SAP-like pentraxin
Map dataThis is the volume of Lumulus Serum Amyloid P-Component
Sample
  • Sample: Limulus SAP-like pentraxin
  • Protein or peptide: SAP-like pentraxin
KeywordsLimulus / pentraxin / serum amyloid p component / phosphoethanolamine binding / cryoEM
Function / homologyPentaxin family / Pentraxin / C-reactive protein / pentaxin family / Pentraxin-related / Pentraxin (PTX) domain profile. / Concanavalin A-like lectin/glucanase domain superfamily / extracellular region / metal ion binding / Pentraxin family member
Function and homology information
Biological speciesLimulus polyphemus (Atlantic horseshoe crab)
Methodsingle particle reconstruction / cryo EM / negative staining / Resolution: 14.0 Å
AuthorsShrive AK / Burns I / Chou HT / Stahlberg H / Armstrong PB / Greenhough TJ
CitationJournal: J Mol Biol / Year: 2009
Title: Crystal structures of Limulus SAP-like pentraxin reveal two molecular aggregations.
Authors: Annette K Shrive / Ian Burns / Hui-Ting Chou / Henning Stahlberg / Peter B Armstrong / Trevor J Greenhough /
Abstract: The serum-amyloid-P-component-like pentraxin from Limulus polyphemus, a recently discovered pentraxin species and important effector protein of the hemolymph immune system, displays two distinct ...The serum-amyloid-P-component-like pentraxin from Limulus polyphemus, a recently discovered pentraxin species and important effector protein of the hemolymph immune system, displays two distinct doubly stacked cyclic molecular aggregations, heptameric and octameric. The refined three-dimensional structures determined by X-ray crystallography, both based on the same cDNA sequence, show that each aggregate is constructed from a similar dimer of protomers, which is repeated to make up the ring structure. The native octameric form has been refined at a resolution of 3 A, the native heptameric form at 2.3 A, and the phosphoethanolamine (PE)-bound octameric form at 2.7 A. The existence of the hitherto undescribed heptameric form was confirmed by single-particle analysis using cryo-electron microscopy. In the native structures, the calcium-binding site is similar to that in human pentraxins, with two calcium ions bound in each subunit. Upon binding PE, however, each subunit binds a third calcium ion, with all three calcium ions contributing to the binding and orientation of the bound phosphate group within the ligand-binding pocket. While the phosphate is well-defined in the electron density, the ethanolamine group is poorly defined, suggesting structural and binding variabilities of this group. Although sequence homology with human serum amyloid P component is relatively low, structural homology is high, with very similar overall folds and a common affinity for PE. This is due, in part, to a "topological" equivalence of side-chain position. Identical side chains that are important in both function and fold, from different regions of the sequence in human and Limulus structures, occupy similar space within the overall subunit fold. Sequence and structure alignment, based on the refined three-dimensional structures presented here and the known horseshoe crab pentraxin sequences, suggest that adaptation and refinement of C-reactive-protein-mediated immune responses in these ancient creatures lacking antibody-based immunity are based on adaptation by gene duplication.
History
DepositionFeb 1, 2009-
Header (metadata) releaseFeb 6, 2009-
Map releaseApr 1, 2009-
UpdateOct 24, 2012-
Current statusOct 24, 2012Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.596563476
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 0.596563476
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

Downloads & links

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Map

FileDownload / File: emd_1598.map.gz / Format: CCP4 / Size: 15.3 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationThis is the volume of Lumulus Serum Amyloid P-Component
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.25 Å/pix.
x 160 pix.
= 200. Å
1.25 Å/pix.
x 160 pix.
= 200. Å
1.25 Å/pix.
x 160 pix.
= 200. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.25 Å
Density
Contour LevelBy AUTHOR: 0.45 / Movie #1: 0.5965635
Minimum - Maximum0.105045 - 0.900693
Average (Standard dev.)0.180809 (±0.138733)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions160160160
Spacing160160160
CellA=B=C: 200 Å
α=β=γ: 90 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.251.251.25
M x/y/z160160160
origin x/y/z0.0000.0000.000
length x/y/z200.000200.000200.000
α/β/γ90.00090.00090.000
start NX/NY/NZ-17-17-200
NX/NY/NZ123123401
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS160160160
D min/max/mean0.1050.9010.181

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Supplemental data

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Sample components

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Entire : Limulus SAP-like pentraxin

EntireName: Limulus SAP-like pentraxin
Components
  • Sample: Limulus SAP-like pentraxin
  • Protein or peptide: SAP-like pentraxin

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Supramolecule #1000: Limulus SAP-like pentraxin

SupramoleculeName: Limulus SAP-like pentraxin / type: sample / ID: 1000
Oligomeric state: Limulus SAP-like pentraxin comprises 14 protomers packed as a doubly stacked ring
Number unique components: 1
Molecular weightTheoretical: 300 KDa

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Macromolecule #1: SAP-like pentraxin

MacromoleculeName: SAP-like pentraxin / type: protein_or_peptide / ID: 1 / Name.synonym: SAP-like pentraxin / Number of copies: 14 / Oligomeric state: 14-mer / Recombinant expression: No
Source (natural)Organism: Limulus polyphemus (Atlantic horseshoe crab) / synonym: Atlantic horseshoe crab / Tissue: hemolymph
Molecular weightTheoretical: 300 KDa

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Experimental details

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Structure determination

Methodnegative staining, cryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration0.1 mg/mL
BufferpH: 7 / Details: 0.1M citrate
StainingType: NEGATIVE
Details: 3ul protein sample laied on the grid for 30 seconds and the grid floated on saturated ammonium molybdate for 1 minute
GridDetails: 400 mesh quantifoil grid
VitrificationCryogen name: ETHANE / Chamber temperature: 100 K / Instrument: OTHER
Method: The grid was blotted for 6 seconds and air-dried for 3 seconds before plunging

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Electron microscopy

MicroscopeJEOL 2100F
Electron beamAcceleration voltage: 120 kV / Electron source: LAB6
Electron opticsCalibrated magnification: 80000 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 80000
Sample stageSpecimen holder: side entry Gatan 626 / Specimen holder model: GATAN LIQUID NITROGEN
TemperatureAverage: 95 K
DetailsImaged in JEOL minimum dose system
Image recordingCategory: FILM / Film or detector model: KODAK SO-163 FILM / Digitization - Scanner: PRIMESCAN / Digitization - Sampling interval: 10 µm / Number real images: 18 / Bits/pixel: 16

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Image processing

Final reconstructionApplied symmetry - Point group: D7 (2x7 fold dihedral) / Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 14.0 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: SPIDER / Details: D7 symmetry was imposed on the reconstruction / Number images used: 12243
DetailsThe particles were selected automatically by the program BOXER and screened manually

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