|Entry||Database: EMDB / ID: EMD-1569|
|Title||Keyhole limpet hemocyanin (KLH): 9A cryoEM structure and molecular model of the didecamer reveal the interfaces and intricate topology of the 160 functional units|
|Sample||Keyhole Limpet Hemocyanin Isoform 1 (KLH1):|
Keyhole Limpet hemocyanin Isoform 1
|Keywords||keyhole limpet hemocyanin / KLH / Gastropoda / Megathura crenulata / oxygen carrier|
|Function / homology|
Function and homology information
oxidoreductase activity / metal ion binding
Tyrosinase copper-binding domain / Uncharacterised domain, di-copper centre / Haemocyanin beta-sandwich domain / Haemocyanin C-terminal domain superfamily / Common central domain of tyrosinase / Haemocyanin beta-sandwich / Tyrosinase CuA-binding region signature. / Tyrosinase and hemocyanins CuB-binding region signature.
|Biological species||Megathura crenulata (invertebrata)|
|Method||single particle reconstruction / cryo EM / Resolution: 9.1 Å|
|Authors||Gatsogiannis C / Markl J|
|Citation||Journal: J. Mol. Biol. / Year: 2009|
Title: Keyhole limpet hemocyanin: 9-A CryoEM structure and molecular model of the KLH1 didecamer reveal the interfaces and intricate topology of the 160 functional units.
Authors: Christos Gatsogiannis / Jürgen Markl /
Abstract: Hemocyanins are blue copper-containing respiratory proteins in the hemolymph of many arthropods and molluscs. Molluscan hemocyanins are decamers, didecamers, or multidecamers of a 340- to 400-kDa ...Hemocyanins are blue copper-containing respiratory proteins in the hemolymph of many arthropods and molluscs. Molluscan hemocyanins are decamers, didecamers, or multidecamers of a 340- to 400-kDa polypeptide subunit containing seven or eight globular functional units (FUs; FU-a to FU-h), each with an oxygen-binding site. The decamers are short 35-nm hollow cylinders, with their lumen narrowed by a collar complex. Our recently published 9-A cryo-electron microscopy/crystal structure hybrid model of a 3.4-MDa cephalopod hemocyanin decamer [Nautilus pompilius hemocyanin (NpH)] revealed the pathway of the seven-FU subunit (340 kDa), 15 types of inter-FU interface, and an asymmetric collar consisting of five "arcs" (FU-g pairs). We now present a comparable hybrid model of an 8-MDa gastropod hemocyanin didecamer assembled from two asymmetric decamers [isoform keyhole limpet hemocyanin (KLH) 1 of the established immunogen KLH]. Compared to NpH, the KLH1 subunit (400 kDa) is C-terminally elongated by FU-h, which is further extended by a unique tail domain. We have found that the wall-and-arc structure of the KLH1 decamer is very similar to that of NpH. We have traced the subunit pathway and how it continues from KLH1-g to KLH1-h to form an annulus of five "slabs" (FU-h pairs) at one cylinder edge. The 15 types of inter-FU interface detected in NpH are also present in KLH1. Moreover, we have identified one arc/slab interface, two slab/slab interfaces, five slab/wall interfaces, and four decamer/decamer interfaces. The 27 interfaces are described on the basis of two subunit conformers, yielding an asymmetric homodimer. Six protrusions from the cryo-electron microscopy structure per subunit are associated with putative attachment sites for N-linked glycans, indicating a total of 120 sugar trees in KLH1. Also, putative binding sites for divalent cations have been detected. In conclusion, the present 9-A data on KLH1 confirm and substantially broaden our recent analysis of the smaller cephalopod hemocyanin and essentially solve the gastropod hemocyanin structure.
|Validation Report||PDB-ID: 4bed|
SummaryFull reportAbout validation report
|Structure viewer||EM map: |
Downloads & links
|File||Download / File: emd_1569.map.gz / Format: CCP4 / Size: 339.5 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)|
|Projections & slices|
Images are generated by Spider.
|Voxel size||X=Y=Z: 1.24 Å|
|Symmetry||Space group: 1|
CCP4 map header:
-Entire Keyhole Limpet Hemocyanin Isoform 1 (KLH1)
|Entire||Name: Keyhole Limpet Hemocyanin Isoform 1 (KLH1) / Number of components: 1 |
Oligomeric State: KLH1 is a didecamer of a 400 kDa subunit. Each subunit is composed by 8 paralogous O2 binding functional units
|Mass||Theoretical: 8 MDa|
-Component #1: protein, Keyhole Limpet hemocyanin Isoform 1
|Protein||Name: Keyhole Limpet hemocyanin Isoform 1 / a.k.a: KLH1 / Recombinant expression: No|
|Mass||Theoretical: 8 MDa|
|Source||Species: Megathura crenulata (invertebrata)|
|Specimen||Specimen state: Particle / Method: cryo EM|
|Sample solution||Specimen conc.: 0.7 mg/mL|
Buffer solution: 50 mM Tris-Hcl, 150 mM NaCl, 5mM CaCl, 5mM MgCl2
|Support film||400 mesh copper|
|Staining||cryoEM, no stain|
|Vitrification||Instrument: HOMEMADE PLUNGER / Cryogen name: ETHANE / Temperature: 86 K / Method: Single side blotting and rapid plunging|
Details: Vitrification instrument: Home made. Vitrification carried out in 25 percent oxygen atmosphere
-Electron microscopy imaging
|Imaging||Microscope: FEI TECNAI 20 / Date: Jul 25, 2007|
|Electron gun||Electron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: OTHER|
|Lens||Magnification: 50000 X (nominal), 50000 X (calibrated) / Cs: 2 mm / Imaging mode: BRIGHT FIELD / Defocus: 800 - 4000 nm|
|Specimen Holder||Holder: Gatan single-tilt cryoholde / Model: GATAN LIQUID NITROGEN / Temperature: 86|
|Camera||Detector: KODAK SO-163 FILM|
|Image acquisition||Number of digital images: 98 / Scanner: PRIMESCAN / Sampling size: 1.24 µm / Bit depth: 8|
|Processing||Method: single particle reconstruction / Number of projections: 4762 / Applied symmetry: D5 (2x5 fold dihedral)|
|3D reconstruction||Software: IMAGIC-5 / CTF correction: CTFFIND3 and TRANSFER, IMAGIC 5 / Resolution: 9.1 Å / Resolution method: FSC 1/2 BIT|
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