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Structural analysis of spider hemocyanin by cryogenic electro microscopy (“cryo-EM”)

Riciluca KCT1,2,3, Afanasyev P3, Cassago A4, van Heel M3, Silva Junior PI1,2, Portugal RV4,

1 Interunidades em Biotecnologia - ICB, Universidade de São Paulo, Brasil; 2 Laboratório Especial de Toxinologia Aplicada CeTICS, Instituto Butantan, Brasil; 3 NeCEN, Leiden University, The Netherlands; 4 LNNano, Laboratório Nacional de Nanotecnologia, CNPEM, Campinas, SP, Brasil

Introduction: Hemocyanins are large copper-proteins that transport O2 in the hemolymph of many arthropods. Hemocyanin fragments have been reported in crustaceans and it was haemocyanin speculated that these fragments are produced as antimicrobial agents. Objectives: We found a 10-aa peptide “rondonin”, in the plasma of the Acanthoscurria rondoniae spider, which was antifungal and non-hemolytic. In order to find the position of this peptide within the hemocyanin we are performing a three-dimensional structure analysis. Methods: A. rondoniae hemolymph was collected and hemocyanin was purified by ultracentrifugation. A three-dimensional structure analysis of the hemocyanin by single-particle cryo-EM are being held. A first dataset of 304 micrographs was acquired on a Jeol JEM-2100 (200kV) microscope with an F-416 CMOS camera (TVIPS; LNNano/Campinas) to obtain a first low-resolution 3D-reconstruction. All the data processing was performed using IMAGIC-4D software. The picked particles from the first dataset were analysed by multivariate statistical analysis. Class averages were used for angular reconstitution to obtain an initial 3D-reconstruction, followed by iterative multi-reference analysis. To achieve a higher resolution, further data collection was performed on a Titan Krios (300kV) instrument using a FEI falcon II camera (NeCEN) in “movie-mode”.   Results and Discussion: We acquired more than 2000 movies and we are currently processing this data in order to obtain a high-resolution structure in which we hope to identify the position of the  “rondonin” peptide. Rondonin is generated by proteolytic cleavage of hemocyanin as a first line of defense against infection by microorganisms. This represents new insight into the functioning of the Arachnida immune system. Preliminary 3D reconstructions of haemocyanin show a 4x6-meric structure. Our results help us understand why Arachnida survived for such a long time on an evolutionary scale.  

Supported by Capes, CNPq, FAPESP

2 - BIOCHEMISTRY 21th Annual Scientific Meeting of Instituto Butantan.