The Pithoviridae giant virus family exhibits the largest viral particle known so far, a prolate spheroid up to 2.5 μm in length and 0.9 μm in diameter. These particles show significant variations in size. Little is known about the structure of the intact virion due to technical limitations with conventional electron cryo-microscopy (cryo-EM) when imaging thick specimens. Here we present the intact structure of the giant Pithovirus sibericum particle at near native conditions using high-voltage electron cryo-tomography (cryo-ET) and energy-filtered cryo-EM. We detected a previously undescribed low-density outer layer covering the tegument and a periodical structuring of the fibres in the striated apical cork. Energy-filtered Zernike phase-contrast cryo-EM images show distinct substructures inside the particles, implicating an internal compartmentalisation. The density of the interior volume of Pithovirus particles is three quarters lower than that of the Mimivirus. However, it is remarkably high given that the 600 kbp Pithovirus genome is only half the size of the Mimivirus genome and is packaged in a volume up to 100 times larger. These observations suggest that the interior is densely packed with macromolecules in addition to the genomic nucleic acid.

Laboratory of Molecular Biophysics Department of Cell and Molecular Biology Uppsala University Husargatan 3 SE 75124 Uppsala Sweden

Laboratory of Molecular Biophysics Department of Cell and Molecular Biology Uppsala University Husargatan 3 SE 75124 Uppsala Sweden Institute of Physics AS CR v v i Na Slovance 2 18221 Prague 8 Czech Republic

National Institute for Physiological Sciences Okazaki Aichi 444 8585 Japan

Structural and Genomic Information Laboratory UMR 7256 Centre National de la Recherche Scientifique and Aix Marseille University Marseille 13288 France

Structural and Genomic Information Laboratory UMR 7256 Centre National de la Recherche Scientifique and Aix Marseille University Marseille 13288 France Assistance Publique des Hôpitaux de Marseille La Timone 13005 Marseille France

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$a Structural variability and complexity of the giant Pithovirus sibericum particle revealed by high-voltage electron cryo-tomography and energy-filtered electron cryo-microscopy / $c K. Okamoto, N. Miyazaki, C. Song, FRNC. Maia, HKN. Reddy, C. Abergel, JM. Claverie, J. Hajdu, M. Svenda, K. Murata,

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$a The Pithoviridae giant virus family exhibits the largest viral particle known so far, a prolate spheroid up to 2.5 μm in length and 0.9 μm in diameter. These particles show significant variations in size. Little is known about the structure of the intact virion due to technical limitations with conventional electron cryo-microscopy (cryo-EM) when imaging thick specimens. Here we present the intact structure of the giant Pithovirus sibericum particle at near native conditions using high-voltage electron cryo-tomography (cryo-ET) and energy-filtered cryo-EM. We detected a previously undescribed low-density outer layer covering the tegument and a periodical structuring of the fibres in the striated apical cork. Energy-filtered Zernike phase-contrast cryo-EM images show distinct substructures inside the particles, implicating an internal compartmentalisation. The density of the interior volume of Pithovirus particles is three quarters lower than that of the Mimivirus. However, it is remarkably high given that the 600 kbp Pithovirus genome is only half the size of the Mimivirus genome and is packaged in a volume up to 100 times larger. These observations suggest that the interior is densely packed with macromolecules in addition to the genomic nucleic acid.

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$a Maia, Filipe R N C $u Laboratory of Molecular Biophysics, Department of Cell and Molecular Biology, Uppsala University, Husargatan 3 (Box 596), SE-75124, Uppsala, Sweden.

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$a Reddy, Hemanth K N $u Laboratory of Molecular Biophysics, Department of Cell and Molecular Biology, Uppsala University, Husargatan 3 (Box 596), SE-75124, Uppsala, Sweden.

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$a Hajdu, Janos $u Laboratory of Molecular Biophysics, Department of Cell and Molecular Biology, Uppsala University, Husargatan 3 (Box 596), SE-75124, Uppsala, Sweden. Institute of Physics AS CR, v.v.i., Na Slovance 2, 18221, Prague 8, Czech Republic.

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$a Svenda, Martin $u Laboratory of Molecular Biophysics, Department of Cell and Molecular Biology, Uppsala University, Husargatan 3 (Box 596), SE-75124, Uppsala, Sweden.

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$a Murata, Kazuyoshi $u National Institute for Physiological Sciences (NIPS), Okazaki, Aichi, 444-8585, Japan. kazum@nips.ac.jp.

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