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Yorodumi- EMDB-1598: Cryo-negative staining electron microscopy reveals the structure ... -
+Open data
-Basic information
Entry | Database: EMDB / ID: EMD-1598 | |||||||||
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Title | Cryo-negative staining electron microscopy reveals the structure and oligomeric state of Limulus SAP-like pentraxin | |||||||||
Map data | This is the volume of Lumulus Serum Amyloid P-Component | |||||||||
Sample |
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Keywords | Limulus / pentraxin / serum amyloid p component / phosphoethanolamine binding / cryoEM | |||||||||
Function / homology | : / Pentaxin 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 species | Limulus polyphemus (Atlantic horseshoe crab) | |||||||||
Method | single particle reconstruction / cryo EM / negative staining / Resolution: 14.0 Å | |||||||||
Authors | Shrive AK / Burns I / Chou HT / Stahlberg H / Armstrong PB / Greenhough TJ | |||||||||
Citation | Journal: 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 |
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-Structure visualization
Movie |
Movie viewer |
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Structure viewer | EM map: SurfViewMolmilJmol/JSmol |
Supplemental images |
-Downloads & links
-EMDB archive
Map data | emd_1598.map.gz | 12 MB | EMDB map data format | |
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Header (meta data) | emd-1598-v30.xml emd-1598.xml | 8.9 KB 8.9 KB | Display Display | EMDB header |
Images | 1598.gif | 57.4 KB | ||
Archive directory | http://ftp.pdbj.org/pub/emdb/structures/EMD-1598 ftp://ftp.pdbj.org/pub/emdb/structures/EMD-1598 | HTTPS FTP |
-Validation report
Summary document | emd_1598_validation.pdf.gz | 206.1 KB | Display | EMDB validaton report |
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Full document | emd_1598_full_validation.pdf.gz | 205.2 KB | Display | |
Data in XML | emd_1598_validation.xml.gz | 5.3 KB | Display | |
Arichive directory | https://ftp.pdbj.org/pub/emdb/validation_reports/EMD-1598 ftp://ftp.pdbj.org/pub/emdb/validation_reports/EMD-1598 | HTTPS FTP |
-Related structure data
-Links
EMDB pages | EMDB (EBI/PDBe) / EMDataResource |
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-Map
File | Download / File: emd_1598.map.gz / Format: CCP4 / Size: 15.3 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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Annotation | This is the volume of Lumulus Serum Amyloid P-Component | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Projections & slices | Image control
Images are generated by Spider. | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Voxel size | X=Y=Z: 1.25 Å | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Details | EMDB XML:
CCP4 map header:
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-Supplemental data
-Sample components
-Entire : Limulus SAP-like pentraxin
Entire | Name: Limulus SAP-like pentraxin |
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Components |
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-Supramolecule #1000: Limulus SAP-like pentraxin
Supramolecule | Name: 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 |
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Molecular weight | Theoretical: 300 KDa |
-Macromolecule #1: SAP-like pentraxin
Macromolecule | Name: 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 |
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Source (natural) | Organism: Limulus polyphemus (Atlantic horseshoe crab) / synonym: Atlantic horseshoe crab / Tissue: hemolymph |
Molecular weight | Theoretical: 300 KDa |
-Experimental details
-Structure determination
Method | negative staining, cryo EM |
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Processing | single particle reconstruction |
Aggregation state | particle |
-Sample preparation
Concentration | 0.1 mg/mL |
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Buffer | pH: 7 / Details: 0.1M citrate |
Staining | Type: NEGATIVE Details: 3ul protein sample laied on the grid for 30 seconds and the grid floated on saturated ammonium molybdate for 1 minute |
Grid | Details: 400 mesh quantifoil grid |
Vitrification | Cryogen name: ETHANE / Chamber temperature: 100 K / Instrument: OTHER Method: The grid was blotted for 6 seconds and air-dried for 3 seconds before plunging |
-Electron microscopy
Microscope | JEOL 2100F |
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Temperature | Average: 95 K |
Details | Imaged in JEOL minimum dose system |
Image recording | Category: FILM / Film or detector model: KODAK SO-163 FILM / Digitization - Scanner: PRIMESCAN / Digitization - Sampling interval: 10 µm / Number real images: 18 / Bits/pixel: 16 |
Electron beam | Acceleration voltage: 120 kV / Electron source: LAB6 |
Electron optics | Calibrated magnification: 80000 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal magnification: 80000 |
Sample stage | Specimen holder: side entry Gatan 626 / Specimen holder model: GATAN LIQUID NITROGEN |
-Image processing
Details | The particles were selected automatically by the program BOXER and screened manually |
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Final reconstruction | Applied 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 |