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- EMDB-1569: Keyhole limpet hemocyanin (KLH): 9A cryoEM structure and molecula... -

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Basic information

Entry
Database: EMDB / ID: EMD-1569
TitleKeyhole limpet hemocyanin (KLH): 9A cryoEM structure and molecular model of the didecamer reveal the interfaces and intricate topology of the 160 functional units
Map dataKeyhole Limpet Hemocyanin Isoform 1
Sample
  • Sample: Keyhole Limpet Hemocyanin Isoform 1 (KLH1)
  • Protein or peptide: Keyhole Limpet hemocyanin Isoform 1
Keywordskeyhole limpet hemocyanin / KLH / Gastropoda / Megathura crenulata / oxygen carrier
Function / homology
Function and homology information


oxidoreductase activity / extracellular region
Similarity search - Function
Haemocyanin beta-sandwich domain / Haemocyanin C-terminal domain superfamily / Haemocyanin beta-sandwich / Tyrosinase CuA-binding region signature. / : / Common central domain of tyrosinase / Tyrosinase and hemocyanins CuB-binding region signature. / Tyrosinase copper-binding domain / Di-copper centre-containing domain superfamily
Similarity search - Domain/homology
Hemocyanin 1 / Hemocyanin 1
Similarity search - Component
Biological speciesMegathura crenulata (invertebrata)
Methodsingle particle reconstruction / cryo EM / negative staining / Resolution: 9.1 Å
AuthorsGatsogiannis C / Markl J
CitationJournal: 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.
History
DepositionOct 6, 2008-
Header (metadata) releaseOct 7, 2008-
Map releaseNov 12, 2010-
UpdateJan 8, 2014-
Current statusJan 8, 2014Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.006
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.006
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-4bed
  • Surface level: 0.006
  • Imaged by UCSF Chimera
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  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-4bed
  • Imaged by Jmol
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

1569.png

Downloads & links

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Map

FileDownload / File: emd_1569.map.gz / Format: CCP4 / Size: 339.5 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationKeyhole Limpet Hemocyanin Isoform 1
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.24 Å/pix.
x 450 pix.
= 558. Å		1.24 Å/pix.
x 450 pix.
= 558. Å		1.24 Å/pix.
x 450 pix.
= 558. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.24 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.006 / Movie #1: 0.006
Minimum - Maximum-0.022887 - 0.036899
Average (Standard dev.)0.000381167 (±0.00267905)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-224-225-225
Dimensions450450450
Spacing450450450
CellA=B=C: 558 Å
α=β=γ: 90 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.241.241.24
M x/y/z450450450
origin x/y/z0.0000.0000.000
length x/y/z558.000558.000558.000
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS-225-224-225
NC/NR/NS450450450
D min/max/mean-0.0230.0370.000

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Supplemental data

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Sample components

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Entire : Keyhole Limpet Hemocyanin Isoform 1 (KLH1)

EntireName: Keyhole Limpet Hemocyanin Isoform 1 (KLH1)
Components
  • Sample: Keyhole Limpet Hemocyanin Isoform 1 (KLH1)
  • Protein or peptide: Keyhole Limpet hemocyanin Isoform 1

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Supramolecule #1000: Keyhole Limpet Hemocyanin Isoform 1 (KLH1)

SupramoleculeName: Keyhole Limpet Hemocyanin Isoform 1 (KLH1) / type: sample / ID: 1000
Oligomeric state: KLH1 is a didecamer of a 400 kDa subunit. Each subunit is composed by 8 paralogous O2 binding functional units
Number unique components: 1
Molecular weightTheoretical: 8 MDa

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Macromolecule #1: Keyhole Limpet hemocyanin Isoform 1

MacromoleculeName: Keyhole Limpet hemocyanin Isoform 1 / type: protein_or_peptide / ID: 1 / Name.synonym: KLH1 / Recombinant expression: No
Source (natural)Organism: Megathura crenulata (invertebrata)
Molecular weightTheoretical: 8 MDa

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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

Concentration0.7 mg/mL
BufferpH: 7.4 / Details: 50 mM Tris-Hcl, 150 mM NaCl, 5mM CaCl, 5mM MgCl2
StainingType: NEGATIVE / Details: cryoEM, no stain
GridDetails: 400 mesh copper
VitrificationCryogen name: ETHANE / Chamber temperature: 86 K / Instrument: HOMEMADE PLUNGER
Details: Vitrification instrument: Home made. Vitrification carried out in 25 percent oxygen atmosphere
Method: Single side blotting and rapid plunging

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Electron microscopy

MicroscopeFEI TECNAI 20
TemperatureAverage: 86 K
DateJul 25, 2007
Image recordingCategory: FILM / Film or detector model: KODAK SO-163 FILM / Digitization - Scanner: PRIMESCAN / Digitization - Sampling interval: 1.24 µm / Number real images: 98 / Bits/pixel: 8
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 50000 / Illumination mode: OTHER / Imaging mode: BRIGHT FIELD / Cs: 2 mm / Nominal defocus max: 4.0 µm / Nominal defocus min: 0.8 µm / Nominal magnification: 50000
Sample stageSpecimen holder: Gatan single-tilt cryoholde / Specimen holder model: GATAN LIQUID NITROGEN

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Image processing

CTF correctionDetails: CTFFIND3 and TRANSFER, IMAGIC 5
Final reconstructionApplied symmetry - Point group: D5 (2x5 fold dihedral) / Resolution.type: BY AUTHOR / Resolution: 9.1 Å / Resolution method: OTHER / Software - Name: IMAGIC-5 / Number images used: 4762

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