# The giant mimivirus 1.2 Mb genome is elegantly organized into a 30 nm diameter helical protein shield

Alejandro Villalta, Alain Schmitt, Leandro F Estrozi, Emmanuelle Rj Quemin, Jean-Marie Alempic, Audrey Lartigue, Vojtěch Pražák, Lucid Belmudes, Daven Vasishtan, Agathe Mg Colmant, et al.

<figure><img src="image_1.png" /></figure>

## To cite this version:

Alejandro Villalta, Alain Schmitt, Leandro F Estrozi, Emmanuelle Rj Quemin, Jean-Marie Alempic, et al.. The giant mimivirus 1.2 Mb genome is elegantly organized into a 30 nm diameter helical protein shield. eLife, 2022, 11, 10.7554/eLife.77607. hal-03740274

HAL Id: hal-03740274

https://amu.hal.science/hal-03740274v1

Submitted on 29 Jul 2022

HAL is a multi-disciplinary open access archive for the deposit and dissemination of scientific research documents, whether they are published or not. The documents may come from teaching and research institutions in France or abroad, or from public or private research centers.

L'archive ouverte pluridisciplinaire HAL, est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

<figure><img src="image_2.png" /></figure>

Distributed under a Creative Commons CC BY 4.0 - Attribution - International License
1 **Title:** The giant mimivirus 1.2 Mb genome is elegantly organized into a 30 nm diameter

2 helical protein shield

3 **Authors:** Alejandro Villalta¹†, Alain Schmitt¹†, Leandro F. Estrozi²†, Emmanuelle R. J.

4 Quemin³#, Jean-Marie Alempic¹, Audrey Lartigue¹, Vojtěch Pražák³,⁴, Lucid Belmudes⁵, Da-

5 ven Vasishtan³,⁴, Agathe M. G. Colmant¹, Flora A. Honoré¹, Yohann Couté⁵, Kay Grüne-

6 wald³,⁴, Chantal Abergel¹*

7 **Affiliations:**

8 ¹Aix-Marseille University, Centre National de la Recherche Scientifique, Information Géno-

9 mique & Structurale, Unité Mixte de Recherche 7256 (Institut de Microbiologie de la Médi-

10 terranée, FR3479, IM2B), 13288 Marseille Cedex 9, France.

11 ²Univ. Grenoble Alpes, CNRS, CEA, Institut de Biologie Structurale (IBS), 38000 Grenoble,

12 France.

13 ³Centre for Structural Systems Biology, Leibniz Institute for Experimental Virology (HPI),

14 University of Hamburg, D-22607 Hamburg, Germany.

15 ⁴Division of Structural Biology, Wellcome Centre for Human Genetics, University of Oxford,

16 Oxford OX3 7BN, United Kingdom.

17 ⁵Univ. Grenoble Alpes, CEA, INSERM, IRIG, BGE, 38000 Grenoble, France

18 *Corresponding author. Email: Chantal.Abergel@igs.cnrs-mrs.fr

19 †These authors contributed equally to the work

20 # present address: Department of Virology, Institute for Integrative Biology of the Cell

21 (I2BC), Centre National de la Recherche Scientifique UMR9198, 1, avenue de la Ter-rasse,

22 91198 Gif-sur-Yvette Cedex, France.

23 **One-Sentence Summary:** Mimivirus genome organization in the icosahedral virion.

24 **Subject Area:** Virology, Cro-electron microscopy

25 **Keywords:** Mimivirus, giant virus, dsDNA, viral genome organization, genomic fiber, cryo-

26 EM, cryo-ET, helical reconstruction, proteomics.

27

28 **Abstract**

29 Mimivirus is the prototype of the *Mimiviridae* family of giant dsDNA viruses. Little is known

30 about the organization of the 1.2 Mb genome inside the membrane-limited nucleoid filling the

31 ~0.5 µm icosahedral capsids. Cryo-electron microscopy, cryo-electron tomography and

32 proteomics revealed that it is encased into a ~30 nm diameter helical protein shell surprisingly

33 composed of two GMC-type oxidoreductases, which also form the glycosylated fibrils

34 decorating the capsid. The genome is arranged in 5- or 6-start left-handed super-helices, with

35 each DNA-strand lining the central channel. This luminal channel of the nucleoprotein fiber is

36 wide enough to accommodate oxidative stress proteins and RNA polymerase subunits

37 identified by proteomics. Such elegant supramolecular organization would represent a

38 remarkable evolutionary strategy for packaging and protecting the genome, in a state ready

39 for immediate transcription upon unwinding in the host cytoplasm. The parsimonious use of

40 the same protein in two unrelated substructures of the virion is unexpected for a giant virus

41 with thousand genes at its disposal.

42
43 **Introduction**

44 *Acanthamoeba* infecting giant viruses were discovered with the isolation of mimivirus (1, 2).
45 Giant viruses now represent a highly diverse group of dsDNA viruses infecting unicellular
giant viruses now represent a highly diverse group of dsDNA viruses infecting unicellular
eukaryotes (3) which play important roles in the environment (4–6). They also challenge the
canonical definitions of viruses (7, 8) as they can encode central translation components (2, 9)
as well as a complete glycosylation machinery (10, 11) among other unique features.

49 Mimivirus has been the most extensively studied giant virus infecting *Acanthamoeba* (12).
50 The virions are 0.75 µm wide and consist of icosahedral capsids of 0.45 µm diameter
51 surrounded by a dense layer of radially arranged fibrils (2). Structural analyses of the virions
52 have provided some insights into the capsid structure (13–17) but given the size of the
53 icosahedral particles (and hence the sample thickness), accessing the internal organization of
54 the core of the virions remains challenging. Consequently, little is known about the packaging
55 of the 1.2 Mb dsDNA genome (18). Inside the capsids, a lipid membrane delineates an
56 internal compartment (~340 nm in diameter, Fig 1A, Figure 1-figure supplement 1), referred
to as the nucleoid, which contains the viral genome, together with all proteins necessary to
58 initiate the replicative cycle within the host cytoplasm (17, 19–21). Acanthamoeba cells
59 engulf mimivirus particles, fooled by their bacteria-like size and the heavily glycosylated
60 decorating fibrils (1, 2, 11). Once in the phagosome, the Stargate portal located at one specific
61 vertex of the icosahedron opens up (22), enabling the viral membrane to fuse with that of the
62 host vacuole to deliver the nucleoid into the host cytoplasm (17, 20). EM studies have shown
63 that next the nucleoid gradually loses its electron dense appearance, transcription begins and
64 the early viral factory is formed (21, 23, 24). Previous Atomic Force Microscopy (AFM)
65 studies of the mimivirus infectious cycle suggested that the DNA forms a highly condensed
66 nucleoprotein complex enclosed within the nucleoid (13). Here we show that opening of the
67 large icosahedral capsid *in vitro* led to the release of rod-shaped structures of about 30 nm
68 width. These structures were further purified and the various conformations characterized
69 using cryo-electron microscopy (cryo-EM), tomography and MS-based proteomics.

70

71 **Results**

72 **Capsid opening induces the release of a ~ 30 nm-wide rod-shaped structure that contains**
73 the dsDNA genome

74 We developed an *in vitro* protocol for particle opening that led to the release of ~ 30 nm-wide
75 rod-shaped fibers of several microns in length (Fig. 1, Fig. 1-figure supplement 1). We coined
76 this structure the mimivirus genomic fiber. Complete expulsion of the opened nucleoid
77 content produced bundled fibers resembling a “ball of yarn” (13) (Fig. 1C,). The capsid
78 opening procedure involves limited proteolysis and avoids harsh conditions, as we found that
79 the structure becomes completely denatured by heat (95°C) and is also sensitive to acidic
80 treatment, thus preventing its detection in such conditions (17). Various conformations of the
81 genomic fiber were observed, sometimes even on the same fiber (Fig. 1D), ranging from the
82 most compact rod-shaped structures (Fig. 1D (left), E-F (top)) to more relaxed structures
83 where DNA strands begin to dissociate (Fig. 1D (right), E-F (bottom)). After optimizing the
84 *in vitro* extraction on different strains of group-A mimiviruses, we focused on an isolate from
85 La Réunion Island (mimivirus reunion), as more capsids were opened by our protocol, leading
86 to higher yields of genomic fibers that were subsequently purified on sucrose gradient. All
87 opened capsids released genomic fibers (Fig. 1C, Fig. 1-figure supplement 1).

88 The first confirmation of the presence of DNA in the genomic fiber was obtained by agarose
89 gel electrophoresis (Fig. 1-figure supplement 3). Electron cryo microscopy (cryo-EM)
bubblegram analysis (25, 26) gave a further indication that the nucleic acid is located in the fiber lumen. Alike other nucleoprotein complexes, fibers are expected to be more susceptible to radiation damage then pure proteinaceous structures. Surprisingly, the specimen could sustain higher electron irradiation before the appearance of bubbles compared to other studies (27): 600 e-/Å² for relaxed helices and up to 900 e-/Å² for long compact ones, while no bubbles could be detected in unfolded ribbons (Fig. 1-figure supplement 4). For comparison, bacteriophage capsids containing free DNA, i.e. not in the form of nucleoproteins, show bubbling for doses of ~30-40 e-/Å² (27).

**Cryo-EM single particle analysis of the different compaction states of the mimivirus genomic fiber**

In order to shed light on the mimivirus genome packaging strategy and to determine the structure of the purified genomic fibers, we performed cryo-EM single-particle analysis. The different conformations of the genomic fiber initially observed by negative staining (Fig. 1) and cryo-EM resulted in a highly heterogeneous dataset for single-particle analysis. In order to separate different conformations, *in silico* sorting through 2D classification (using Relion (28, 29)) was performed. Next, we carried out cluster analysis relying on the widths of the helical segments and correlations (real and reciprocal spaces) between the experimental 2D class patterns (Fig. 1-figures supplement 5 & 6). Three independent clusters (Cl) could be distinguished, corresponding to the compact (Cl1), intermediate (Cl2) and relaxed (Cl3) fiber conformations (Fig. 1-figure supplement 6) with the latter being the widest. For each cluster, we determined their helical symmetry parameters by image power spectra analyses and performed structure determination and refinement (Fig. 2, Fig. 2-figures supplement 1-3).

For both Cl1a and Cl3a conformations, after 3D-refinement, we obtained helical structures of 3.7 Å resolution (FSC threshold 0.5, masked), corresponding to 5-start left-handed helices made of a ~8 nm-thick proteinaceous external shell (Fig. 2-figures supplement 1-2). For the most compact conformation (Cl1a) 5 dsDNA strands were lining the interior of the protein shell leaving a ~9 nm wide central channel (Fig. 2-3, supplementary file 1). The dsDNA strands appear as curved cylinders in the helical structure, the characteristic shape of the DNA (minor and major groove) becoming only visible after focused refinement of a single strand of dsDNA (Fig. 3, Fig. 3-figures supplement 1-2). In the relaxed subcluster Cl3a, the DNA strands at the interface to the ~17 nm-wide central channel are not clearly recognizable (Fig. 2, supplementary file 1 & Fig. 2-figure supplement 2), most likely because they are at least partially detached inside the broken expanded fiber. The breaks after relaxation of the helix might be the result of the extraction and purification treatment, while DNA will remain in the central channel, at least in the early phase of Acanthamoeba infection.

Finally, the 4 Å resolution Cl2 map obtained after 3D refinement (Fig. 2-figure supplement 3) corresponds to a 6-start left-handed helix made of a ~8 nm-thick proteinaceous external shell, with 6 dsDNA strands lining the shell interior and leaving a ~12 nm wide inner channel (Fig. 2, supplementary file 1).

**The most abundant proteins in the genomic fiber are GMC-oxidoreductases, the same that compose the fibrils decorating mimivirus capsid**

MS-based proteomic analyses performed on three biological replicates identified two GMC-oxidoreductases as the main components of the purified genomic fiber (qu_946 and qu_143 in mimivirus reunion corresponding to L894/93 and R135 respectively in mimivirus prototype) (supplementary file 2). The two mimivirus reunion proteins share 69% identity (81% similarity). The available mimivirus R135 GMC-oxidoreductase dimeric structure (30) (PDB
137 4Z24, lacking the 50 amino acid long cysteine-rich N-terminal domain) was fitted into the
138 EM maps (Figs. 2-3). This is quite unexpected, since GMC-oxidoreductases are already
139 known to compose the fibrils surrounding mimivirus capsids (11, 31). The corresponding
140 genes are highly expressed during the late phase of the infection cycle at the time of virion
141 assembly. Notably, the proteomic analyses provided different sequence coverages for the
142 GMC-oxidoreductases depending on whether samples were intact virions or purified genomic
143 fiber preparations, with substantial under-representation of the N-terminal domain in the
144 genomic fiber (Fig. 2-figure supplement 4). Accordingly, the maturation of the GMC-
145 oxidoreductases involved in genome packaging must be mediated by one of the many
146 proteases encoded by the virus or the host cell. Interestingly, mimivirus M4 (31), a laboratory
147 strain having lost the genes responsible for the synthesis of the two polysaccharides
decorating mimivirus fibrils (11) also lacks the GMC-oxidoreductase genes. Additional
148 studies on this specific variant will be key to establish if it exhibits a similar genomic fiber,
149 and if yes, which proteins are composing it.
150

151 **Analysis of the genomic fiber structure**

152 The EM maps and FSC curves of C11a are shown in Fig. 2-figure supplement 1. An additional
153 step of refinement focused on the asymmetric unit further improved the local resolution to 3.3
154 Å as indicated by the corresponding FSC (Fig. 3 and Fig. 2-figure supplement 1). After fitting
155 the most abundant GMC-oxidoreductase qu_946 (SWISS-MODEL model (32)) in the final
156 map of C11a, five additional N-terminal residues in each monomer were manually built using
157 the uninterpreted density available. This strikingly brings the cysteines of each monomer
158 (C51 in qu_946) close enough to allow a disulfide bridge, directly after the 50 amino acids
159 domain not covered in the proteomic analysis of the genomic fiber (Fig. 3G, Fig. 2-figure
160 supplement 4). The N-terminal chain, being more disordered than the rest of the structure, it is
161 absent in the focused refined map, and also absent in the Cl3a map of the relaxed helix,
162 suggesting that a break of the disulfide bridge could be involved in the observed unwinding
163 process. Models of the three helical assemblies and asymmetric unit were further refined
164 using the real-space refinement program in PHENIX 1.18.2 (33). In the 3.3 Å resolution map
165 of the asymmetric unit, most side chains and notably the FAD co-factor are accommodated by
166 density suggesting that the oxidoreductase enzyme could be active (Fig. 3D). Density that can
167 be attributed to the FAD cofactor is also present in the Cl2 and Cl3a maps. The atomic models
168 of C11a and Cl3a dimers are superimposable with a core RMSD of 0.68 Å based on Cα atoms.
169

170 Inspection of individual genomic fibers in the tomograms confirmed the co-existence of both
171 5- and 6-start left-handed helices containing DNA (Fig. 1-figure supplement 2 & Fig. 3-
172 animation 1, Fig.1-video 1). Further, some intermediate and relaxed structures were also
173 observed in which the DNA segments appeared detached from the protein shell and
174 sometimes completely absent from the central channel of the broken fibers. Both GMC-
175 oxidoreductases (qu_946 and qu_143) can be fitted in the 5- and 6-start maps.

176 In relaxed or broken fibers, large electron dense structures that might correspond to proteins
177 inside the lumen were sometimes visible (Fig. 1-figure supplement 2C & E & Fig.1-video 3)
178 as well as dissociating DNA fragments, either in the central channel (Fig. 1-figure supplement
179 2B & Fig.1-video 2) or at the breakage points of the fibers in its periphery (Fig. 1-figure
180 supplement 2D & Fig.1-video 4). Densities corresponding to the dimer subunits composing
181 the protein shell were also commonly observed on dissociated DNA strands (Fig. 1E-F, Fig.
182 1-figure supplement 2 and Fig.1-video 1-4).

183 In the C11a and Cl2 helices, the monomers in each dimer are interacting with two different
dsDNA strands. As a result, the DNA strands are interspersed between two dimers, each also
corresponding to a different strand of the protein shell helix (Fig. 3). Based on the periodic contacts between protein shell and DNA strands, these interactions might involve, in the case of the C11a helix, one aspartate (D82 relative to the N-terminal Methionine in qu_946), one glutamate (E321), two lysines (K344, K685), one arginine (R324) and a histidine (H343) or one asparagine (N80), two lysines (K319, K342), one arginine (R322) and one tyrosine (Y687), in the case of the qu_143 (Fig. 3E-F, supplementary file 3). Intra- and inter-strands contacts between each dimer are presented in supplementary file 3 for qu_143 and qu_946 in C11a, C12 and C13 maps.

Despite the conformational heterogeneity and the flexibility of the rod-shaped structure, we were able to build three atomic models of the mimivirus genomic fiber, in compact (5- and 6-start) and relaxed (5-start) states. Higher resolution data would still be needed to determine the precise structure of the dsDNA corresponding to the viral genome (Fig. 3B, H), however, the lower resolution for this part of the map even in focused refinement runs (Fig. 3H) might also mean that the DNA does not always bind in the same orientation.

## 199 Rough estimation of genome compaction to fit into the nucleoid

Since there is a mixture of 5 and 6 strands of DNA in the genomic fiber, this could correspond to 5 or 6 genomes per fiber or to a single folded genome. Assuming that the length of DNA in B-form is ~34 Å for 10 bp, the mimivirus linear genome of $1.2 \times 10^6$ bp would extend over ~400 µm and occupy a volume of $1.3 \times 10^6$ nm³ (~300 µm and ~1x10⁶ nm³ if in A-form) (34). The volume of the nucleoid (19) (~340 nm in diameter) is approximately $2.1 \times 10^7$ nm³ and could accommodate over 12 copies of viral genomes in a naked state, but only 40 µm of the ~30 nm wide flexible genomic fiber. Obviously, the mimivirus genome cannot be simply arranged linearly in the genomic fiber and must undergo further compaction to accommodate the 1.2 Mb genome in a ~40 µm long genomic fiber. As a result, the complete mimivirus genome, folded at least 5 times, fits into the helical shell. This structure surprisingly resembles a nucleocapsid, such as the archaea infecting APBV1 nucleocapsid (35).

## 211 Additional proteins, including RNA polymerase subunits, are enriched in the genomic fiber

The proteomic analysis of fiber preparations revealed the presence of additional proteins including several RNA polymerase subunits: Rpb1 and Rpb2 (qu_530/532 and qu_261/259/257/255), Rpb3/11 (qu_493), Rpb5 (qu_245), RpbN (qu_379) and Rpb9 (qu_219), in addition to a kinesin (qu_313), a regulator of chromosome condensation (qu_366), a helicase (qu_572), to be possibly associated with the genome (supplementary file 2). In addition to the two GMC-oxidoreductases, at least three oxidative stress proteins were also identified together with hypothetical proteins (supplementary file 2). RNA polymerase subunits start being expressed 1 h post infection with a peak after 5h and are expressed until the end the infection cycle. GMC-oxidoreductases, kinesin, regulator of chromosome condensation are all expressed after 5 hours of infection until the end of the cycle.

As expected, the core protein (qu_431) composing the nucleoid and the major capsid proteins (MCP, qu_446) were significantly decreased in the genomic fiber proteome compared to intact virions. In fact, qu_431 and qu_446 represent respectively 4.5% and 9.4% of the total protein abundance in virions whereas they only account for 0.4% and 0.7% of the total protein abundance in the genomic fiber, suggesting that they could be contaminants in this preparation. On the contrary, we calculated enrichment factors of more than five hundred (qu_946) and twenty-six (qu_143) in the genomic fiber samples compared to the intact virion. Finally, the most abundant RNA polymerase subunit (qu_245) is increased by a factor of eight in the genomic fiber compared to intact virion (if the six different subunits identified are used, the global enrichment is seven-fold). Furthermore, upon inspection of the negative
233 staining micrographs, macromolecules strikingly resembling the characteristic structure of the
234 poxviruses RNA polymerase (36) were frequently observed scattered around the unwinding
235 fiber and sometimes sitting on DNA strands near broken fibers (Fig. 4). Together with the
236 tomograms showing large electron dense structures in the lumen, some RNA polymerase
237 units could occupy the center of the genomic fiber.

238 Discussion

239 Several DNA compaction solutions have been described. For instance, the DNA of
240 filamentous viruses infecting archaea is wrapped by proteins to form a ribbon which in turn
241 folds into a helical rod forming a cavity in its lumen (37, 38). In contrast, the chromatin of
242 cellular eukaryotes consists of DNA wrapped around histone complexes (39). It was recently
243 shown that the virally encoded histone doublets of the *Marseilleviridae* can form nucleosomes
244 (40, 41) and such organization would be consistent with previous evolutionary hypotheses
245 linking giant DNA viruses with the emergence of the eukaryotic nucleus (42–46).
246 Herpesviruses (47, 48), bacteriophages (49, 50) and APBV1 archaeal virus (35) package their
247 dsDNA genome as naked helices or coils. Yet, APBV1 nucleocapsid structure strikingly
248 resemble the mimivirus genomic fiber with a proteinaceous shell enclosing the folded dsDNA
249 genome. Consequently, mimivirus genomic fiber is a nucleocapsid further bundled as a ball of
250 yarn into the nucleoid, itself encased in the large icosahedral capsids. The structure of the
251 mimivirus genomic fiber described herein supports a complex assembly process where the
252 DNA must be folded into 5 or 6 strands prior to or concomitant with packaging, a step that
253 may involve the repeat containing regulator of chromosome condensation (qu_366) identified
254 in the proteomic analysis of the genomic fiber. The proteinaceous shell, *via* contacting
255 residues between the dsDNA and the GMC-oxidoreductases, would guide the folding of the
256 dsDNA strands into the structure prior loading into the nucleoid. The lumen of the fiber being
257 large enough to accommodate the mimivirus RNA polymerase, we hypothesize that it could
258 be sitting on the highly conserved promoter sequence of early genes (51). This central
259 position would support the involvement of the RNA polymerase in genome packaging into
260 the nucleoid and could determine the channel width *via* its anchoring on the genome (Fig.
261 4D). According to this scenario, the available space (although tight) for the RNA polymerase
262 inside the genomic fiber lumen suggests it could be sterically locked inside the compact form
263 of the genomic fiber and could start moving and transcribing upon helix relaxation, initiating
264 the replicative cycle and the establishment of the cytoplasmic viral factory. The genome and
265 the transcription machinery would thus be compacted together into a proteinaceous shield,
266 ready for transcription upon relaxation (Fig. 2-video 1). This organization would represent a
267 remarkable evolutionary strategy for packaging and protecting the viral genome, in a state
268 ready for immediate transcription upon unwinding in the host cytoplasm. This is conceptually
269 reminiscent of icosahedral and filamentous dsRNA viruses which pack and protect their
270 genomes together with the replicative RNA polymerase into an inner core (52–54). As a
271 result, replication and transcription take place within the protein shield and viral genomes
272 remain protected during their entire infectious cycle. In the case of dsDNA viruses however,
273 the double helix must additionally open up to allow transcription to proceed, possibly
274 involving the helicase identified in our proteomic study (supplementary file 2). Finally, in
275 addition to their structural roles, the FAD containing GMC-oxidoreductases making the
276 proteinaceous shield, together with other oxidative stress proteins (supplementary file 2),
277 could alleviate the oxidative stress to which the virions are exposed while entering the cell by
278 phagocytosis.

279 Mimivirus virion thus appears as a Russian doll, with its icosahedral capsids covered with
280 heavily glycosylated fibrils, two internal membranes, one lining the capsid shell, the other
281 encasing the nucleoid, in which the genomic fiber is finally folded. To our knowledge, the
structure of the genomic fiber used by mimivirus to package and protect its genome in the
nucleoid represents the first description of the genome organization of a giant virus. Since the
genomic fiber appears to be expelled from the nucleoid as a flexible and subsequently straight
structure starting decompaction upon release, we suspect that an active, energy-dependent,
process is required to bundle it into the nucleoid during virion assembly. Such an efficient
structure is most likely shared by other members of the Mimiviridae family infecting
*Acanthamoeba* and could be used by other dsDNA viruses relying on exclusively cytoplasmic
replication like poxviruses to immediately express early genes upon entry into the infected
cell (55, 56). Finally, the parsimonious use of moonlighting GMC-oxidoreductases playing a
central role in two functionally unrelated substructures of the mimivirus particle - i) as a
component of the heavily glycosylated peripheral fibril layer and ii) as a proteinaceous shield
to package the dsDNA into the genomic fibers questions the evolutionary incentive leading to
such an organization for a virus encoding close to a thousand proteins.

## 295 Materials and Methods

296 Nucleoid extraction

297 Mimivirus reunion virions defibrillated, as described previously (11, 19), were centrifuged at
298 10,000 x g for 10 min, resuspended in 40 mM TES pH 2 and incubated for 1h at 30°C to
299 extract the nucleoid from the opened capsids.

300 Extraction and purification of the mimivirus genomic fiber

301 The genomic fiber was extracted from 12 mL of purified mimivirus reunion virions at 1.5 x
302 10¹⁰ particles/mL, split into 12x1 mL samples processed in parallel. Trypsin (Sigma T8003)
303 in 40 mM Tris-HCl pH 7.5 buffer was added at a final concentration of 50 µg/mL and the
304 virus-enzyme mix was incubated for 2h at 30°C in a heating dry block (Grant Bio PCH-1).
305 DTT was then added at a final concentration of 10 mM and incubated at 30°C for 16h.
306 Finally, 0.001% Triton X-100 was added to the mix and incubated for 4h at 30°C. Each tube
307 was vortexed for 20 s with 1.5 mm diameter stainless steel beads (CIMAP) to separate the
308 fibers from the viral particles and centrifuged at 5,000 x g for 15 min to pellet the opened
capsids. The supernatant was recovered, and the fibers were concentrated by centrifugation at
309 15,000 x g for 4h at 4°C. Most of the supernatant was discarded leaving 12x~200 µL of
310 concentrated fibers that were pooled and layered on top of ultracentrifuge tubes of 4 mL
311 (polypropylene centrifuge tubes, Beckman Coulter) containing a discontinuous sucrose
312 gradient (40%, 50%, 60%, 70% w/v in 40 mM Tris-HCl pH 7.5 buffer). The gradients were
313 centrifuged at 200,000 x g for 16h at 4 °C. Since no visible band was observed, successive 0.5
314 mL fractions were recovered from the bottom of the tube, the first one supposedly
315 corresponding to 70% sucrose. Each fraction was dialyzed using 20 kDa Slide-A-Lyzers
316 (ThermoFisher) against 40 mM Tris-HCl pH 7.5 to remove the sucrose. These fractions were
317 further concentrated by centrifugation at 15,000 x g, at 4°C for 4h and most of the supernatant
318 was removed, leaving ~100 µL of sample at the bottom of each tube. At each step of the
319 extraction procedure the sample was imaged by negative staining transmission electron
320 microscopy (TEM) to assess the integrity of the genomic fiber (Fig. 1-figure supplement 1).
321 Each fraction of the gradient was finally controlled by negative staining TEM. For proteomic
322 analysis, an additional step of concentration was performed by speedvac (Savant
323 SPD131DDA, Thermo Scientific).

325 Negative stain TEM

326 300 mesh ultra-thin carbon-coated copper grids (Electron Microscopy Sciences, EMS) were
327 prepared for negative staining by adsorbing 4-7 µL of the sample for 3 min., followed by two
328 washes with water before staining for 2 min in 2% uranyl acetate. The grids were imaged
329 either on a FEI Tecnai G2 microscope operated at 200 keV and equipped with an Olympus
330 Veleta 2k camera (IBDM microscopy platform, Marseille, France); a FEI Tecnai G2
331 microscope operated at 200 keV and equipped with a Gatan OneView camera (IMM,
332 microscopy platform, France) or a FEI Talos L120c operated at 120 keV and equipped with a
333 Ceta 16M camera (CSSB multi-user cryo-EM facility, Germany, Fig. 1, Fig. 1-figure
334 supplement 1).

335 **Cryo-electron tomography**

336 Sample preparation

337 For cryo-ET of the mimivirus genomic fiber, samples were prepared as described above for
338 single-particle analysis except that 5 nm colloidal gold fiducial markers (UMC, Utrecht) were
339 added to the sample right before plunge freezing at a ratio of 1:2 (sample: fiducial markers).

340 Data acquisition

341 Tilt series were acquired using SerialEM (57) on a Titan Krios (Thermo Scientific)
342 microscope operated at 300 keV and equipped with a K3 direct electron detector and a GIF
343 BioQuantum energy filter (Gatan). We used the dose-symmetric tilt-scheme (58) starting at 0°
344 with a 3° increment to +/-60° at a nominal magnification of 64,000x, a pixel size of 1.4 Å and
345 a total fluence of 150 e-/Å² over the 41 tilts (i.e. ~3.7 e-/Å²/tilt for an exposure time of 0.8 s
346 fractionated into 0.2 s frames (supplementary file 4).

347 Data processing

348 Tilt series were aligned and reconstructed using the IMOD (59). For visualization purposes,
349 we applied a binning of 8 and SIRT-like filtering from IMOD (59) as well as a bandpass
350 filter bsoft (60). The tomograms have been deposited on EMPIAR, accession number 1131
351 and videos were prepared with Fiji (Fig. 1E-F, Fig. 1-figure supplement 2 & Fig.1-video 1-
352 4).

353 Agarose gel electrophoresis and DNA dosage to assess the presence of DNA into the fiber

354 Genomic DNA was extracted from 10¹⁰ virus particles using the PureLink TM Genomic DNA
355 mini kit (Invitrogen) according to the manufacturer's protocol. Purified genomic fiber was
356 obtained following the method described above. The purified fiber was treated by adding
357 proteinase K (PK) (Takara ST 0341) to 20 µL of sample (200 ng as estimated by dsDNA
358 Qubit fluorometric quantification) at a final concentration of 1 mg/mL and incubating the
359 reaction mix at 55°C for 30 min. DNase treatment was done by adding DNase (Sigma
360 10104159001) and MgCl₂ to a final concentration of 0.18 mg/mL and 5 mM, respectively, in
361 20 µL of sample and incubated at 37°C for 30 min prior to PK treatment. For RNase
362 treatment, RNase (Sigma SLBW2866) was added to 20 µL (200 ng) of sample solution to a
363 final concentration of 1 mg/mL and incubated at 37°C for 30 min prior to PK treatment. All
364 the samples were then loaded on a 1 % agarose gel and stained with ethidium bromide after
365 migration. The bands above the 20 kbp marker correspond to the stacked dsDNA fragments
366 of various lengths compatible with the negative staining images of the broken fibers where
367 long DNA fragment are still attached to the helical structure. The mimivirus purified genomic
368 DNA used as a control migrates at the same position (Fig. 1-figure supplement 3).

369 Cryo-EM bubblegram analysis

370 Samples were prepared as described for single-particle analysis. Dose series were acquired on
371 a Titan Krios (Thermo Scientific) microscope operated at 300 keV and equipped with a K3
direct electron detector and a GIF BioQuantum (Gatan) energy filter. Micrographs were
372 recorded using SerialEM (57) at a nominal magnification of 81,000x, a pixel size of 1.09 Å
373
and a rate of 15 e-/pixel/s (Fig. 1-figure supplement 4 & supplementary file 4). Dose series were acquired by successive exposures of 6 s, resulting in an irradiation of 75 e-/Å² per exposure. Micrographs were acquired with 0.1 s frames and aligned in SerialEM (57). In a typical bubblegram experiment, 12 to 15 successive exposures were acquired in an area of interest with cumulative irradiations of 900 to 1,125 e-/Å² total (Fig. 1-figure supplement 4).

379 **Single-particle analysis by cryo-EM**

380 **Sample preparation**

For single-particle analysis, 3 µL of the purified sample were applied to glow-discharged Quantifoil R 2/1 Cu grids, blotted for 2 s using a Vitrobot Mk IV (Thermo Scientific) and applying the following parameters: 4°C, 100% humidity, blotting force 0, and plunge frozen in liquid ethane/propane cooled to liquid nitrogen temperature.

385 **Data acquisition**

Grids were imaged using a Titan Krios (Thermo Scientific) microscope operated at 300 keV and equipped with a K2 direct electron detector and a GIF BioQuantum energy filter (Gatan). 7,656 movie frames were collected using the EPU software (Thermo Scientific) at a nominal magnification of 130,000x with a pixel size of 1.09 Å and a defocus range of -1 to -3 µm. Micrographs were acquired using EPU (Thermo Scientific) with 8 s exposure time, fractionated into 40 frames and 7.5 e-/pixel/s (total fluence of 50.5 e-/Å²) (supplementary file 4).

393 **2D classification and clustering of 2D classes**

All movie frames were aligned using MotionCor2 (61) and used for contrast transfer function (CTF) estimation with CTFFIND-4.1 (62). Helical segments of the purified genomic fibers, manually picked with Relion 3.0 (28, 29)(28, 29)(28, 29)(28, 29)(28, 29), were initially extracted with different box sizes, 400 pixels for 3D reconstructions, 500 pixels for initial 2D classifications and clustering and 700 pixels to estimate the initial values of the helical parameters. Particles were subjected to reference-free 2D classification in Relion 3.1.0 (28, 29), where multiple conformations of the fiber were identified (Fig. 1-figures supplement 5 & 6).

We then performed additional cluster analysis of the 194 initial 2D classes provided by Relion (Fig. 1-figure supplement 5) to aim for more homogeneous clusters, eventually corresponding to different states (Fig. 1-figure supplement 6). A custom 2-step clustering script was written in python with the use of Numpy(63) and Scikit-learn (64) libraries. First, a few main clusters were identified by applying a DBSCAN (65) clustering algorithm on the previously estimated fiber external width values (W1). The widths values, estimated by adjusting a parameterized cross-section model on each 2D stack, range from roughly 280 Å to 340 Å. The cross-section model fitting process is based on adjusting a section profile (S) described in Equation 1 on the longitudinally integrated 2D-class profile (Fig. 1-figure supplement 6A). The cross-section model is composed of a positive cosine component, (parameterized by the center position µ, its width σ₁ and amplitude a₁) associated with the fiber external shell, a negative cosine component (parameterized by the center position µ, its width σ₂ and amplitude a₂) associated with the central hollow lumen (W2), and a constant p₀, accounting for the background level, as

$$S(\mu, \sigma_1, a_1, \sigma_2, a_2, p_0, x) = a_1 \cos \frac{x - \mu}{\sigma_1} - a_2 \cos \frac{x - \mu}{\sigma_2} + p_0 \qquad \text{Equation 1}$$

Then, as a second step, each main cluster was subdivided into several sub-clusters by
417 applying a KMEANS(63) clustering algorithm on a pairwise similarity matrix. This similarity
418 metric was based on a 2D image cross-correlation scheme, invariant to image shifts, and
419 mirroring (67). The number of sub-clusters was manually chosen by visual inspection. For the
420 most populated 2D classes corresponding to the most compact conformations, the number of
421 sub-clusters was small: n=2 sub-clusters, C11a and C11b, and n=1 sub-cluster for the
422 intermediate class, C12. However, the number of sub-clusters was higher (n=5) for the relaxed
423 conformation (C13 from green to purple in Fig. 1-figure supplement 6), highlighting the
424 overall heterogeneity of our dataset with compact, intermediate, relaxed states and even loss
425 of one protein strands (C13b) and unwound ribbons.

426 *Identification of candidate helical parameters*

427 Fourier transform analysis methods have been used to identify helical parameters candidates
428 (68–70) for the C11a, C12 and C13a clusters. For each cluster, we first estimated the repeat
429 distance by applying the method consisting in a longitudinal autocorrelation with a windowed
430 segment of the real space 2D class of fixed size (100 Å) (69). Then, a precise identification of
431 the power spectrum maxima could be achieved on a high signal-to-noise ratio power
432 spectrum, obtained by averaging all the constituting segments in the Fourier domain, which
433 helps lowering the noise, and fill in the CTF zeros regions. The best candidates were validated
434 with Helixplorer (http://rico.ibs.fr/helixplorer/).

435 For the C11a cluster, the parameters of a 1-start helix have been identified with a rise of 7.937
436 Å and a twist of 221.05°. For the C12 cluster, the candidate parameters are a rise of 20.383 Å
437 and a twist of 49.49° and C3 cyclic symmetry. For the C13a cluster, the candidate parameters
438 correspond to a rise of 31.159 Å and a twist of 24° and D5 symmetry (Fig. 1-figure
439 supplement 6C-E).

440 Cryo-EM data processing and 3D reconstruction

441- C11a-C13a

442 After helical parameters determination, two last 2D classifications were performed on
443 segments extracted with a box size of 400 pixels (decimated to 200 pixels) using the proper
444 rises for the most compact C11a (7.93 Å, 113,026 segments, overlap ~98.2%) and the relaxed
445 C13a (31.16 Å, 16,831 segments, overlap ~92.9%). Values of the helical parameters (rise and
446 twist) were then used for Relion 3D classification (28, 29), with a +/-10% freedom search
447 range, using a featureless cylinder as initial reference (diameter of 300 Å for the compact
448 particles C11a and 340 Å for the relaxed particles C13a). The superimposable 3D classes
449 (same helical parameters, same helix orientation) were then selected, reducing the data set to
450 95,722 segments for the compact fiber (C11a) and to 15,289 segments for the relaxed fiber
451 (C13a). After re-extraction of the selected segments without scaling, further 3D refinement
452 was performed with a 3D classification output low pass filtered to 15 Å as reference. With
453 this, the maps were resolved enough (C11a: 4.4 Å, C13a: 4.8; FSC threshold 0.5) to identify
454 secondary structure elements (with visible cylinders corresponding to the helices) (Fig. 2-
455 figures supplement 1-2).

456- C12

457 The twelve 2D classes corresponding to segments of the C12 conformation, were extracted
458 with a box size of 400 pixels (decimated to 200 pixels, rise 20.4 Å, 5,775 segments, overlap
459 ~94.9%). They were used in Relion for 3D refinement with the helical parameters values
460 identified previously, with a +/-10% freedom search range, using a 330 Å large featureless
461 cylinder as initial reference and resulted in a 7.1 Å map (FSC threshold 0.5) and C3 cyclic
462 symmetry (Fig. 2-figure supplement 3).
463 *Focused refinement of a single oxidoreductase dimer*

464 The C11a 3D map was used to make a mask corresponding to a single dimer of
465 oxidoreductases through the segmentation module (71) of the program Chimera (72) (this
466 mask was deposited as part of the EMDB D_1292117739). With Relion and this mask we
467 performed partial signal subtraction (73) to remove the information of the other dimers from
468 the experimental images generating a new stack of subtracted images. This was followed by
469 focused refinement with an initial model created (command "relion_reconstruct") from the
470 subtracted dataset and the corresponding orientation/centering parameters from the partial
471 signal subtraction. CTF parameters refinement was performed followed by a last 3D
472 refinement step and post-processing (B-factor applied -45). This led to the best resolved 3.3 Å
473 3D map (FSC threshold 0.5, masked, Fig. 2-figure supplement 1) that was used to build the
474 atomic model of the GMC-oxidoreductase and its ligands (supplementary file 1, Fig. 3). As
475 expected, densities corresponding to DNA were still visible in the final 3D map because the
476 3D density used during partial signal subtraction contained no high-resolution information
477 about the DNA structure. A Relion project directory summarizing all the steps to perform the
478 focused refinement of the asymmetric unit (oxidoreductase dimer) is available under the link:
479 https://src.koda.cnrs.fr/igs/genfiber_c11a_focusrefine_relion_pipe

480 DNA focused refinement

481 The 3D EM maps of C11a and C12 were used to make masks for each DNA strand separately
482 with ChimeraX (74). Each strand was used in a partial signal subtraction removing
483 information from all proteins keeping only information near the presumed DNA regions on
484 the corresponding experimental images. The subtracted (DNA only) datasets were merged
485 and subjected to 2D classification to allow visual assessment of the quality of the subtracted
486 images (Fig. 3-figure supplement 1). 3D refinement was performed followed by 3D
487 classification and final refinement of the best 3D classes (Fig. 3E, Fig. 3-figure supplement
488 2).
489 For the C13a compaction state (where some barely visible densities could correspond to
490 remaining DNA) a cylinder with a diameter corresponding to the lumen of the relaxed
491 structure (140 Å) was used as mask for partial signal subtraction in an attempt to enhance the
492 information from the presumed DNA regions. However, the signal remained too weak to be
493 recognized as such.

494

495 Automatic picking of the three different conformations (C11a, C12, C13a)

496 Projections from the different helical maps were used as new input for automatic picking of
497 each C11a, C12 and C13a clusters to get more homogeneous datasets for each conformation
498 (75). For each dataset a final round of extraction (box size C11a: 380 pixels, 121,429
499 segments; C12: 400 pixels, 8479 segments; C13a: 400 pixels, 11,958 segments) and 3D
500 refinement with solvent flattening (Central z length 30%) was performed using the
501 appropriate helical parameters and additional symmetries (none for C11a, C3 for C12 and D5
502 for C13a). This led to the improved maps presented in Figs. 2-3, Fig. 2-figures supplement 1-3
503 (C11a: 3.7 Å; C12: 4.0 Å; C13a: 3.7 Å; FSC threshold 0.5, masked). Post-processing was
504 performed with B-factor -80 (supplementary file 1, Fig. 2-figures supplement 1-3).

505 *Structures refinement*

506 The resolution of the EM map enabled to fit the R135 dimeric structure (30) (PDB 4Z24) into
507 the maps using UCSF Chimera 1.13.1 (72). The qu_143 and qu_946 models were obtained
508 using SWISS-MODEL (76) (closest PDB homologue: 4Z24). It is only at that stage that the
best fitted qu_946 model was manually inspected and additional N-terminal residues built using the extra density available in the cryo-EM map of the 5-start compact reconstruction (Fig. 3). The entire protein shell built using the corresponding helical parameters were finally fitted into the C11a and C13a maps and were further refined against the map using the real-space refinement program in PHENIX 1.18.2 (33) (Fig. 2-figures supplement 1-2). The qu_143 and qu_946 models were also used to build the entire shell using the C12 helical parameters and symmetry and were further refined using the real-space refinement program in PHENIX 1.18.2 (33) (Fig. 2-figure supplement 3). Validations were also performed into PHENIX 1.18.2 (33) using the comprehensive validation program and statistics in supplementary file 1 correspond to the qu_143 model. The qu_946 model was manually corrected and ultimately refined and validated into PHENIX 1.18.2 (33) using the highest resolution focused refined C11a map. In that map the 2 first amino acid are disordered including the cysteine.

## 522 Mass spectrometry-based proteomic analysis of mimivirus virion and genomic fiber

Proteins extracted from total virions and purified fiber were solubilized with Laemmli buffer (4 volumes of sample with 1 volume of Laemmli 5X - 125 mM Tris-HCl pH 6.8, 10% SDS, 20% glycerol, 25% β-mercaptoethanol and traces of bromophenol blue) and heated for 10 min at 95 °C. Three independent infections using 3 different batches of virions were performed and the genomic fiber was extracted from the resulting viral particles to analyze three biological replicates. Extracted proteins were stacked in the top of a SDS-PAGE gel (4-12% NuPAGE, Life Technologies), stained with Coomassie blue R-250 (Bio-Rad) before in-gel digestion using modified trypsin (Promega, sequencing grade) as previously described (77). Resulting peptides were analyzed by online nanoliquid chromatography coupled to tandem MS (UltiMate 3000 RSLCnano and Q-Exactive Plus, Thermo Scientific). Peptides were sampled on a 300 µm x 5 mm PepMap C18 precolumn and separated on a 75 µm x 250 mm C18 column (Reprosil-Pur 120 C18-AQ, 1.9 µm, Dr. Maisch) using a 60-min gradient for fiber preparations and a 140-min gradient for virion. MS and MS/MS data were acquired using Xcalibur (Thermo Scientific). Peptides and proteins were identified using Mascot (version 2.7.0) through concomitant searches against mimivirus reunion, classical contaminant databases (homemade) and the corresponding reversed databases. The Proline software (78) was used to filter the results: conservation of rank 1 peptides, peptide score ≥ 25, peptide length ≥ 6, peptide-spectrum-match identification false discovery rate < 1% as calculated on scores by employing the reverse database strategy, and minimum of 1 specific peptide per identified protein group. Proline was then used to perform a compilation and MS1-based quantification of the identified protein groups. Intensity-based absolute quantification (iBAQ) (79) values were calculated from MS intensities of identified peptides. The viral proteins detected in a minimum of two replicates are reported with their molecular weight, number of identified peptides, sequence coverage and iBAQ values in each replicate. The number of copies per fiber for each protein was calculated according to their iBAQ value based on the GMC-oxidoreductases iBAQ value (see below, supplementary file 2). Mapping of the identified peptides for the GMC-oxidoreductases are presented in Fig. 2-figure supplement 4.

## 551 Protein/DNA ratio validation

To compare the theoretical composition of the genomic fiber accommodating a complete genome with the experimental concentrations in protein and DNA of the sample, we performed DNA and protein quantification (Qubit fluorometric quantification, Thermo Fischer Scientific) on two independent samples of the purified genomic fiber. This returned a concentration of 1.5 ng/µL for the dsDNA and 24 ng/µL for the proteins in one sample and 10
ng/µL for the dsDNA and 150 ng/µL for the protein in the second sample (deposited on agarose gel, Fig. 1-figure supplement 3). Considering that all proteins correspond to the GMC-oxidoreductases subunits (~71 kDa), in the sample there is a molecular ratio of 7.2 dsDNA base pairs per GMC-oxidoreductases subunit in the first sample and 7.68 in the second. Based on our model, a 5-start genomic fiber containing the complete mimivirus genome (1,196,989 bp) should be composed of ~95,000 GMC-oxidoreductases subunits (~93,000 for a 6-start). This gives a molecular ratio of 12.5 dsDNA base pairs per GMC-oxidoreductase subunit, which would be more than experimentally measured. However, in the cryo-EM dataset, there are at most 50% of genomic fibers containing the DNA genome (mostly Cl1), while some DNA strands can be observed attached to the relaxed genomic fiber Cl3 but the vast majority of released DNA was lost during purification of the genomic fiber. Applying an estimated loss of 50 % to the total DNA compared to the measured values, we obtain a ratio, which is in the same order of magnitude of the ones measured in the purified genomic fiber samples.

## 571 Model visualization

Molecular graphics and analyses were performed with UCSF Chimera 1.13.1 (72) and UCSF ChimeraX 1.1 (74), developed by the Resource for Biocomputing, Visualization, and Informatics at the University of California, San Francisco, with support from National Institutes of Health R01-GM129325 and the Office of Cyber Infrastructure and Computational Biology, National Institute of Allergy and Infectious Diseases.

## 577 Analysis of macromolecules in NS-TEM images of mimivirus genomic fiber

Manually picked positions of the particles of interest have been used to automatically extract 193 Å square area from the micrographs (Fig. 4C (E)). Then, manual clipping of the particle from its noisy background has been achieved using GIMP's intelligent scissors and a smooth transparent to black mask, producing the clean extraction image (Fig. 4C (CE)). This clipping step was applied in order to improve the semi-automatic identification of the closest orientation in all RNA polymerase projections. The projection views of the different RNA polymerase were produced by converting the PDB model into a volume density through EMAN2's e2pdb2mrc software, and converting the volume into a 2D projection using a dedicated python script, ultimately applying a gaussian blur filter (σ=2.9Å) in order to roughly simulate the whole imaging transfer function. The projections dataset of all orientations of the vaccinia virus RNA polymerase structure was produced with a 5° rotation step in all angles (PDB: 6RIC, equivalent subunits identified by our MS-based proteomics of purified mimivirus genomic fibers). Preferred projections (Fig. 4C (P)) orientations were manually assessed.

## 593 Acknowledgements

The cryo-EM work was performed at the multi-user Cryo-EM Facility at CSSB. We thank Jean-Michel Claverie for his comments on the manuscript and discussions all along the project. We thank Irina Gutsche, Ambroise Desfosses, Eric Durand and Juan Reguera for their helpful support on structural work. We thank Carolin Seuring for support and technical help. Processing was performed on the DESY Maxwell cluster, at IBS and IGS. We thank Wolfgang Lugmayr for assistance and Sebastien Santini and Guy Schoehn for support. The preliminary electron microscopy experiments were performed on the PiCSL-FBI core facility (Nicolas Brouilly, Fabrice Richard and Aïcha Aouane, IBDM, AMU-Marseille), member of the France-BioImaging national research infrastructure and on the IMM imaging platform (Artemis Kosta).
607 **References**

608 1. B. La Scola, S. Audic, C. Robert, L. Jungang, X. de Lamballerie, M. Drancourt, R. Birtles, J.-M.

609 Claverie, D. Raoult, A giant virus in amoebae. *Science*. **299**, 2033 (2003).

610 2. D. Raoult, S. Audic, C. Robert, C. Abergel, P. Renesto, H. Ogata, B. La Scola, M. Suzan, J.-M.

611 Claverie, The 1.2-megabase genome sequence of Mimivirus. *Science*. **306**, 1344–1350 (2004).

612 3. C. Abergel, M. Legendre, J.-M. Claverie, The rapidly expanding universe of giant viruses:

613 Mimivirus, Pandoravirus, Pithovirus and Mollivirus. *FEMS Microbiol. Rev.* **39**, 779–796 (2015).

614 4. F. Schulz, S. Roux, D. Paez-Espino, S. Jungbluth, D. A. Walsh, V. J. Denef, K. D. McMahon, K. T.

615 Konstantinidis, E. A. Eloe-Fadrosh, N. C. Kyrpides, T. Woyke, Giant virus diversity and host

616 interactions through global metagenomics. *Nature*. **578**, 432–436 (2020).

617 5. M. Moniruzzaman, A. R. Weinheimer, C. A. Martinez-Gutierrez, F. O. Aylward, Widespread

618 endogenization of giant viruses shapes genomes of green algae. *Nature*. **588**, 141–145 (2020).

619 6. H. Kaneko, R. Blanc-Mathieu, H. Endo, S. Chaffron, T. O. Delmont, M. Gaia, N. Henry, R.

620 Hernández-Velázquez, C. H. Nguyen, H. Mamitsuka, P. Forterre, O. Jaillon, C. de Vargas, M. B.

621 Sullivan, C. A. Suttle, L. Guidi, H. Ogata, Eukaryotic virus composition can predict the efficiency

622 of carbon export in the global ocean. *iScience*. **24**, 102002 (2021).

623 7. P. Forterre, Giant viruses: conflicts in revisiting the virus concept. *Intervirology*. **53**, 362–378

624 (2010).

625 8. J.-M. Claverie, C. Abergel, Mimivirus: the emerging paradox of quasi-autonomous viruses.

626 *Trends Genet*. **26**, 431–437 (2010).

627 9. C. Abergel, J. Rudinger-Thirion, R. Giegé, J.-M. Claverie, Virus-encoded aminoacyl-tRNA

628 synthetases: structural and functional characterization of mimivirus TyrRS and MetRS. *J. Virol*.

629 **81**, 12406–12417 (2007).

630 10. F. Piacente, C. De Castro, S. Jeudy, M. Gaglianone, M. E. Laugieri, A. Notaro, A. Salis, G.

631 Damonte, C. Abergel, M. G. Tonetti, The rare sugar N-acetylated viosamine is a major

632 component of Mimivirus fibers. *J. Biol. Chem.* **292**, 7385–7394 (2017).

633 11. A. Notaro, Y. Couté, L. Belmudes, M. E. Laugeri, A. Salis, G. Damonte, A. Molinaro, M. G.

634 Tonetti, C. Abergel, C. De Castro, Expanding the Occurrence of Polysaccharides to the Viral

635 World: The Case of Mimivirus. *Angew. Chem. Int. Ed.* **60**, 19897–19904 (2021).

636 12. P. Colson, B. La Scola, A. Levasseur, G. Caetano-Anollés, D. Raoult, Mimivirus: leading the way in

637 the discovery of giant viruses of amoebae. *Nat. Rev. Microbiol.* **15**, 243–254 (2017).

638 13. Y. G. Kuznetsov, T. Klose, M. Rossmann, A. McPherson, Morphogenesis of mimivirus and its

639 viral factories: an atomic force microscopy study of infected cells. *J. Virol.* **87**, 11200–11213

640 (2013).
641 14. C. Xiao, Y. G. Kuznetsov, S. Sun, S. L. Hafenstein, V. A. Kostyuchenko, P. R. Chipman, M. Suzan-Monti, D. Raoult, A. McPherson, M. G. Rossmann, Structural Studies of the Giant Mimivirus. *PLoS Biol.* **7**, e1000092 (2009).

644 15. T. Klose, Y. G. Kuznetsov, C. Xiao, S. Sun, A. McPherson, M. G. Rossmann, The three-dimensional structure of Mimivirus. *Intervirology*. **53**, 268-273 (2010).

646 16. T. Ekeberg, M. Svenda, C. Abergel, F. R. N. C. Maia, V. Seltzer, J.-M. Claverie, M. Hantke, O. Jönsson, C. Nettelblad, G. van der Schot, M. Liang, D. P. DePonte, A. Barty, M. M. Seibert, B. Iwan, I. Andersson, N. D. Loh, A. V. Martin, H. Chapman, C. Bostedt, J. D. Bozek, K. R. Ferguson, J. Krzywinski, S. W. Epp, D. Rolles, A. Rudenko, R. Hartmann, N. Kimmel, J. Hajdu, Three-dimensional reconstruction of the giant mimivirus particle with an x-ray free-electron laser. *Phys. Rev. Lett.* **114**, 098102 (2015).

652 17. J. R. Schrad, J. S. Abrahão, J. R. Cortines, K. N. Parent, Structural and Proteomic Characterization of the Initiation of Giant Virus Infection. *Cell*. **181**, 1046-1061.e6 (2020).

654 18. V. Chelikani, T. Ranjan, K. Kondabagil, Revisiting the genome packaging in viruses with lessons from the "Giants." *Virology*. **466-467**, 15-26 (2014).

656 19. Y. G. Kuznetsov, C. Xiao, S. Sun, D. Raoult, M. Rossmann, A. McPherson, Atomic force microscopy investigation of the giant mimivirus. *Virology*. **404**, 127-137 (2010).

658 20. J.-M. Claverie, R. Grzela, A. Lartigue, A. Bernadac, S. Nitsche, J. Vacelet, H. Ogata, C. Abergel, Mimivirus and Mimiviridae: giant viruses with an increasing number of potential hosts, including corals and sponges. *J. Invertebr. Pathol.* **101**, 172-180 (2009).

661 21. D. Arslan, M. Legendre, V. Seltzer, C. Abergel, J.-M. Claverie, Distant Mimivirus relative with a larger genome highlights the fundamental features of Megaviridae. *Proc. Natl. Acad. Sci. U. S. A.* **108**, 17486-17491 (2011).

664 22. N. Zauberman, Y. Mutsafi, D. B. Halevy, E. Shimoni, E. Klein, C. Xiao, S. Sun, A. Minsky, Distinct DNA exit and packaging portals in the virus Acanthamoeba polyphaga mimivirus. *PLoS Biol.* **6**, e114 (2008).

667 23. M. Suzan-Monti, B. L. Scola, L. Barrassi, L. Espinosa, D. Raoult, Ultrastructural Characterization of the Giant Volcano-like Virus Factory of Acanthamoeba polyphaga Mimivirus. *PLoS ONE*. **2**, e328 (2007).

670 24. Y. Mutsafi, Y. Fridmann-Sirkis, E. Milrot, L. Hevroni, A. Minsky, Infection cycles of large DNA viruses: emerging themes and underlying questions. *Virology*. **466-467**, 3-14 (2014).

672 25. W. Wu, J. A. Thomas, N. Cheng, L. W. Black, A. C. Steven, Bubblegrams Reveal the Inner Body of Bacteriophage KZ. *Science*. **335**, 182-182 (2012).

674 26. N. Cheng, W. Wu, N. R. Watts, A. C. Steven, Exploiting radiation damage to map proteins in nucleoprotein complexes: The internal structure of bacteriophage T7. *J. Struct. Biol.* **185**, 250-256 (2014).

677 27. M. Mishyna, O. Volokh, Ya. Danilova, N. Gerasimova, E. Pechnikova, O. S. Sokolova, Effects of radiation damage in studies of protein-DNA complexes by cryo-EM. *Micron*. **96**, 57-64 (2017).

679 28. S. He, S. H. W. Scheres, Helical reconstruction in RELION. *J. Struct. Biol.* **198**, 163-176 (2017).
680 29. S. H. W. Scheres, RELION: Implementation of a Bayesian approach to cryo-EM structure determination. *J. Struct. Biol.* **180**, 519–530 (2012).

682 30. T. Klose, D. A. Herbst, H. Zhu, J. P. Max, H. I. Kenttämaa, M. G. Rossmann, A Mimivirus Enzyme that Participates in Viral Entry. *Struct. Lond. Engl. 1993.* **23**, 1058–1065 (2015).

684 31. M. Boyer, S. Azza, L. Barrassi, T. Klose, A. Campocasso, I. Pagnier, G. Fournous, A. Borg, C. Robert, X. Zhang, C. Desnues, B. Henrissat, M. G. Rossmann, B. La Scola, D. Raoult, Mimivirus shows dramatic genome reduction after intraamoebal culture. *Proc. Natl. Acad. Sci. U. S. A.* **108**, 10296–10301 (2011).

688 32. A. Waterhouse, M. Bertoni, S. Bienert, G. Studer, G. Tauriello, R. Gumienny, F. T. Heer, T. A. P. de Beer, C. Rempfer, L. Bordoli, R. Lepore, T. Schwede, SWISS-MODEL: homology modelling of protein structures and complexes. *Nucleic Acids Res.* **46**, W296–W303 (2018).

691 33. D. Liebschner, P. V. Afonine, M. L. Baker, G. Bunkóczi, V. B. Chen, T. I. Croll, B. Hintze, L.-W. Hung, S. Jain, A. J. McCoy, N. W. Moriarty, R. D. Oeffner, B. K. Poon, M. G. Prisant, R. J. Read, J. S. Richardson, D. C. Richardson, M. D. Sammito, O. V. Sobolev, D. H. Stockwell, T. C. Terwilliger, A. G. Urzhumtsev, L. L. Videau, C. J. Williams, P. D. Adams, Macromolecular structure determination using X-rays, neutrons and electrons: recent developments in *Phenix*. *Acta Crystallogr. Sect. Struct. Biol.* **75**, 861–877 (2019).

697 34. S. Li, W. K. Olson, X.-J. Lu, Web 3DNA 2.0 for the analysis, visualization, and modeling of 3D nucleic acid structures. *Nucleic Acids Res.* **47**, W26–W34 (2019).

699 35. D. Ptchelkine, A. Gillum, T. Mochizuki, S. Lucas-Staat, Y. Liu, M. Krupovic, S. E. V. Phillips, D. Prangishvili, J. T. Huiskonen, Unique architecture of thermophilic archaeal virus APBV1 and its genome packaging. *Nat. Commun.* **8**, 1436 (2017).

702 36. C. Grimm, H. S. Hillen, K. Bedenk, J. Bartuli, S. Neyer, Q. Zhang, A. Hüttenhofer, M. Erlacher, C. Dienemann, A. Schlosser, H. Urlaub, B. Böttcher, A. A. Szalay, P. Cramer, U. Fischer, Structural Basis of Poxvirus Transcription: Vaccinia RNA Polymerase Complexes. *Cell.* **179**, 1537–1550.e19 (2019).

706 37. F. DiMaio, X. Yu, E. Rensen, M. Krupovic, D. Prangishvili, E. H. Egelman, Virology. A virus that infects a hyperthermophile encapsidates A-form DNA. *Science.* **348**, 914–917 (2015).

708 38. F. Wang, D. P. Baquero, L. C. Beltran, Z. Su, T. Osinski, W. Zheng, D. Prangishvili, M. Krupovic, E. H. Egelman, Structures of filamentous viruses infecting hyperthermophilic archaea explain DNA stabilization in extreme environments. *Proc. Natl. Acad. Sci. U. S. A.* **117**, 19643–19652 (2020).

711 39. P. J. J. Robinson, L. Fairall, V. A. T. Huynh, D. Rhodes, EM measurements define the dimensions of the “30-nm” chromatin fiber: evidence for a compact, interdigitated structure. *Proc. Natl. Acad. Sci. U. S. A.* **103**, 6506–6511 (2006).

714 40. Y. Liu, H. Bisio, C. M. Toner, S. Jeudy, N. Philippe, K. Zhou, S. Bowerman, A. White, G. Edwards, C. Abergel, K. Luger, Virus-encoded histone doublets are essential and form nucleosome-like structures. *Cell.* **184**, 4237–4250.e19 (2021).

717 41. M. I. Valencia-Sánchez, S. Abini-Agbomson, M. Wang, R. Lee, N. Vasilyev, J. Zhang, P. De Ioannes, B. La Scola, P. Talbert, S. Henikoff, E. Nudler, A. Erives, K.-J. Armache, The structure of a virus-encoded nucleosome. *Nat. Struct. Mol. Biol.* **28**, 413–417 (2021).
720 42. P. J. Bell, Viral eukaryogenesis: was the ancestor of the nucleus a complex DNA virus? *J. Mol. Evol.* **53**, 251–256 (2001).

722 43. P. J. L. Bell, Evidence supporting a viral origin of the eukaryotic nucleus. *Virus Res.* **289**, 198168 (2020).

724 44. V. Chaikeeratisak, K. Nguyen, K. Khanna, A. F. Brilot, M. L. Erb, J. K. C. Coker, A. Vavilina, G. L. Newton, R. Buschauer, K. Pogliano, E. Villa, D. A. Agard, J. Pogliano, Assembly of a nucleus-like structure during viral replication in bacteria. *Science.* **355**, 194–197 (2017).

727 45. J.-M. Claverie, Viruses take center stage in cellular evolution. *Genome Biol.* **7**, 110 (2006).

728 46. M. Takemura, Poxviruses and the origin of the eukaryotic nucleus. *J. Mol. Evol.* **52**, 419–425 (2001).

730 47. D. Gong, X. Dai, J. Jih, Y.-T. Liu, G.-Q. Bi, R. Sun, Z. H. Zhou, DNA-Packing Portal and Capsid-Associated Tegument Complexes in the Tumor Herpesvirus KSHV. *Cell.* **178**, 1329-1343.e12 (2019).

733 48. Y.-T. Liu, J. Jih, X. Dai, G.-Q. Bi, Z. H. Zhou, Cryo-EM structures of herpes simplex virus type 1 portal vertex and packaged genome. *Nature.* **570**, 257–261 (2019).

735 49. L. Sun, X. Zhang, S. Gao, P. A. Rao, V. Padilla-Sanchez, Z. Chen, S. Sun, Y. Xiang, S. Subramaniam, V. B. Rao, M. G. Rossmann, Cryo-EM structure of the bacteriophage T4 portal protein assembly at near-atomic resolution. *Nat. Commun.* **6**, 7548 (2015).

738 50. V. B. Rao, M. Feiss, Mechanisms of DNA Packaging by Large Double-Stranded DNA Viruses. *Annu. Rev. Virol.* **2**, 351–378 (2015).

740 51. K. Suhre, S. Audic, J.-M. Claverie, Mimivirus gene promoters exhibit an unprecedented conservation among all eukaryotes. *Proc. Natl. Acad. Sci. U. S. A.* **102**, 14689–14693 (2005).

742 52. S. Toriyama, An RNA-dependent RNA Polymerase Associated with the Filamentous Nucleoproteins of Rice Stripe Virus. *J. Gen. Virol.* **67**, 1247–1255 (1986).

744 53. A. M. Collier, O. L. Lyytinen, Y. R. Guo, Y. Toh, M. M. Poranen, Y. J. Tao, Initiation of RNA Polymerization and Polymerase Encapsulation by a Small dsRNA Virus. *PLOS Pathog.* **12**, e1005523 (2016).

747 54. K. Ding, C. C. Celma, X. Zhang, T. Chang, W. Shen, I. Atanasov, P. Roy, Z. H. Zhou, In situ structures of rotavirus polymerase in action and mechanism of mRNA transcription and release. *Nat. Commun.* **10**, 2216 (2019).

750 55. A. J. Malkin, A. McPherson, P. D. Gershon, Structure of Intracellular Mature Vaccinia Virus Visualized by In Situ Atomic Force Microscopy. *J. Virol.* **77**, 6332–6340 (2003).

752 56. R. Blanc-Mathieu, H. Dahle, A. Hofgaard, D. Brandt, H. Ban, J. Kalinowski, H. Ogata, R.-A. Sandaa, A Persistent Giant Algal Virus, with a Unique Morphology, Encodes an Unprecedented Number of Genes Involved in Energy Metabolism. *J. Virol.* **95**, e02446-20 (2021).

755 57. D. N. Mastronarde, Automated electron microscope tomography using robust prediction of specimen movements. *J. Struct. Biol.* **152**, 36–51 (2005).
757 58. W. J. H. Hagen, W. Wan, J. A. G. Briggs, Implementation of a cryo-electron tomography tilt-scheme optimized for high resolution subtomogram averaging. *J. Struct. Biol.* **197**, 191–198 (2017).

760 59. J. R. Kremer, D. N. Mastronarde, J. R. McIntosh, Computer visualization of three-dimensional image data using IMOD. *J. Struct. Biol.* **116**, 71–76 (1996).

762 60. J. B. Heymann, D. M. Belnap, Bsoft: image processing and molecular modeling for electron microscopy. *J. Struct. Biol.* **157**, 3–18 (2007).

764 61. S. Q. Zheng, E. Palovcak, J.-P. Armache, K. A. Verba, Y. Cheng, D. A. Agard, MotionCor2: anisotropic correction of beam-induced motion for improved cryo-electron microscopy. *Nat. Methods.* **14**, 331–332 (2017).

767 62. A. Rohou, N. Grigorieff, CTFFIND4: Fast and accurate defocus estimation from electron micrographs. *J. Struct. Biol.* **192**, 216–221 (2015).

769 63. C. R. Harris, K. J. Millman, S. J. van der Walt, R. Gommers, P. Virtanen, D. Cournapeau, E. Wieser, J. Taylor, S. Berg, N. J. Smith, R. Kern, M. Picus, S. Hoyer, M. H. van Kerkwijk, M. Brett, A. Haldane, J. F. del Río, M. Wiebe, P. Peterson, P. Gérard-Marchant, K. Sheppard, T. Reddy, W. Weckesser, H. Abbasi, C. Gohlke, T. E. Oliphant, Array programming with NumPy. *Nature.* **585**, 357–362 (2020).

774 64. F. Pedregosa, G. Varoquaux, A. Gramfort, V. Michel, B. Thirion, O. Grisel, M. Blondel, P. Prettenhofer, R. Weiss, V. Dubourg, J. Vanderplas, A. Passos, D. Cournapeau, M. Brucher, M. Perrot, É. Duchesnay, Scikit-learn: Machine Learning in Python. *J. Mach. Learn. Res.* **12**, 2825–2830 (2011).

778 65. M. Hahsler, M. Piekenbrock, D. Doran, **dbscan**: Fast Density-Based Clustering with *R. J. Stat. Softw.* **91** (2019), doi:10.18637/jss.v091.i01.

780 66. S. Mannor, X. Jin, J. Han, X. Jin, J. Han, X. Jin, J. Han, X. Zhang, "K-Means Clustering" in *Encyclopedia of Machine Learning*, C. Sammut, G. I. Webb, Eds. (Springer US, Boston, MA, 2011); http://link.springer.com/10.1007/978-0-387-30164-8_425), pp. 563–564.

783 67. M. Guizar-Sicairos, S. T. Thurman, J. R. Fienup, Efficient subpixel image registration algorithms. *Opt. Lett.* **33**, 156 (2008).

785 68. N. Coudray, R. Lasala, Z. Zhang, K. M. Clark, M. E. Dumont, D. L. Stokes, Deducing the symmetry of helical assemblies: Applications to membrane proteins. *J. Struct. Biol.* **195**, 167–178 (2016).

787 69. R. Diaz, W. J. Rice, D. L. Stokes, Fourier-Bessel reconstruction of helical assemblies. *Methods Enzymol.* **482**, 131–165 (2010).

789 70. C. Sachse, Single-particle based helical reconstruction—how to make the most of real and Fourier space. *AIMS Biophys.* **2**, 219–244 (2015).

791 71. G. D. Pintilie, J. Zhang, T. D. Goddard, W. Chiu, D. C. Gossard, Quantitative analysis of cryo-EM density map segmentation by watershed and scale-space filtering, and fitting of structures by alignment to regions. *J. Struct. Biol.* **170**, 427–438 (2010).
794 72. E. F. Pettersen, T. D. Goddard, C. C. Huang, G. S. Couch, D. M. Greenblatt, E. C. Meng, T. E. Ferrin, UCSF Chimera-A visualization system for exploratory research and analysis. *J. Comput. Chem.* **25**, 1605–1612 (2004).

797 73. X. Bai, E. Rajendra, G. Yang, Y. Shi, S. H. Scheres, Sampling the conformational space of the catalytic subunit of human γ-secretase. *eLife*. **4**, e11182 (2015).

799 74. T. D. Goddard, C. C. Huang, E. C. Meng, E. F. Pettersen, G. S. Couch, J. H. Morris, T. E. Ferrin, UCSF ChimeraX: Meeting modern challenges in visualization and analysis: UCSF ChimeraX Visualization System. *Protein Sci.* **27**, 14–25 (2018).

802 75. L. F. Estrozi, J. Navaza, Fast projection matching for cryo-electron microscopy image reconstruction. *J. Struct. Biol.* **162**, 324–334 (2008).

804 76. A. Waterhouse, M. Bertoni, S. Bienert, G. Studer, G. Tauriello, R. Gumienny, F. T. Heer, T. A. P. de Beer, C. Rempfer, L. Bordoli, R. Lepore, T. Schwede, SWISS-MODEL: homology modelling of protein structures and complexes. *Nucleic Acids Res.* **46**, W296–W303 (2018).

807 77. M. G. Casabona, Y. Vandenbrouck, I. Attree, Y. Couté, Proteomic characterization of Pseudomonas aeruginosa PAO1 inner membrane. *Proteomics*. **13**, 2419–2423 (2013).

809 78. D. Bouyssié, A.-M. Hesse, E. Mouton-Barbosa, M. Rompais, C. Macron, C. Carapito, A. Gonzalez de Peredo, Y. Couté, V. Dupierris, A. Burel, J.-P. Menetrey, A. Kalaitzakis, J. Poisat, A. Romdhani, O. Burlet-Schiltz, S. Cianférani, J. Garin, C. Bruley, Proline: an efficient and user-friendly software suite for large-scale proteomics. *Bioinforma. Oxf. Engl.* **36**, 3148–3155 (2020).

813 79. B. Schwanhäusser, D. Busse, N. Li, G. Dittmar, J. Schuchhardt, J. Wolf, W. Chen, M. Selbach, Global quantification of mammalian gene expression control. *Nature*. **473**, 337–342 (2011).

815

816 **Funding:**

817 This project has received funding from the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation program (grant agreement No 832601).

820 This work was also partially supported by the French National Research Agency ANR-16-CE11-0033-01.

822 Proteomic experiments were partly supported by ProFI (ANR-10-INBS-08-01) and GRAL, a program from the Chemistry Biology Health (CBH) Graduate School of University Grenoble Alpes (ANR-17-EURE-0003).

825 Cryo-EM data collection was supported by DFG grants (INST 152/772-1|152/774-1|152/775-1|152/776-1).

827 Work in the laboratory of Kay Grünewald is funded by the Wellcome Trust (107806/Z/15/Z), the Leibniz Society, the City of Hamburg and the BMBF (05K18BHA).

829 Emmanuelle Quemin received support from the Alexander von Humboldt foundation to (individual research fellowship No. FRA 1200789 HFST-P).

831 France-BioImaging national research infrastructure (ANR-10-INBS-04).

832
**Data and materials availability:** Mimivirus reunion genome has been deposited under the following accession number: BankIt2382307 Seq1 MW004169. 3D reconstruction maps and the corresponding PDB have been deposited to EMDB (Deposition number C11a: 7YX4, EMD-14354; C11a focused refined: 7PTV, EMD-13641; C13a: 7YX5, EMD-14355; C12: 7YX3, EMD-14353). The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD021585 and 10.6019/PXD021585. The tomograms have been deposited in EMPIAR under the accession number 1131.

841

## Figures

843

<figure><img src="image_3.png" /></figure>

844

<caption>**Figure 1:** The mimivirus genomic fiber
A] Micrograph of an ultrathin section of resin-embedded infected cells showing the DNA tightly packed inside mimivirus capsids (C) with electron dense material inside the nucleoid (N). The string-like features, most likely enhanced by the dehydration caused by the fixation and embedding protocol, correspond to the genomic fiber (F) packed into the nucleoid.
B] Micrograph of negative stained mimivirus capsid (C) opened *in vitro* with the genomic fiber (F) still being encased into the membrane limited nucleoid (N).
C] Multiple strands of the flexible genomic fiber (F) are released from the capsid (C) upon proteolytic treatment.
D] Micrograph of negative stained purified mimivirus genomic fibers showing two conformations (the right fiber resembling the one in [B] and free DNA strands (white arrowheads)).
E] Slices through two electron cryo tomograms of the isolated helical protein shell of the purified genomic fibers in compact (top) or relaxed</caption>

845

846

847

848

849

850

851

852

853

854

855
conformation (bottom) in the process of losing one protein strand (Fig. 1-figure supplement 2, Fig.1-video 1 & 4). F] Different slices through the two tomograms shown in [E] reveal DNA strands lining the helical protein shell of the purified genomic fibers in compact (top) or relaxed conformation (bottom). Examples of DNA strands extending out at the breaking points of the genomic fiber are marked by black arrowheads. Note in the top panel, individual DNA strands coated by proteins (red arc). The slicing planes at which the mimivirus genomic fibers were viewed are indicated on diagrams on the top right corner as blue dashed lines and the internal colored segments correspond to DNA strands lining the protein shell. The thickness of the tomographic slices is 1.1 nm and the distance between tomographic slices in panels [E] and [F] is 4.4 nm. Scale bars as indicated.

<figure><img src="image_4.png" /><figcaption>**Figure 2:** Structures of the mimivirus genomic fiber for Cl1a [A-C], Cl3a [D-F], and Cl2 [G-I]: A, D, G] Electron microscopy (EM) maps of Cl1a (A) and Cl3a (D) are shown with each monomer of one GMC-oxidoreductase dimer colored in green and orange and 3 adjacent dimers in yellow, to indicate the large conformational change taking place between the two fiber states. The transition from Cl1a to Cl3a (5-start helix) corresponds to a rotation of each individual unit (corresponding to a GMC- oxidoreductase dimer) by ~ -10° relative to the fiber longitudinal axis and a change in the steepness of the helical rise by ~ -11°. Scale bars 50 Å. Compared to Cl1a, the Cl2 6-start helix (G) shows a difference of ~2° relative to the fiber longitudinal axis and ~2° in the steepness of the helical rise. Scale bars 50 Å. B, E, H] Cross-sectional (bottom) and longitudinal (top) sections through the middle of final Cl1a (B) Cl3a (E) and Cl2 (H) EM maps. Scale bars 50 Å. C, F, I] Longitudinal (top) and orthogonal (bottom) views of final Cl1a (C) and Cl3a (F) and Cl2 (I) EM maps color-coded according to each start of the 5-start helix. Densities for some asymmetric units in the front have been removed on the side view map to show the 5 DNA strands lining the protein shell interior. Scale bars 50 Å.</figcaption></figure>
<figure><img src="image_5.png" /><figcaption>**Figure 3: Maps of the compact (Cl1a and Cl2) genomic fiber structures:** A] Cl1a EM map corresponding to the protein shell prior focused refinement is shown as a transparent surface and the 5 DNA strands as solid surface. One protein dimer strand is shown yellow except for one asymmetric unit (transparent yellow) to illustrate the dimer fit. The position of a second dimer (green) from the adjacent dimer strand is shown to emphasize that the DNA strand (gold) is lining the interface between two dimers. B] Cartoon representation of GMC-oxidoreductase qu_946 dimers fitted into one of the Cl1a 5-start helix strands in the 3.7 Å resolution map. The map is shown at a threshold highlighting the periodicity of contacts between the dsDNA and the protein shell. C, D] Charged distribution on surface representation of Cl1a protein shell made of qu_946 dimers (C) or qu_143 dimers (D). E, F] Cartoon representation of qu_946 (E) and qu_143 (F) fitted into the Cl1a cryo-EM maps</figcaption></figure>
894 highlighting the interacting residues (given as stick models) between each monomer and one
dsDNA strand. The isosurface threshold chosen allows visualization of density for the
895 manually built N-terminal residues, including terminal cysteines (stick model), of two
896 neighboring monomers that could form a terminal disulfide bridge. G] Zoom into the 3.3 Å
897 resolution focused refined C11a map illustrating the fit of the side chains and the FAD ligand
898 (Fig. 3-video 1). H] Cartoon representation of the DNA fitted in the focused refined DNA
899 only map (Fig. 3-figure supplement 2). I] Focused refined C11a map colored by monomer,
900 next to a cartoon representation of the qu_946 dimer (α-helices in red, β-strands in blue and
901 coils in yellow). Secondary structure elements are annotated in both representations (H: helix,
902 B: beta-strand).
903

<figure><img src="image_6.png" /><figcaption>**Figure 4: RNA polymerase could be associated to the genomic fiber.** A] Micrograph of negative stained fiber with released DNA still being connected to a relaxed and broken fiber and adjacent scattered macromolecular complexes that might resemble RNA polymerases. B] Strikingly, some of them (black arrows) appear to sit on a DNA strand (white arrow). C] E, particle extracted from the NS-TEM image, P, projections of vaccinia virus RNA polymerase (6RIC and 6RUI (36)) structure in preferred orientation, CE, clean extraction (see Material and methods section). White and black boxed corresponds to images with white and black asterisk (CE), respectively. Scale bar 50 Å. Negative staining imaging may dehydrate the objects and change macromolecules volumes.</figcaption></figure>

904

905

906

907

908

909

910

911

912

913

914

915

**Supplementary Figures legend**

916

**Fig. 1-figure supplement 1: Negative staining micrographs of opened Mimivirus reunion virions before purification of the genomic fiber.** (A) Spherical nucleoids (N) released from

917
the capsids upon specific treatment (see methods). (B) Dissolved nucleoids expelled from the opened capsids after regular treatment (the region marked with dashed lines is enlarged in Fig 1C), all of the opened capsids (>25%) are releasing the condensed genomic fiber. (C) Multiple broken genomic fibers can be seen next to debris prior to purification and an isolated Stargate (S). (D-E) Multiple segments of the flexible genomic fiber are released upon proteolytic treatment of the capsids. (F) Example of a long, flexible, genomic fiber released from an open capsid (C).

**Fig. 1-figure supplement 2: Cryo-ET of Mimivirus genomic fiber.** (Upper panel) Slices through tomograms exhibiting different features of compact, unwinding and broken genomic fibers of Mimivirus. (A) Slices through a cryo-tomogram focused on a long and broken fiber. Features of the protein shell are visible in tangential slices (top and bottom), while filamentous densities corresponding to DNA strands become visible when slicing deeper inside the fiber. Scale bar, 100 nm. (B) A straight filament running along the fiber symmetry axis has been observed in several cases (blue rectangle), which may correspond to a single or a bundle of DNA strands dissociated from the protein shell. (C) Large amorphous electron dense structures have been observed inside the lumen of the fiber (highlighted with purple circles). (D) DNA strands are often observed emanating from breaks along the genomic fiber, partially disassembling (indicated with yellow arrowheads). (E) Detail of the central slices through the genomic fibers showed in the third depicted slice in A-D. Thickness of the slices is 1.1 nm. Distance between the tomographic slices (from top to bottom) is 4.4 nm between the first and second and the fourth and fifth, and 6.6 nm between the third and second or fourth. The scheme on the left represents a cross-section of a cartoon representation of the 5-start genomic fiber depicted as a cylinder containing 5 strands of coloured DNA internally lining the helical protein shell. It also indicates the plane through which the genomic fiber is viewed as 2D slices extracted from the tomograms in Fig.1-video 1 (A), Fig.1-video 2 (B), Fig.1-video 3 (C), and Fig.1-video 4 (D). Scale bars (B, C, D), 50 nm.

**Fig. 1-figure supplement 3: Agarose gel electrophoresis of the purified genomic fiber compared to the viral genomic DNA:** M: Molecular weight markers (1 kbp DNA Ladder Plus, Euromedex). Lane 1, 100 ng of Mimivirus genomic DNA, Lane 2: 200 ng of untreated genomic fiber, Lane 3: 200 ng of Proteinase K-(PK) treated genomic fiber, Lane 4: 200 ng of DNase- and PK-treated genomic fiber, Lane 5: 200 ng of RNase- and PK-treated genomic fiber.

**Fig. 1-figure supplement 4: Bubblegrams on the Mimivirus genomic fiber.** Field of view for genomic fibers either (from top to bottom): long and bent, long and straight, long and broken, or a mix of short and broken, subjected to a dose series. The total electron flux applied is specified for each field of view. First column: fibers showed after the first exposure of 75 e⁻/Å². Second column: representative micrographs indicating the point at which the first sign of radiation damage as “bubbles” inside the fiber (red arrowheads) were detected. Nucleoproteins with high nucleic acid content are often more susceptible to bubbling than pure proteinaceous structures. In unfolded ribbons (black arrowhead), no bubbles are detected at an accumulated flux of up to 1,125 e⁻/Å². Scale bars, 100 nm. Third column: enlarged view where the effect of radiation damage as trapped “bubbles” is more clearly visible.

**Fig. 1-figure supplement 5.** 200 2D classes (6 empty) were obtained after reference-free 2D classification of fibers acquired (see methods) for single-particle analysis and extracted with a box size of 500 pixels in Relion 3.1.0 after motion correction, CTF estimation and manual
965
966

picking. The 2D classes are representative of the different relaxation states of the Mimivirus genomic fiber observed in our highly heterogeneous dataset.

967 **Fig. 1-figure supplement 6: Clustering analysis of the 2D classes. (A)** Cross-section model adjusted to one 2D class, representative of cluster C11b, to estimate the parameters of the widths W1 (external) and W2 (internal). **(B)** Automatic sorting of the 2D classes using the fiber width W1 and pairwise correlations of the 2D classes resulting in 3 main clusters (compact, C11 in dark blue; intermediate, C12 in cyan and relaxed, C13 in green and orange). **970** **Power spectra indexing with Helixplorer** (http://rico.ibs.fr/helixplorer/) for the candidate helical parameters estimation. **(C)** C11a: 5-start, cyclic symmetry C1 (https://tinyurl.com/mrxnyjwn), **(D)** C12: 6-start, cyclic symmetry C3 (https://tinyurl.com/yckmk79b) et **(E)** C13a: 5-start, dihedral symmetry D5 (https://tinyurl.com/38v5f37m).

971 **Fig. 2-figure supplement 1: Workflow of the C11a compact helix reconstruction process and fitting.** Segment extraction was performed with a box size of 400 pixels (pix) binned twice (box size 200 pix, 2.18 Å/pix). The distance between consecutive boxes was equal to the axial rise calculated by indexation on the power spectrum. From 2D classification with 200 classes, 30 classes were selected for C11a after clustering (see methods and Fig. S5). First, 3D classification was carried out using the segments from 30 2D classes, helical symmetry parameters from the power spectrum indexation and a 300 Å featureless cylinder as 3D reference. 3D-refinement of the 4 boxed 3D-classes was achieved using one low pass filtered 3D class as reference on the unbinned segments. One step of focused refinement (with solvent flattening) was performed using a low pass filtered 3D reconstruction (before solvent flattening) as reference. 3D-refinement was then performed. The resulting map was then used as model for automatic picking. A last 3D-refinement cycle was performed using as reference a featureless cylinder. The bottom right graph presents the Fourier shell correlation (FSC) curves for the final 3D reconstructions (helical assembly and asymmetric unit).

991 **Fig. 2-figure supplement 2: Workflow of the C13a relaxed helix reconstruction process and fitting.** Segment extraction was performed with a box size of 400 pixels (pix) binned twice (box size 200 pix, 2.18 Å/pix) using the rise obtained from the power spectrum indexation (see methods). From 2D classifications with 200 classes, 42 classes were selected for C13a after clustering (see methods and Fig. S5). First, 3D classification was carried out using the 42 2D classes, with D5 symmetry, rise and twist from the power spectrum indexation and a featureless cylinder of 340 Å as reference. 3D-refinement of the two 3D-classes (red boxes) was achieved using one low pass filtered 3D class as reference on the unbinned segments. The resulting map was used as model for automatic picking and segment extraction. Final 3D-refinement was performed on unbinned segments using as initial reference a featureless cylinder with a last step of solvent flattening.

1002 **Fig. 2-figure supplement 3: Workflow of the C12 helix reconstruction process and fitting.** Segment extraction was performed with a box size of 400 pixels (pix) binned twice (box size 200 pix, 2.18 Å/pix) using the rise obtained from the power spectrum indexation (see methods). From 2D classifications with 200 classes, 12 classes were selected for C12 based on correlation (see methods and Fig. S5). First, 3D-refinements combining 12 2D classes with solvent flattening, were performed with the C3 symmetry, rise and twist from the power spectrum indexation and a 330 Å featureless cylinder as reference. The resulting map was used as model for automatic picking and segment extraction on the unbinned segments. Final 3D-refinement was performed on unbinned segments using as initial reference a featureless cylinder with a last step of solvent flattening.

1012 **Fig. 2-figure supplement 4: Comparison of sequence coverages** for qu_946 (A) and
1013 qu_143 (B) obtained by MS-based proteomic analysis of genomic fibers and total virions.
1014 Identified peptides are highlighted in red, blue and grey when identified respectively in three,
1015 two and one replicate of the purified genomic fiber.

1016 **Fig. 3-figure supplement 1: 2D Classification of subtracted segments.** 50 2D classes (2
1017 empty) were obtained after reference-free 2D classification in Relion 3.1.0 of segments (box
1018 size of 400 pixels) of the C11a genomic fibers after signal subtraction to keep only the
1019 information of the DNA strands inside the fiber. The 2D classes show the enhancement of the
1020 signal corresponding to the DNA.

1021 **Fig. 3-figure supplement 2: Workflow of the DNA strand focused refinement process.**
1022 One strand segmented from the 5-start compacted map (C11a) was low-pass filtered at 20 Å
1023 and used as reference for 3D-refinement of the dataset corresponding to the merged dataset of
1024 the individually subtracted 5 strands. The result of the refinement was low-pass filtered at 30
1025 Å and used as reference for 3D-classification. The best 3D class was further refined leading to
1026 a focused refined map of the DNA strand.

1027 **Supplementary file 1: Mimivirus Reunion genomic fibers data statistics (33)**

1028 **Supplementary file 2: Mass spectrometry-based proteomic analysis** of A] three
1029 independent preparations of mimivirus genomic fiber and B] one sample of purified
1030 mimivirus virions. (ND: Not detected). RNA polymerase subunits are marked in red.

1031 **Supplementary file 3: Contacting residues for each GMC-oxidoreductases in the different maps.** Conserved or divergent amino-acids are color coded in green or in red,
1032 respectively.

1034 **Supplementary file 4: Data acquisition parameters for Cryo-EM**

1035 **Supplementary Material**

1036 Figures supplement (Fig. 1-supplement figure 1-6, Fig. 2-supplement figure 1-4, Fig. 3-
1037 supplement figure 1-2)

1038 Supplementary files 1-4

1039 Animation supplement (Fig.3-animation 1)

1040 Video supplement (Fig.1-video 1-4, Fig.2-video 1, Fig. 3-video 1)

1041