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- EMDB-1304: Limulus polyphemus hemocyanin: 10 A cryo-EM structure, sequence a... -

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Basic information

Entry
Database: EMDB / ID: EMD-1304
TitleLimulus polyphemus hemocyanin: 10 A cryo-EM structure, sequence analysis, molecular modelling and rigid-body fitting reveal the interfaces between the eight hexamers.
Map dataThis is a map of the 8x6mer hemocyanin from the arthropod Limulus polyphemus. Mass correlated threshold: 0.001.
Sample
  • Sample: 8x6mer arthropod hemocyanin
  • Protein or peptide: subunit type I
  • Protein or peptide: subunit type IIA
  • Protein or peptide: subunit type II
  • Protein or peptide: subunit type IIIA
  • Protein or peptide: subunit type IIIB
  • Protein or peptide: subunit type IV
  • Protein or peptide: subunit type V
  • Protein or peptide: subunit type VI
Biological speciesLimulus polyphemus (Atlantic horseshoe crab)
Methodsingle particle reconstruction / cryo EM / negative staining / Resolution: 9.6 Å
AuthorsMartin AG / Depoix F / Stohr M / Meissner U / Hagner-Holler S / Hammouti K / Burmester T / Heyd J / Wriggers W / Markl J
CitationJournal: J Mol Biol / Year: 2007
Title: Limulus polyphemus hemocyanin: 10 A cryo-EM structure, sequence analysis, molecular modelling and rigid-body fitting reveal the interfaces between the eight hexamers.
Authors: Andreas G Martin / Frank Depoix / Michael Stohr / Ulrich Meissner / Silke Hagner-Holler / Kada Hammouti / Thorsten Burmester / Jochen Heyd / Willy Wriggers / Jürgen Markl /
Abstract: The blue copper protein hemocyanin from the horseshoe crab Limulus polyphemus is among the largest respiratory proteins found in nature (3.5 MDa) and exhibits a highly cooperative oxygen binding. Its ...The blue copper protein hemocyanin from the horseshoe crab Limulus polyphemus is among the largest respiratory proteins found in nature (3.5 MDa) and exhibits a highly cooperative oxygen binding. Its 48 subunits are arranged as eight hexamers (1x6mers) that form the native 8x6mer in a nested hierarchy of 2x6mers and 4x6mers. This quaternary structure is established by eight subunit types (termed I, IIA, II, IIIA, IIIB, IV, V, and VI), of which only type II has been sequenced. Crystal structures of the 1x6mer are available, but for the 8x6mer only a 40 A 3D reconstruction exists. Consequently, the structural parameters of the 8x6mer are not firmly established, and the molecular interfaces between the eight hexamers are still to be defined. This, however, is crucial for understanding how allosteric transitions are mediated between the different levels of hierarchy. Here, we show the 10 A structure (FSC(1/2-bit) criterion) of the oxygenated 8x6mer from cryo-electron microscopy (cryo-EM) and single-particle analysis. Moreover, we show its molecular model as obtained by DNA sequencing of subunits II, IIIA, IV and VI, and molecular modelling and rigid-body fitting of all subunit types. Remarkably, the latter enabled us to improve the resolution of the cryo-EM structure from 11 A to the final 10 A. The 10 A structure allows firm assessment of various structural parameters of the 8x6mer, the 4x6mer and the 2x6mer, and reveals a total of 46 inter-hexamer bridges. These group as 11 types of interface: four at the 2x6mer level (II-II, II-IV, V-VI, IV-VI), three form the 4x6mer (V-V, V-VI, VI-IIIB/IV/V), and four are required to assemble the 8x6mer (IIIA-IIIA, IIIA-IIIB, II-IV, IV-IV). The molecular model shows the amino acid residues involved, and reveals that several of the interfaces are intriguingly histidine-rich and likely to transfer allosteric signals between the different levels of the nested hierarchy.
History
DepositionDec 5, 2006-
Header (metadata) releaseDec 5, 2006-
Map releaseJan 12, 2011-
UpdateMay 26, 2011-
Current statusMay 26, 2011Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.02
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 0.02
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

EMD-1304.png

Downloads & links

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Map

FileDownload / File: emd_1304.map.gz / Format: CCP4 / Size: 62.5 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationThis is a map of the 8x6mer hemocyanin from the arthropod Limulus polyphemus. Mass correlated threshold: 0.001.
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.8 Å/pix.
x 256 pix.
= 460.8 Å		1.8 Å/pix.
x 256 pix.
= 460.8 Å		1.8 Å/pix.
x 256 pix.
= 460.8 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.8 Å
Density

Histogram

Histogram (log scale)
Contour Level1: 0.029 / Movie #1: 0.02
Minimum - Maximum-0.24175 - 0.278623
Average (Standard dev.)0.000411826 (±0.0201117)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 460.8 Å
α=β=γ: 90 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.81.81.8
M x/y/z256256256
origin x/y/z0.0000.0000.000
length x/y/z460.800460.800460.800
α/β/γ90.00090.00090.000
start NX/NY/NZ-184-184-183
NX/NY/NZ368368368
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS256256256
D min/max/mean-0.2420.2790.000

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Supplemental data

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Sample components

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Entire : 8x6mer arthropod hemocyanin

EntireName: 8x6mer arthropod hemocyanin
Components
  • Sample: 8x6mer arthropod hemocyanin
  • Protein or peptide: subunit type I
  • Protein or peptide: subunit type IIA
  • Protein or peptide: subunit type II
  • Protein or peptide: subunit type IIIA
  • Protein or peptide: subunit type IIIB
  • Protein or peptide: subunit type IV
  • Protein or peptide: subunit type V
  • Protein or peptide: subunit type VI

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Supramolecule #1000: 8x6mer arthropod hemocyanin

SupramoleculeName: 8x6mer arthropod hemocyanin / type: sample / ID: 1000 / Oligomeric state: hetero-oligomeric 8x6mer / Number unique components: 8
Molecular weightTheoretical: 3.5 MDa / Method: sequence analysis

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Macromolecule #1: subunit type I

MacromoleculeName: subunit type I / type: protein_or_peptide / ID: 1 / Recombinant expression: No
Source (natural)Organism: Limulus polyphemus (Atlantic horseshoe crab)

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Macromolecule #2: subunit type IIA

MacromoleculeName: subunit type IIA / type: protein_or_peptide / ID: 2 / Recombinant expression: No
Source (natural)Organism: Limulus polyphemus (Atlantic horseshoe crab)

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Macromolecule #3: subunit type II

MacromoleculeName: subunit type II / type: protein_or_peptide / ID: 3 / Recombinant expression: No
Source (natural)Organism: Limulus polyphemus (Atlantic horseshoe crab)

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Macromolecule #4: subunit type IIIA

MacromoleculeName: subunit type IIIA / type: protein_or_peptide / ID: 4 / Recombinant expression: Yes
Source (natural)Organism: Limulus polyphemus (Atlantic horseshoe crab)

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Macromolecule #5: subunit type IIIB

MacromoleculeName: subunit type IIIB / type: protein_or_peptide / ID: 5 / Recombinant expression: No
Source (natural)Organism: Limulus polyphemus (Atlantic horseshoe crab)

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Macromolecule #6: subunit type IV

MacromoleculeName: subunit type IV / type: protein_or_peptide / ID: 6 / Recombinant expression: No
Source (natural)Organism: Limulus polyphemus (Atlantic horseshoe crab)

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Macromolecule #7: subunit type V

MacromoleculeName: subunit type V / type: protein_or_peptide / ID: 7 / Recombinant expression: No
Source (natural)Organism: Limulus polyphemus (Atlantic horseshoe crab)

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Macromolecule #8: subunit type VI

MacromoleculeName: subunit type VI / type: protein_or_peptide / ID: 8 / Recombinant expression: No
Source (natural)Organism: Limulus polyphemus (Atlantic horseshoe crab)

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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: 7.8 / Details: 100mM Tris-HCL,10mM MgCl2, 10mM CaCl2
StainingType: NEGATIVE / Details: cryo-EM, no stain
GridDetails: 400 mesh copper
VitrificationCryogen name: ETHANE / Chamber temperature: 86 K / Instrument: HOMEMADE PLUNGER
Details: Vitrification instrument: self constructed plunger. Vitrification carried out in 25% oxygen - 75% nitrogen atmosphere
Method: Single side blotting and rapid plunging

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Electron microscopy

MicroscopeFEI TECNAI F30
TemperatureAverage: 86 K
Image recordingCategory: FILM / Film or detector model: KODAK SO-163 FILM / Digitization - Scanner: PRIMESCAN / Digitization - Sampling interval: 1.8 µm / Number real images: 29 / Bits/pixel: 8
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 59000 / Illumination mode: OTHER / Imaging mode: BRIGHT FIELD / Cs: 1.2 mm / Nominal defocus max: 5.0 µm / Nominal defocus min: 1.0 µm / Nominal magnification: 59000
Sample stageSpecimen holder: Gatan single-tilt cryoholder / Specimen holder model: GATAN LIQUID NITROGEN
Experimental equipment
Model: Tecnai F30 / Image courtesy: FEI Company

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Image processing

DetailsThe particles were selected using the automatic selection program boxer.
CTF correctionDetails: CTFFIND3 and TRANSFER, IMAGIC5
Final reconstructionApplied symmetry - Point group: D2 (2x2 fold dihedral) / Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 9.6 Å / Resolution method: OTHER / Software - Name: IMAGIC-5 / Number images used: 12069
Final two d classificationNumber classes: 1140

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