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- PDB-4bed: Keyhole limpet hemocyanin (KLH): 9A cryoEM structure and molecula... -

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Database: PDB / ID: 4bed
TitleKeyhole limpet hemocyanin (KLH): 9A cryoEM structure and molecular model of the KLH1 didecamer reveal the interfaces and intricate topology of the 160 functional units
Components(HEMOCYANIN KLH1) x 2
KeywordsOXYGEN TRANSPORT / KEYHOLE LIMPET HEMOCYANIN / KLH / GASTROPODA / OXYGEN CARRIER
Function / homology
Function and homology information


oxygen carrier activity / oxidoreductase activity / extracellular region / metal ion binding
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
CU2-O2 CLUSTER / Hemocyanin 1 / Hemocyanin 1
Similarity search - Component
Biological speciesMEGATHURA CRENULATA (invertebrata)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 9 Å
AuthorsGatsogiannis, 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.
#1: Journal: Biochim Biophys Acta / Year: 2013
Title: Evolution of molluscan hemocyanin structures.
Authors: Jürgen Markl /
Abstract: Hemocyanin transports oxygen in the hemolymph of many molluscs and arthropods and is therefore a central physiological factor in these animals. Molluscan hemocyanin molecules are oligomers composed ...Hemocyanin transports oxygen in the hemolymph of many molluscs and arthropods and is therefore a central physiological factor in these animals. Molluscan hemocyanin molecules are oligomers composed of many protein subunits that in turn encompass subsets of distinct functional units. The structure and evolution of molluscan hemocyanin have been studied for decades, but it required the recent progress in DNA sequencing, X-ray crystallography and 3D electron microscopy to produce a detailed view of their structure and evolution. The basic quaternary structure is a cylindrical decamer 35nm in diameter, consisting of wall and collar (typically at one end of the cylinder). Depending on the animal species, decamers, didecamers and multidecamers occur in the hemolymph. Whereas the wall architecture of the decamer seems to be invariant, four different types of collar have been identified in different molluscan taxa. Correspondingly, there exist four subunit types that differ in their collar functional units and range from 350 to 550kDa. Thus, molluscan hemocyanin subunits are among the largest polypeptides in nature. In this report, recent 3D reconstructions are used to explain and visualize the different functional units, subunits and quaternary structures of molluscan hemocyanins. Moreover, on the basis of DNA analyses and structural considerations, their possible evolution is traced. This article is part of a Special Issue entitled: Oxygen Binding and Sensing Proteins.
History
DepositionMar 8, 2013Deposition site: PDBE / Processing site: PDBE
Revision 1.0Dec 25, 2013Provider: repository / Type: Initial release
Revision 2.0Aug 30, 2017Group: Advisory / Atomic model / Category: atom_site / pdbx_unobs_or_zero_occ_atoms
Item: _atom_site.Cartn_x / _atom_site.Cartn_y ..._atom_site.Cartn_x / _atom_site.Cartn_y / _atom_site.Cartn_z / _atom_site.auth_atom_id / _atom_site.label_atom_id / _atom_site.type_symbol
Revision 2.1Oct 3, 2018Group: Data collection / Category: em_software / Item: _em_software.image_processing_id

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Assembly

Deposited unit
A: HEMOCYANIN KLH1
B: HEMOCYANIN KLH1
C: HEMOCYANIN KLH1
D: HEMOCYANIN KLH1
hetero molecules


Theoretical massNumber of molelcules
Total (without water)784,80120
Polymers782,2564
Non-polymers2,54516
Water0
1
A: HEMOCYANIN KLH1
B: HEMOCYANIN KLH1
C: HEMOCYANIN KLH1
D: HEMOCYANIN KLH1
hetero molecules
x 10


Theoretical massNumber of molelcules
Total (without water)7,848,013200
Polymers7,822,55840
Non-polymers25,455160
Water0
TypeNameSymmetry operationNumber
point symmetry operation10
2


  • Idetical with deposited unit in distinct coordinate
  • point asymmetric unit
TypeNameSymmetry operationNumber
point symmetry operation1
3


  • Idetical with deposited unit in distinct coordinate
  • point asymmetric unit, std point frame
TypeNameSymmetry operationNumber
transform to point frame1
SymmetryPoint symmetry: (Schoenflies symbol: D (2xn fold dihedral))

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Components

#1: Protein HEMOCYANIN KLH1 / KEYHOLE LIMPET HEMOCYANIN ISOFORM 1 FRAGMENT ENCOMPASSING THE FUNCTIONAL UNITS A TO D


Mass: 191623.828 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Source: (natural) MEGATHURA CRENULATA (invertebrata) / References: UniProt: Q53IP9, UniProt: Q10583*PLUS
#2: Protein HEMOCYANIN KLH1 / KEYHOLE LIMPET HEMOCYANIN ISOFORM 1 FRAGMENT ENCOMPASSING THE FUNCTIONAL UNITS E TO H


Mass: 199504.094 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Source: (natural) MEGATHURA CRENULATA (invertebrata) / References: UniProt: Q53IP9, UniProt: Q10583*PLUS
#3: Chemical
ChemComp-CUO / CU2-O2 CLUSTER / CU-O2-CU LINKAGE


Mass: 159.091 Da / Num. of mol.: 16 / Source method: obtained synthetically / Formula: Cu2O2
Sequence detailsTHIS PDB ENTRY IS BASED ON AN UPDATED SEQUENCE

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: KEYHOLE LIMPET HEMOCYANIN ISOFORM 1 (KLH1) / Type: COMPLEX
Buffer solutionName: 50 MM TRIS-HCL, 150 MM NACL, 5MM CACL, 5MM MGCL2 / pH: 7.4 / Details: 50 MM TRIS-HCL, 150 MM NACL, 5MM CACL, 5MM MGCL2
SpecimenConc.: 0.7 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: HOLEY CARBON
VitrificationInstrument: HOMEMADE PLUNGER / Cryogen name: ETHANE
Details: VITRIFICATION INSTRUMENT - HOME MADE. VITRIFICATION CARRIED OUT IN 25 PERCENT OXYGEN ATMOSPHERE. SINGLE- SIDED BLOTTING

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Electron microscopy imaging

Experimental equipment
Model: Tecnai F20 / Image courtesy: FEI Company
MicroscopyModel: FEI TECNAI F20 / Date: Jul 25, 2007
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 50000 X / Calibrated magnification: 50000 X / Nominal defocus max: 4000 nm / Nominal defocus min: 800 nm / Cs: 2 mm
Specimen holderTemperature: 68 K
Image recordingFilm or detector model: KODAK SO-163 FILM
Image scansNum. digital images: 98

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Processing

EM softwareName: IMAGIC / Version: 5 / Category: 3D reconstruction
CTF correctionDetails: PER MICROGRAPH
SymmetryPoint symmetry: D5 (2x5 fold dihedral)
3D reconstructionResolution: 9 Å / Num. of particles: 4762 / Nominal pixel size: 1 Å / Actual pixel size: 1 Å
Details: KLH1 IS A CYLINDRICAL 8 MDA PROTEIN WITH D5 SYMMETRY AND COMPOSED OF 20 IDENTICAL 400 KDA POLYPEPTIDE SUBUNITS. THE ASYMMETRIC UNIT IS A SUBUNIT DIMER (800 KDA). THE TWO CONSTITUENT ...Details: KLH1 IS A CYLINDRICAL 8 MDA PROTEIN WITH D5 SYMMETRY AND COMPOSED OF 20 IDENTICAL 400 KDA POLYPEPTIDE SUBUNITS. THE ASYMMETRIC UNIT IS A SUBUNIT DIMER (800 KDA). THE TWO CONSTITUENT POLYPEPTIDES ARE REPRESENTED HERE BY FOUR CHAINS (A, B, C, D). THIS ALLOWS A QUICK VISUALIZATION OF TWO ALTERNATIVE SUBUNIT PATHWAYS THAT HAVE BEEN PROPOSED: A-B + C-D (THIS STUDY) AND A-D + C-B (PDB-ID 3J32). THE 400 KDA POLYPEPTIDE IS FOLDED INTO EIGHT FUNCTIONAL UNITS, TERMED KLH1-A TO KLH1-H (1-421, 422-835, 836-1255, 1256-1664, 1665-2081, 2082-2488, 2489-2905, 2906-3398). THEY ARE CONCATENATED VIA LONG LINKERS. FOR THIS MODEL, THE STRUCTURE OF KLH1-H WAS AVAILABLE (PDB-ID 3QJO). HOMOLOGY MODELING OF THE OTHER FUNCTIONAL UNITS WAS DONE USING PDB-ID 1JS8 AS TEMPLATE. EACH FUNCTIONAL UNIT IS ABLE TO BIND A DIOXYGEN MOLECULE BETWEEN TWO COPPER IONS COORDINATED BY SIX HISTIDINES: 42, 60, 69, 179, 183, 210; 462, 482, 491, 602, 606, 633; 876, 896, 905, 1015, 1019, 1046; 1293, 1311, 1320, 1424, 1428, 1455; 1705, 1725, 1734, 1847, 1851, 1878; 2122, 2141, 2150, 2260, 2264, 2291; 2542, 2561, 2570, 2670, 2674, 2701; 2946, 2965, 2974, 3075, 3079, 3106. SUBMISSION BASED ON EXPERIMENTAL DATA FROM EMDB EMD-1569. (DEPOSITION ID: 6428).
Symmetry type: POINT
Atomic model buildingProtocol: RIGID BODY FIT / Details: METHOD--RIGID-BODY FITTING AND LOOP REFINEMENT
RefinementHighest resolution: 9 Å
Refinement stepCycle: LAST / Highest resolution: 9 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms55228 0 64 0 55292

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