Unique features of mRNA translation initiation in trypanosomatids

Abstract

The 43S preinitiation complex (PIC) assembly requires establishment of numerous interactions among eukaryotic initiation factors (eIFs), Met-tRNAiMet and the small ribosomal subunit (40S). Owing to several differences in the structure and composition of kinetoplastidian 40S compared to their mammalian counterparts, translation initiation in trypanosomatids is suspected to display substantial variability. Here, we determined the structure of the 43S PIC from Trypanosoma cruzi, the Chagas disease parasite, showing numerous specific features, such as different eIF3 structure and interactions with the large rRNA expansion segments 9 S , 7 S and 6 S , and the association of a kinetoplastid-specific ~245 kDa DDX60-like helicase. We also revealed a previously undetermined binding site of the eIF5 C-terminal domain, and terminal tails of eIF2β, eIF1, eIF1A and eIF3 c and d subunits, uncovering molecular details of their critical activities.

Full text

Cryo-EM structure of the RNA-rich plant
mitochondrial ribosome
Florent Waltz1* & Heddy Soufari1*, Anthony Bochler1, Philippe Giegé2+ & Yaser Hashem1+
1 Institut Européen de Chimie et Biologie, U1212 Inserm, Université de Bordeaux, 2 rue R. Escarpit, F-
33600 Pessac, France
2Institut de biologie de moléculaire des plantes, UPR 2357 du CNRS, Université de Strasbourg, 12 rue
du général Zimmer, F-67084 Strasbourg, France
*equally contributing authors
+corresponding authors
The vast majority of eukaryotic cells contain mitochondria, essential powerhouses and
metabolic hubs1. These organelles have a bacterial origin and were acquired during an early
endosymbiosis event2. Mitochondria possess specialized gene expression systems composed
of various molecular machines including the mitochondrial ribosomes (mitoribosomes).
Mitoribosomes are in charge of translating the few essential mRNAs still encoded by
mitochondrial genomes3. While chloroplast ribosomes strongly resemble those of bacteria4,5,
mitoribosomes have diverged significantly during evolution and present strikingly different
structures across eukaryotic species6–10. In contrast to animals and trypanosomatides, plants
mitoribosomes have unusually expanded ribosomal RNAs and conserved the short 5S rRNA,
which is usually missing in mitoribosomes11. We have previously characterized the
composition of the plant mitoribosome6 revealing a dozen plant-specific proteins, in addition to
the common conserved mitoribosomal proteins. In spite of the tremendous recent advances in
the field, plant mitoribosomes remained elusive to high-resolution structural investigations,
and the plant-specific ribosomal features of unknown structures. Here, we present a cryo-
electron microscopy study of the plant 78S mitoribosome from cauliflower at near-atomic
resolution. We show that most of the plant-specific ribosomal proteins are pentatricopeptide
repeat proteins (PPR) that deeply interact with the plant-specific rRNA expansion segments.
These additional rRNA segments and proteins reshape the overall structure of the plant
mitochondrial ribosome, and we discuss their involvement in the membrane association and
mRNA recruitment prior to translation initiation. Finally, our structure unveils an rRNA-
constructive phase of mitoribosome evolution across eukaryotes.
Previously, we determined the full composition as well as the overall architecture of the
Arabidopsis thaliana mitoribosome6. However, due to the difficulty to purify large amounts of A.
thaliana mitoribosomes, mainly because of the low quantities of plant material usable for mitochondrial
extraction, only a low-resolution cryo-EM reconstruction was derived. In order to obtain a high-
resolution structure of the plant mitochondrial ribosome, we purified mitoribosome from a closely
related specie, Brassica oleracea var. botrytis, or cauliflower (both Arabidopsis and cauliflower belong
to the group of Brassicaceae plants), as previously described6(see Methods). We have recorded cryo-
EM images for ribosomal complexes purified from two different sucrose gradient peaks (see Methods),
corresponding to the small ribosomal subunit (SSU) and the full 78S mitoribosome. After extensive
particle sorting (see Methods) we have obtained cryo-EM reconstructions for both types of complexes.
The SSU reconstruction displayed an average resolution of 4.36Å (Extended Data Fig. 1). After multi-
body refinement (3 bodies) and particle polishing in RELION312 (see Methods), reconstructions were
derived of the body of the SSU at 3.77Å, the head at 3.9Å and the head extension at 10.5 Å. The
combined structure revealed the full plant mitoribosome SSU (Fig. 1a-d). As for the full mitoribosome,
multi-body refinement of the LSU, SSU head and SSU body generated reconstructions at 3.50Å,
3.74Å and 3.66Å, respectively (Fig. 1e-g, Extended Data Fig. 1).
Density segmentations of our various cryo-EM reconstructions revealed the fine architecture of
the plant mitoribosome showing a large rRNA core in interaction with numerous ribosomal proteins.
Among those ribosomal proteins, 8 densities located at the surface of both subunits (3 on the LSU and
5 on the SSU) unambiguously display alpha-helical motifs characteristic of pentatricopeptide repeat
proteins (Fig. 1a-g). Most of these ribosomal PPRs (rPPRs) are in direct interaction with large rRNA
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expansion segments (ESs) such as the head extension of the SSU (Fig. 1a-d). However, some of
these SSU rPPRs appear to present a higher level of flexibility in the context of the full 78S, as they
appear more scant compared to the SSU-only reconstruction, along with the ESs that they interact
with. Consequently, we have focused our structural analysis of these SSU-rPPRs and their associated
ESs on the SSU-only reconstruction.
Our cryo-EM reconstructions at near-atomic resolutions, along with our previous extensive
MS/MS analysis6, allowed us to build a near-complete atomic models of the 78S mitoribosome. In
contrast to its mammalian10,13 and trypanosoma8 counterparts, the plant mitoribosome is characterized
by its largely expanded rRNAs, completely reshaping the overall structure of this mitoribosome. The
26S, 18S and 5S rRNAs are respectively 3,169, 1,935 and 118 nucleotides (nt) long, thus making the
plant mitochondria SSU and LSU rRNAs 20% and 9% larger than their prokaryote counterparts,
respectively6 (Fig. 1h-i). Nevertheless, while plant mitoribosomes contain more rRNA in general as
compared to bacteria, they have lost a few rRNA helices present in bacteria (Fig. 1j-k).
As for the ribosomal proteins (45 in the LSU, 37 in the SSU), most of them are either
universally conserved or mitochondria-specific constituting the common protein-core of almost all the
known mitoribosomes, e.g. mS23, mS26 and mS29 on the SSU or mL46 and mL59/64 on the central
protuberance of the LSU, thus confirming an acquisition of these proteins early during eukaryotes
evolution.
The structure of the SSU revealed the exact nature of its several specific features, namely its
large and elongated head additional domain, the body protuberance and its elongated foot (Fig. 1).
The body protuberance is mainly formed by the mitoribosome-specific r-protein mS47, shared with
yeast7 and trypanosoma8. Interestingly, mS47 is only absent in mammals, suggesting a loss of this
protein during animal evolution. The body protuberance also contains one additional proteins (mS45)
and extensions of uS4m (Extended Data Fig. 7), as well as additional protein densities. The foot of the
small subunit is mainly reshaped by rRNA ESs and deletions, stabilized by plant-specific r-proteins,
namely ribosomal PPR (rPPR) proteins. The SSU-characteristic helix 44 is 47 nt longer, thus slightly
extending the SSU and forming part of the foot extension (Figs. 2 and 3e). This extension is stabilized
by a rPPR protein itself connected to ES-h6 forming a three-way junction also stabilized by an
additional rPPR protein. We could not identify these rPPRs with certainty based on their sequence
because of the lack of resolution at these regions, however they can only correspond to rPPR1, 3a or
3b, identified in our previous work6, based on their number of repeats, they are here referred as
rPPR*. In contrast, h8, h10 and h17 are reduced compared to their bacterial counterpart (Fig. 1j),
leaving spaces for the plant specific additional proteins.
Our analysis of the large SSU head extension revealed that it is indeed primarily shaped by a
370nt rRNA novel domain inserted in h39. Due to its high flexibility, it was refined with an average
resolution around 10Å. Indeed, due to its movement relative to the head, but also the head movement
relative to the body, the overall movement of the head extension is of large amplitude (~30°)(Extended
Data Fig. 2), impairing the local resolution of this area. Nevertheless, the composition of the head
extension can be determined unambiguously, as our data identifies secondary structure elements for
both rRNA and rPPRs (Fig. 3c). It is mainly composed of rRNA, rooting from h39. From there, the
extension forms a four-ways junction stabilized by a long PPR protein (rPPR6 or mS80), locking the
whole additional domain in a position perpendicular to the intersubunit side. Past the four-ways
junction, two of the rRNAs helices organize into two parallel segments, forming the core of the
extension - one of the two helices ends in a three-way junction shaping the tip of the head-extension.
The two parallel RNA helices are themselves contacted by a rPPR (Fig. 3c). However, local resolution
is too low to clearly determine its exact identity (rPPR*). Interestingly, the protein bTHXm, previously
identified by mass-spectrometry6 was found buried deep inside the small subunit head. This protein is
only found in the plant mitoribosome as well as in chlororibosomes4,5 and ribosomes from the Thermus
genus14 .
On the back of the SSU, a large cleft extends the exit of the mRNA channel. This cleft is
delimited by mS26, uS8m and h26 on one side and by mS47 and the rPPR mS83 on the other side
(Fig. 4a). Similarly to all known PPRs, mS83 is predicted to be an RNA binder. In plants, the
processes underlying the recruitment and correct positioning of mRNAs during translation initiation is
unknown. Similar to other known mitochondrial translation systems, the Shine-Dalgarno (SD) and the
anti-Shine-Dalgarno sequences are absent from both plant mRNAs and SSU rRNAs. Moreover,
mRNAs have long 5’ untranslated regions (UTRs), similarly to yeast mitochondrial mRNAs7.
Interestingly, half of the plant mitochondrial mRNA 5’UTRs harbors an A/purine rich sequence AxAAA
located about 19nt upstream of the AUG (Extended Data Fig. 8). This distance correlates with the size
of the extended mRNA exit channel and would put the purine-rich sequence in close vicinity to the
rPPR protein mS83. We thus hypothesize that this plant-specific cleft may act as a recruitment
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platform for incoming mRNAs and / or additional factors. mS83 might recognize the AxAAA motif, thus
recapitulating a SD/antiSD-like recognition system, using an RNA-protein interaction instead of an
RNA-RNA interaction (Fig. 4a). An example of such possible rPPR-mediated initiation system may be
found in mammalian mitochondria where the rPPR mS39 located on the SSU was proposed to
accommodate the 5’UTR-less mRNAs from their 3’ end through a U-rich motif15. It is important to note
that this A-rich cis-element is not found in all plant mitochondrial mRNAs, thus suggesting that this
proposed mechanism for translation initiation would not be universal in plant mitochondria. Likewise, in
chloroplasts, a third of the mRNAs do not possess SD-sequence, suggesting that at least two different
mechanisms co-exist for translation initiation16.
Similar to the SSU, the overall shape of the LSU is also strongly remodeled, even though a
large portion of the core components are conserved (Fig. 2). Indeed, core functional components,
such as the peptidyl-transferase center (PTC), the L7/L12 stalk and the central protuberance (CP) are
similar to those found in bacteria. This conservation is in line with the globally bacterial-like
intersubunit bridges (Fig. 2b). However, the LSU is reshaped by the conserved mitochondria-specific
proteins e.g. mL41 or mL59/mL64 connecting the body of the LSU to the CP. Unlike all other
mitoribosomes described to date, the plant mitoribosome CP includes a 5S rRNA. The core structure
of the CP is quite conserved compared to prokaryotes. However, mitochondria-specific ribosomal
proteins, namely mL40, mL59/64 and mL46, complement the classical bacterial-like CP and bind on
top of the r-proteins bL27m, uL5m and uL18m. Interestingly, the same mitochondria-specific ribosomal
proteins forming part of the plant CP are also present in other mitoribosomes even though they have
lost their 5S rRNA, indicating that the acquisition of these mitoribosome-specific proteins occurred
prior to the loss of the 5S in other mitoribosomes (Extended Data Fig. 5).
Interestingly, the universally conserved uL2 is split in two parts in the plant mitoribosome, its
N-terminal part is encoded by a mitochondria encoded gene whereas its C-terminal part is encoded by
a nucleus encoded gene, which could constitute a way of mito-nuclear crosstalk (Extended Data Fig.
7). In yeast7, mammals10,13 and trypanosoma8 mitoribosomes, the peptide exit channel is highly
remodeled by species-specific proteins (e.g mL45 or mL71). In plants however, the major part of the
peptide channel and its exit are rather bacterial-like, with only minimal rearrangement of the
surrounding rRNAs helices and a small extension of uL29m (Fig. 4b). In contrast to human and
yeast17, a significant portion of the mitochondria encoded proteins are soluble proteins in plants (e.g. 7
soluble r-proteins are mitochondria encoded in Arabidopsis), thus it is conceivable that the plant
mitoribosome does not systematically requires an association with the inner mitochondrial membrane,
as it is the case for human and yeast18,19. However, it is likely that, at least in some cases, a non-
ribosomal protein could link the mitoribosome to the mitochondrial insertase Oxa1. This hypothesis is
supported by the observation that Oxa1 copurifies with immuno-precipitated plant mitoribosomes6(Fig.
4b). The main plant-specific features of the LSU are the plant-specific proteins and rRNA ESs
completely reshaping the back of the LSU, below the L1-stalk region. Indeed, running along the back
of the LSU, from uL15m and H31 and contacting the largely extended and remodeled domain I rRNAs
(ES-H21-22 and ES-H16-18), the 19-repeats rPPR protein mL102 (rPPR5) stabilizes these additional
rRNAs extensions (Fig. 3d). Moreover, the domain III is extensively remodeled and holds several
expansion segments, therefore helices of this domain were renamed pH53-59 (Fig. 1h, Extended Data
Fig. 4). Indeed, this remodeled domain III has two main helices that largely extend in the solvent and
are stabilized by two PPR proteins (rPPR4 and 9 or mL101 and mL104) (Fig 3a-b). These two rPPRs
appear rigid and present numerous interactions with the rRNA. Thus, rPPR9 encapsulates the tip of
helix H10, and stabilizes the end of pH59, one the newly formed helices of domain III. rPPR9 also
directly contacts rPPR4 that wraps around a single stranded rRNA extension (1645-1644nt) and
contacts the two major helices of domain III pH55 and pH57 (Fig. 3b). Interestingly, the rPPRs
described here, along with those of the SSU, seem to hold a different mode of RNA binding as
compared to the RNA recognition process of canonical PPR proteins. Indeed, PPR proteins usually
bind ssRNA through a combinatorial recognition mechanism mainly involving two specific residues in
each repeat (5 and 35) allowing each repeat to bind a specific nucleotide20, similar to other helical
repeat modular proteins21. However, the structure obtained here revealed that several rPPR proteins
bind the convex surface of double stranded rRNA, mainly through positively charged residues
contacting the phosphate backbone of the rRNAs. This novel mode of RNA binding evidenced for
rPPR proteins (Fig. 3) extends our understanding of the diversity of functions and modes of action held
by PPR proteins. The position of this remodeled domain III on the full 78S suggests that rPPRs 4 and
9 might be involved in the attachment to the inner mitochondrial membrane, as they appear to strongly
stabilize the structure of the whole domain (Fig. 4b).
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In conclusion, the plant mitoribosome with its large rRNA ESs illustrates yet another route
taken during mitoribosome evolution. It represents an augmented prokaryote-type ribosome. Based on
a bacterial scaffold, it has both expanded rRNAs and an expanded set of proteins that were
specifically recruited during eukaryote evolution in the plant clade. The structure of the plant
mitoribosome could reflect the so-called “constructive phase” of mitoribosome evolution where both
rRNAs and protein sets were augmented, in strong contrast with mammals and trypanosoma where
rRNAs were considerably reduced3 (Fig. 4c). The structure presented here thus provides further
insights into the evolution of mitoribosomes and the elaboration of independent new strategies to
perform and regulate translation.
METHODS
Methods and any associated references are available in the online version of the paper.
ACKNOWLEDGEMENTS
This work has benefitted from the facilities and expertise of the Biophysical and Structural Chemistry
platform (BPCS) at IECB, CNRS UMS3033, Inserm US001, University of Bordeaux. We thank A.
Bezault for assistance with the Talos Arctica electron microscope. We thank L. Kuhn , J. Chicher and
P. Hamman of the Strasbourg Espanade proteomic analysis for the proteomic analysis. We thank M.
Sissler for her useful comments during the article redaction.
This work was supported by the “Centre National de la Recherche Scientifique”, the University of
Strasbourg, by Agence Nationale de la Recherche (ANR) grants [MITRA, ANR-16-CE11-0024-02]] to
PG and YH and by the LabEx consortium “MitoCross” in the frame of the French National Program
“Investissement d’Avenir” [ANR-11-LABX-0057_MITOCROSS, as well as by a European Research
Council Starting Grant (TransTryp ID:759120) to YH.
DATA AVAILABILITY
The cryo-EM maps of the mitoribosome have been deposited at the Electron Microscopy Data Bank
(EMDB) with accession codes XXXX. Corresponding atomic models have been deposited in the
Protein Data Bank (PDB) with PDB codes XXXX.
AUTHOR CONTRIBUTIONS
PG, FW, and YH designed and coordinated the experiments. FW purified the mitochondria and
mitochondrial ribosomes. HS acquired the cryo-EM data. HS and YH processed the cryo-EM results.
HS, AB and FW built the atomic models. FW, HS and YH interpreted the structure. PG, FW, HS and
YH wrote and edited the manuscript.
COMPETING FINANCIAL INTERESTS
The authors declare no competing financial interests.
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head
90°
head
h44
90°
beak side platform sideintersubunit side
beak
body
protuberance
foot extension
head extension head extension
h44
solvent side
h44
head
beak
90°
SSU rRNA
SSU rp
LSU rRNA
LSU rp
rPPRs
SSU rRNA
SSU rp
LSU rRNA
LSU rp
rPPRs
SSU rRNA
SSU rp
rPPRs
SSU rRNA
SSU rp
rPPRs
90° 90°
beak side platform sideLSU side
head
head
h44
head
h44
CP
P-stalk
L1-stalk CP
L1-stalk
CP
ES-h39
ES-h33
ES-h16
ES-h6
ES-h44
Domain III ES-H16
ES-H21-22
h33
h17
h10 h8
H1
H63
H9 H78
a b c d
e
fg
hi j k
head
h44
intersubunit side
beak
LSU side
CP
P-stalk
L1-stalk
head
h44
intersubunit side
beak
LSU side
CP
P-stalk
L1-stalk
Plant-specic rRNA extensions Plant-specic rRNA deletionsConserved rRNA
Fig1. Overall structure of the plant mitochondrial ribosome
Composite cryo-EM map of the SSU alone (a-d) and the complete mitoribosome (e-g). rRNAs are colored in light
blue (LSU) and yellow (SSU) and ribosomal proteins in blue (LSU) or orange (SSU). rPPR proteins are shown in
red. h-k Arabidopsis mitoribosome rRNAs compared to E.coli ribosome. hand jcomparison of the SSU, iand k
comparison of the LSU, extensions in Arabidopsis rRNAs are shown in green, reductions are shown in blue. CP
represents the central protuberance.
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180°
mS80 mS80
uS14m uS19m
uS13m
mS35
mS37
uS7m
uS11m
bS21m
mS26
uS8m
mS83
mS47
bS6m
bS18m
mS23
uS15m
uS17m
mS34
mS29 uS9m
mS31/mS46
uS3m
uS2m
uS10m
mS33
uS12m
uS4m
mS47
uS5m
rPPR*
rPPR*
mS41
bS16m
mS45
rPPR*
rPPR*
rPPR*
mL87
mL87
bL35m
uL6m
mL53
mL43
uL13m
uL22m
uL20m
uL30m
uL4m
bL21m
mL59/64 mL46
uL2m
bL9m
bL28m
mL102
uL4m
uL24m
mL41
uL29m
uL23m
mL101
mL104 bL17m
uL18m
mL60
mL102
uL25m
uL16m bL27m
uL15m
uL11m
uL5m
mL46
mL40
uL10m
mL101
uL14m
mL80
bL19m
mB8
mB3
B3m
B8m
B2am
mB3
B2a-bm
B3m
B8m
bacterial bridges
mitochondrial bridges
plant-mitochondrial bridges
bacterial r-prot
mitochondrial r-prot
plant-specic r-prot
bacterial r-prot
mitochondrial r-prot
plant-specic r-prot
B7am
B2bm
B7bm
B2cm
B5m
B4m
mB13
CP
L1 stalk
head
body
Intersubunit side SSU Intersubunit side LSU
a
b
Fig2. Atomic model of the plant mitoribosome
aThe overall model of the plant mitoribosome with individual proteins annotated. rRNAs are colored in blue (LSU)
and light brown (SSU) and ribosomal proteins in shades of blue and green (LSU) or shades of yellow and red
(SSU), according to their conservation. bIntersubunit interfaces with conserved and novel observed intersubunit
bridges highlighted. Bacterial ones are colored in purple, mitoribosomes ones in pink and plant ones in green.
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SSU & LSU rp
SSU rRNA
LSU rRNA
rPPRs
platform side
h44
h6
ES-h39
H10
H31
1913-1921nt
1635-1646nt
1862-1864nt
ES-h6
ES-H16
H16
ES-h44
CP
a b c
d
e
rPPR*
rPPR*
rPPR*
rPPR4
rPPR5
rPPR6
rPPR9
uL15m
Fig3. The plant mitoribosome PPR proteins
Overall view of the mitoribosome with PPR proteins highlighted in red, with zoomed-in view of each rPPR proteins
(a-e) and their rRNA target and mode of binding. In aand drPPR4 and rPPR5 stabilize single stranded segments,
whereas in b,cand erPPR proteins mainly interact with the backbone of the rRNAs.
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uL4m
uL22m
uL23m
uL29m
uL24m
PTC
Channel exit
OM
IM
a b
mS47
mS26
uS8m
h26
bS18m
mS83
mS23
SSU
LSU
LSU
SSU
r-prot
rRNA
Ancestral
alpha-proteobacterial
ribosome
LSU
SSU
LSU
SSU
LSU
SSU
LSU
SSU
1:Constructive evolution of rRNAs and proteins 2:Destructive evolution of rRNAs and constructive of proteins
Plants Yeast KinetoplastidsMammals
c
Oxa1
Linker
Fig4. Specic features of the plant mitoribosome
aView of the SSU back channel and its proteins constituents. The possible mRNA path is highlighted by the
dashed blue line and the blue star indicated the position of the AxAAA motif present in the 5’UTR of half of plant
mitochondrial mRNAs. bHighlight of the plant mitoribosome peptide channel and possible mode of membrane
attachment of the plant mitoribosome. Similar to yeast with Mba122 plant mitoribosome would require an accessory
protein factor (green linker) to tether the mitoribosome to the Oxa1 insertase. The green line represents the
possible surface of interaction with the inner membrane (IM) of the mitochondria revealing an extensive surface
mainly formed by the remodeled Domain III of the LSU. cComparison of the dierent mitoribosomes described to
date. Schemes of the ancestral alpha-proteobacterial is represented compared to the actual mitoribosomes. Light
blue represent core bacterial rRNAs and yellow core bacterial r-proteins, blue and orange represent mitochondria
specic rRNAs and r-proteins acquisition. Two main dierent evolutive paths have been taken by mitoribosomes,
a mainly constructive one (1) represented by the yeast and plant mitoribosomes, where few proteins were lost and
novel r-proteins were acquired as long as extended rRNAs, whereas in human and trypanosoma (2) rRNAs were
highly reduced and more novel r-prot were acquired.
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ONLINE METHODS
Mitochondrial ribosome purification:
Cauliflower (Brassica oleracea var. botrytis) was used here as it is best suited for large scale
biochemical, structural analyses as compared to Arabidopsis. Cauliflower belongs to the same family of
plants as Arabidopsis, Brassicaceae, thus making it an optimal model for this study, allowing the
preparation of large quantities of highly pure mitochondria. However, the genome of Cauliflower is not
sequenced, and the closest fully sequenced member of the family (Brassica oleracea subsp. oleracea)
is poorly annotated. Still, protein sequence identities between members of the Brassicaceae family are
higher than 90%, thus facilitating proteomics identification of cauliflower proteins. Combining the
proteomics results from Waltz 2019 and those obtained in this study it is evident that no difference in
terms of protein composition could be observed between Cauliflower and Arabidopsis. Hence, to
facilitate comprehension and analysis we positioned Arabidopsis proteins in the cauliflower map.
For the mitochondria purification, fresh cauliflower inflorescence tissue was blended in extraction buffer
containing 0.3 M mannitol, 30 mM sodium pyrophosphate (10.H2O), 0.5 % BSA, 0.8 % (w/v)
polyvinylpyrrolidone-25, 2mM beta-mercaptoethanol, 1 mM EDTA, 20 mM ascorbate and 5 mM
cysteine, pH 7.5. Lysate was filtered and clarified by centrifugation at 1.500 g, 10 min at 4°C.
Supernatant was kept and centrifuged at 18.000 g, 15 min at 4°C. Organelle pellet was re-suspended
in wash buffer (0.3 M mannitol and 10 mM phosphate buffer, 1 mM EDTA, pH 7.5) and the precedent
centrifugations were repeated once. The resulting organelle pellet was re-suspended in wash buffer and
loaded on a single-step 30 % Percoll gradient (in wash buffer without EDTA) and run for 1h30 at 40.000
g. Mitochondria are were retrieved, washed two times before being flash frozen in liquid nitrogen.
For mitoribosome purification, mitochondria were re-suspended in Lysis buffer (20 mM HEPES-KOH,
pH 7.6, 100 mM KCl, 30 mM MgCl2, 1 mM DTT, 1.6 % Triton X-100, 100 µg/mL chloramphenicol,
supplemented with proteases inhibitors (Complete EDTA-free)) to a concentration of 1 mg/mL and
incubated for 15 min in 4°C. Lysate was clarified by centrifugation at 30.000 g, 20 min at 4°C. The
supernatant was loaded on a 40% sucrose cushion in Monosome buffer (same as lysis buffer without
Triton X-100 and 50 µg/mL chloramphenicol) and centrifuged at 235.000 g, 3h, 4°C. The crude
ribosomes pellet was re-suspended in Monosome buffer and loaded on a 10-30 % sucrose gradient in
the same buffer and run for 16 h at 65,000 g. Fractions corresponding to mitoribosomes were collected,
pelleted and re-suspended in Monosome buffer.
Grid preparation
4 µL of the samples at a concentration of 2 µg/µl was applied onto Quantifoil R2/2 300-mesh holey
carbon grid, which had been coated with thin home-made continuous carbon film and glow-discharged.
The sample was incubated on the grid for 30 sec and then blotted with filter paper for 2.5 sec in a
temperature and humidity controlled Vitrobot Mark IV (T = 4°C, humidity 100%, blot force 5) followed by
vitrification in liquid ethane pre-cooled by liquid nitrogen.
Single particle cryo-electron microscopy data collection
For the two data-sets (Full and dissociated complexes), data collection was performed on a Talos Artica
instrument (FEI Company) at 200 kV using the EPU software (FEI Company) for automated data
acquisition. Data were collected at a nominal underfocus of -0.5 to -2.7 µm at a magnification of 120,000
X yielding a pixel size of 1.21 Å. Micrographs were recorded as movie stack on a Falcon II direct electron
detector (FEI Compagny), each movie stack were fractionated into 20 frames for a total exposure of 1
sec corresponding to an electron dose of 60 ē/Å2.
Electron microscopy image processing
Drift and gain correction and dose weighting were performed using MotionCor225. A dose weighted
average image of the whole stack was used to determine the contrast transfer function with the software
Gctf26. The following process has been achieved using RELION 3.027. Particles were picked using a
Laplacian of gaussian function (min diameter 260 Å, max diameter 460 Å). For the full mitoribosome,
after 2D classification, 153,608 particles were extracted with a box size of 400 pixels and binned four
fold for 3D classification into 10 classes. Four classes depicting high-resolution features have been
selected for refinement. The complex has been focused refined with a mask on the LSU, the body and
the head of the SSU, yielding respectively 3.50, 3.66 and 3.74 Å resolution. Determination of the local
resolution of the final density map was performed using ResMap28.
.CC-BY-NC-ND 4.0 International licenseauthor/funder. It is made available under a
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For the dissociated subunits, following a 2D classification, 132,130 particles for the LSU and 120,350
particles for the SSU were extracted with a box size of 400 pixels and binned four fold for 3D
classification into 8 classes for each subunits. Five subclasses depicting high-resolution features have
been selected for the SSU refinement with 73,670 particles. After Bayesian polishing a multi-body
refinement has been performed using mask on the body, the head and the RNA expansion on the head,
yielding respectively 3.77, 3.9 and 10.5 Å resolution. Three classes have been selected for the LSU
refinement yielding a resolution of 3.96 Å. Determination of the local resolution of the final density map
was performed using ResMap28.
Structure building and model refinement
The atomic model of the plant mitoribosome was built into the high-resolution maps using Coot, Phoenix
and Chimera. Atomic models from E.coli ribosome (5kcr)29, yeast mitoribosome (5mrc)7, human
mitoribosome (6gaw)15 and trypanosoma mitoribosome (6hiv)8 were used as starting points for protein
identification and modelisation. The online SWISS-MODEL service was used to generate initial models
for bacterial and mitochondria conserved r-proteins. Models were then rigid body fitted to the density in
Chimera30 and all subsequent modeling was done in Coot31.
For the LSU and SSU ribosomal RNA, the 16S and 23S from E.coli were docked into the maps and
used as templates from positioning and reconstruction. A multiple sequence alignment of several plant
mitochondrial ribosomes and E.coli ribosome was performed in order to determine the additional or
depleted domains of A.thaliana mitochondrial ribosome. Ponctual differences were done in Chimera
using the “swapna” command line.
In order to build the additional rRNA domains of A.thaliana, the co-variation algorithm LocARNA
webservice (http://rna.informatik.uni-freiburg.de) was used to determine secondary structure of these
domains. At that point, the secondary structure prediction was used to build the 3D model in Chimera
using the “build structure” tools followed by manual adjustments.
Proteins with clear homologs in either mammalian or yeast mitoribosomes as long as E.coli ribosome
were build using Phyre2 and SWISS-Model.
The global atomic model was refined with VMD using the Molecular Dynamic Flexible Fitting (MDFF)
then with PHENIX using a combination of real and reciprocal space refinement for proteins and
ERRASER for RNA.
Proteomic and statistical analyses of mitochondrial ribosome composition
Mass spectrometry analyses of the ribosome fractions were performed at the Strasbourg-Esplanade
proteomic platform and performed as previously6. In brief, proteins were trypsin digested, mass
spectrometry analyses and quantitative proteomics were carried out by nano LC-ESI-MS/MS analysis
on AB Sciex TripleTOF mass spectrometers and quantitative label-free analysis was performed through
in-house bioinformatics pipelines.
Figure preparation
Figures featuring cryo-EM densities as well as atomic models were visualized with UCSF ChimeraX32.
.CC-BY-NC-ND 4.0 International licenseauthor/funder. It is made available under a
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c
3D classication
65,280 particles
3D classication
61,900 particles
3D classication
73,670 particles
3D Renement
3D Renement
LSU 3D Renement
3.86Å
3.50Å
3.74Å 3.66Å
3.96Å
4.36Å
3.77Å
3.9Å
10.57Å
120,350
particles
132,130
particles
107,710
particles
2D Classication 2D Classication
DATA-SET 1
Full
DATA-SET 2
Dissociated
Focused
renement
Multi-body
renement
Full SSU
a
b
8Å7
6
54
3
8Å7
6
54
3
0.00 0.05 0.10 0.15 0.20 0.25 0.30 0.35 0.40 0.45
-0.2
0.0
0.2
0.4
0.6
0.8
1.0
resolution (1/A)
Fourier ShellCorrelation
Full mitoribosome(3.86Å)
Focused SSU body(3.66Å)
Focused SSU head (3.74Å)
Focused LSU (3.50Å)
0.00 0.05 0.10 0.15 0.20 0.25 0.30 0.35 0.40 0.45
-0.2
0.0
0.2
0.4
0.6
0.8
1.0
resolution (1/A)
Fourier ShellCorrelation
SSU beforemultibody (4.36Å)
SSU body (3.77Å)
SSU head (3.9Å)
SSU head extension (10.57Å)
Extended Data Fig. 1 Data processing workow
Graphical summary of the processing workow described in Methods, with 2D classes presented in afor both
datasets and 3D processing, presented in b, with ResMap of the full mitoribosome and SSU only before further
processing. cFSC curves of the full mitoribosome and SSU before and after focused classication and multibody
renement.
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30 °
ab
head
body
head
head extension
body
90°
Extended Data Fig. 2 Multibody renement, additional SSU head domain movement amplitude
aViews of the two extreme states of the head and head extension, relative to the body of the SSU, calculated
using the multibody renement implemented in RELION312, showing the movement in two dierent planes. bAll
ten states reveals a movement amplitude of 30°.
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1010 1560
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Extended Data Fig. 3 Secondary structure diagram of the plant 18S rRNA
2D representation of the 18S rRNA colored by domain. The rRNA expansions specic to the plant mitoribosome
are highlighted in cyan. Extensions that could not be modelled are indicated by dashed lines. A simplied
secondary structure diagram of the E. coli 16S rRNA is also shown in the black frame, helices not present in the
plant mitoribosome are shown in gray. Secondary structure templates were obtained from the RiboVision suite
(http://apollo.chemistry.gatech.edu/RiboVision)
.CC-BY-NC-ND 4.0 International licenseauthor/funder. It is made available under a
The copyright holder for this preprint (which was not peer-reviewed) is the. https://doi.org/10.1101/777342doi: bioRxiv preprint

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