Publications

2021

Yuzuru Itoh Y, Juni Andrell, Austin Choi, Uwe Richter, Priyanka Maiti, Robert Best, Antonio Barrientos, Brendan Battersby, Alexey Amunts
Mechanism of membrane-tethered mitochondrial protein synthesis
Science, 371, 846-849
Abstract
Mitochondrial ribosomes (mitoribosomes) are tethered to the mitochondrial inner membrane to facilitate the cotranslational membrane insertion of the synthesized proteins. We report cryo–electron microscopy structures of human mitoribosomes with nascent polypeptide, bound to the insertase oxidase assembly 1–like (OXA1L) through three distinct contact sites. OXA1L binding is correlated with a series of conformational changes in the mitoribosomal large subunit that catalyze the delivery of newly synthesized polypeptides. The mechanism relies on the folding of mL45 inside the exit tunnel, forming two specific constriction sites that would limit helix formation of the nascent chain. A gap is formed between the exit and the membrane, making the newly synthesized proteins accessible. Our data elucidate the basis by which mitoribosomes interact with the OXA1L insertase to couple protein synthesis and membrane delivery.

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Victor Tobiasson, Ondrej Gahura, Shintaro Aibara, Rozbeh Baradaran, Alena Zíková, Alexey Amunts
Interconnected assembly factors regulate the biogenesis of mitoribosomal large subunit
The EMBO J, e106292
Abstract
Mitoribosomes consist of ribosomal RNA and protein components, coordinated assembly of which is critical for function. We used mitoribosomes from Trypanosoma brucei with reduced RNA and increased protein mass to provide insights into the biogenesis of the mitoribosomal large subunit. Structural characterization of a stable assembly intermediate revealed 22 assembly factors, some of which have orthologues/counterparts/homologues in mammalian genomes. These assembly factors form a protein network that spans a distance of 180 Å, shielding the ribosomal RNA surface. The central protuberance and L7/L12 stalk are not assembled entirely and require removal of assembly factors and remodeling of the mitoribosomal proteins to become functional. The conserved proteins GTPBP7 and mt‐EngA are bound together at the subunit interface in proximity to the peptidyl transferase center. A mitochondrial acyl‐carrier protein plays a role in docking the L1 stalk, which needs to be repositioned during maturation. Additional enzymatically deactivated factors scaffold the assembly while the exit tunnel is blocked. Together, this extensive network of accessory factors stabilizes the immature sites and connects the functionally important regions of the mitoribosomal large subunit.

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Alexander Mühleip, Rasmus Kock Flygaard, Jana Ovciarikova, Alice Lacombe, Paula Fernandes, Lilach Sheiner, Alexey Amunts
ATP synthase hexamer assemblies shape cristae of Toxoplasma mitochondria
Nature Communications, 12, 120
Abstract
Mitochondrial ATP synthase plays a key role in inducing membrane curvature to establish cristae. In Apicomplexa causing diseases such as malaria and toxoplasmosis, an unusual cristae morphology has been observed, but its structural basis is unknown. Here, we report that the apicomplexan ATP synthase assembles into cyclic hexamers, essential to shape their distinct cristae. Cryo-EM was used to determine the structure of the hexamer, which is held together by interactions between parasite-specific subunits in the lumenal region. Overall, we identified 17 apicomplexan-specific subunits, and a minimal and nuclear-encoded subunit-a. The hexamer consists of three dimers with an extensive dimer interface that includes bound cardiolipins and the inhibitor IF1. Cryo-ET and subtomogram averaging revealed that hexamers arrange into ~20-megadalton pentagonal pyramids in the curved apical membrane regions. Knockout of the linker protein ATPTG11 resulted in the loss of pentagonal pyramids with concomitant aberrantly shaped cristae. Together, this demonstrates that the unique macromolecular arrangement is critical for the maintenance of cristae morphology in Apicomplexa.

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2020

Rasmus Kock Flygaard, Alexander Mühleip, Victor Tobiasson, Alexey Amunts
Type III ATP synthase is a symmetry-deviated dimer that induces membrane curvature through tetramerization
Nature Communications, 11, 5342
Abstract
Mitochondrial ATP synthases form functional homodimers to induce cristae curvature that is a universal property of mitochondria. To expand on the understanding of this fundamental phenomenon, we characterized the unique type III mitochondrial ATP synthase in its dimeric and tetrameric form. The cryo-EM structure of a ciliate ATP synthase dimer reveals an unusual U-shaped assembly of 81 proteins, including a substoichiometrically bound ATPTT2, 40 lipids, and co-factors NAD and CoQ. A single copy of subunit ATPTT2 functions as a membrane anchor for the dimeric inhibitor IF1. Type III specific linker proteins stably tie the ATP synthase monomers in parallel to each other. The intricate dimer architecture is scaffolded by an extended subunit-a that provides a template for both intra- and inter-dimer interactions. The latter results in the formation of tetramer assemblies, the membrane part of which we determined to 3.1 Å resolution. The structure of the type III ATP synthase tetramer and its associated lipids suggests that it is the intact unit propagating the membrane curvature.

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Yuzuru Itoh, Andreas Naschberger, Narges Mortezaei, Johannes M. Herrmann, Alexey Amunts
Analysis of translating mitoribosome reveals functional characteristics of translation in mitochondria of fungi
Nature Communications, 11, 5187
Abstract
Mitoribosomes are specialized protein synthesis machineries in mitochondria. However, how mRNA binds to its dedicated channel, and tRNA moves as the mitoribosomal subunit rotate with respect to each other is not understood. We report models of the translating fungal mitoribosome with mRNA, tRNA and nascent polypeptide, as well as an assembly intermediate. Nicotinamide adenine dinucleotide (NAD) is found in the central protuberance of the large subunit, and the ATPase inhibitory factor 1 (IF1) in the small subunit. The models of the active mitoribosome explain how mRNA binds through a dedicated protein platform on the small subunit, tRNA is translocated with the help of the protein mL108, bridging it with L1 stalk on the large subunit, and nascent polypeptide paths through a newly shaped exit tunnel involving a series of structural rearrangements. An assembly intermediate is modeled with the maturation factor Atp25, providing insight into the biogenesis of the mitoribosomal large subunit and translation regulation.

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Shintaro Aibara, Vivek Singh, Angelika Modelska, Alexey Amunts
Structural basis of mitochondrial translation
eLife, 9 , e58362
Abstract
Translation of mitochondrial messenger RNA (mt-mRNA) is performed by distinct mitoribosomes comprising at least 36 mitochondria-specific proteins. How these mitoribosomal proteins assist in the binding of mt-mRNA and to what extent they are involved in the translocation of transfer RNA (mt-tRNA) is unclear. To visualize the process of translation in human mitochondria, we report ~3.0 Å resolution structure of the human mitoribosome, including the L7/L12 stalk, and eight structures of its functional complexes with mt-mRNA, mt-tRNAs, recycling factor and additional trans factors. The study reveals a transacting protein module LRPPRC-SLIRP that delivers mt-mRNA to the mitoribosomal small subunit through a dedicated platform formed by the mitochondria-specific protein mS39. Mitoribosomal proteins of the large subunit mL40, mL48, and mL64 coordinate translocation of mt-tRNA. The comparison between those structures shows dynamic interactions between the mitoribosome and its ligands, suggesting a sequential mechanism of conformational changes.

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Victor Tobiasson, Alexey Amunts
Ciliate mitoribosome illuminates evolutionary steps of mitochondrial translation
eLife, 9 , e59264
Abstract
To understand the steps involved in the evolution of translation, we used Tetrahymena thermophila, a ciliate with high coding capacity of the mitochondrial genome, as the model organism and characterized its mitochondrial ribosome (mitoribosome) using cryo-EM. The structure of the mitoribosome reveals an assembly of 94-ribosomal proteins and four-rRNAs with an additional protein mass of ~700 kDa on the small subunit, while the large subunit lacks 5S rRNA. The structure also shows that the small subunit head is constrained, tRNA binding sites are formed by mitochondria-specific protein elements, conserved protein bS1 is excluded, and bacterial RNA polymerase binding site is blocked. We provide evidence for anintrinsic protein targeting system through visualization of mitochondria-specific mL105 by the exit tunnel that would facilitate the recruitment of a nascent polypeptide. Functional protein uS3m is encoded by three complementary genes from the nucleus and mitochondrion, establishing a link between genetic drift and mitochondrial translation. Finally, we reannotated nine open reading frames in the mitochondrial genome that code for mitoribosomal proteins.

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Anas Khawaja, Yuzuru Itoh, Cristina Remes, Henrik Spåhr, Olessya Yukhnovets, Henning Höfig, Alexey Amunts, Joanna Rorbach
Distinct pre-initiation steps in human mitochondrial translation
Nature Communications, 11 (1), pp. 1-10
Abstract
Translation initiation in human mitochondria relies upon specialized mitoribosomes and initiation factors, mtIF2 and mtIF3, which have diverged from their bacterial counterparts. Here we report two distinct mitochondrial pre-initiation assembly steps involving those factors. Single-particle cryo-EM revealed that in the first step, interactions between mitochondria-specific protein mS37 and mtIF3 keep the small mitoribosomal subunit in a conformation favorable for a subsequent accommodation of mtIF2 in the second step. Combination with fluorescence cross-correlation spectroscopy analyses suggests that mtIF3 promotes complex assembly without mRNA or initiator tRNA binding, where exclusion is achieved by the N-terminal and C-terminal domains of mtIF3. Finally, the association of large mitoribosomal subunit is required for initiator tRNA and leaderless mRNA recruitment to form a stable initiation complex. These data reveal fundamental aspects of mammalian protein synthesis that are specific to mitochondria.

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Ming Chen, Annemarie Perez-Boerema, Laixing Zhang, Yanxue Li, Maojun Yang, Shizhong Li, Alexey Amunts
Distinct structural modulation of photosystem I and lipid environment stabilizes its tetrameric assembly
Nature Plants, 6 (3), pp. 314-320
Abstract
Photosystem I (PSI) is able to form different oligomeric states across various species. To reveal the structural basis for PSI dimerization and tetramerization, we structurally investigated PSI from the cyanobacterium Anabaena. This revealed a disrupted trimerization domain due to lack of the terminal residues of PsaL in the lumen, which resulted in PSI dimers with loose connections between monomers and weaker energy-coupled chlorophylls than in the trimer. At the dimer surface, specific phospholipids, cofactors and interactions in combination facilitated recruitment of another dimer to form a tetramer. Taken together, the relaxed luminal connections and lipid specificity at the dimer interface account for membrane curvature. PSI tetramer assembly appears to increase the surface area of the thylakoid membrane, which would contribute to PSI crowding.

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Annemarie Perez-Boerema, Daniel Klaiman, Ido Caspy, Sigal Y Netzer-El, Alexey Amunts, Nathan Nelson
Structure of a minimal photosystem I from the green alga Dunaliella salina
Nature Plants, 6 (3), pp. 321-327
Abstract
Solar energy harnessed by oxygenic photosynthesis supports most of the life forms on Earth. In eukaryotes, photosynthesis occurs in chloroplasts and is achieved by membrane-embedded macromolecular complexes that contain core and peripheral antennae with multiple pigments. The structure of photosystem I (PSI) comprises the core and light-harvesting (LHCI) complexes, which together form PSI–LHCI. Here we determined the structure of PSI–LHCI from the salt-tolerant green alga Dunaliella salina using X-ray crystallography and electron cryo-microscopy. Our results reveal a previously undescribed configuration of the PSI core. It is composed of only 7 subunits, compared with 14–16 subunits in plants and the alga Chlamydomonas reinhardtii, and forms the smallest known PSI. The LHCI is poorly conserved at the sequence level and binds to pigments that form new energy pathways, and the interactions between the individual Lhca1–4 proteins are weakened. Overall, the data indicate the PSI of D. salina represents a different type of the molecular organization that provides important information for reconstructing the plasticity and evolution of PSI.

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2019

Alexander Mühleip, Sarah E McComas, Alexey Amunts
Structure of a mitochondrial ATP synthase with bound native cardiolipin
eLife, 8 , e51179
Abstract
The mitochondrial ATP synthase fuels eukaryotic cells with chemical energy. Here we report the cryo-EM structure of a divergent ATP synthase dimer from mitochondria of Euglena gracilis, a member of the phylum Euglenozoa that also includes human parasites. It features 29 different subunits, 8 of which are newly identified. The membrane region was determined to 2.8 Å resolution, enabling the identification of 37 associated lipids, including 25 cardiolipins, which provides insight into protein-lipid interactions and their functional roles. The rotor-stator interface comprises four membrane-embedded horizontal helices, including a distinct subunit a. The dimer interface is formed entirely by phylum-specific components, and a peripherally associated subcomplex contributes to the membrane curvature. The central and peripheral stalks directly interact with each other. Last, the ATPase inhibitory factor 1 (IF1) binds in a mode that is different from human, but conserved in Trypanosomatids.

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E. Bugiardini, A. Mitchell, I. Rosa, H. Horning-Do, A. Pitmann, O. Poole, J. Holton, S. Shah, C. Woodward, I. Hargreaves, R. Quinlivan, A. Amunts , R. Wiesner, H. Houlden, I. Holt, M. Hanna, R. Pitceathly, A. Spinazzola
MRPS25 mutations impair mitochondrial translation and cause encephalomyopathy
Hum Mol Genet. 28(16): 2711-2719
Abstract
Mitochondrial disorders are clinically and genetically heterogeneous and are associated with a variety of disease mechanisms. Defects of mitochondrial protein synthesis account for the largest subgroup of disorders manifesting with impaired respiratory chain capacity; yet, only a few have been linked to dysfunction in the protein components of the mitochondrial ribosomes. Here, we report a subject presenting with dyskinetic cerebral palsy and partial agenesis of the corpus callosum, while histochemical and biochemical analyses of skeletal muscle revealed signs of mitochondrial myopathy. Using exome sequencing, we identified a homozygous variant c.215C>T in MRPS25, which encodes for a structural component of the 28S small subunit of the mitochondrial ribosome (mS25). The variant segregated with the disease and substitutes a highly conserved proline residue with leucine (p.P72L) that, based on the high-resolution structure of the 28S ribosome, is predicted to compromise inter-protein contacts and destabilize the small subunit. Concordant with the in silico analysis, patient’s fibroblasts showed decreased levels of MRPS25 and other components of the 28S subunit. Moreover, assembled 28S subunits were scarce in the fibroblasts with mutant mS25 leading to impaired mitochondrial translation and decreased levels of multiple respiratory chain subunits. Crucially, these abnormalities were rescued by transgenic expression of wild-type MRPS25 in the mutant fibroblasts. Collectively, our data demonstrate the pathogenicity of the p.P72L variant and identify MRPS25 mutations as a new cause of mitochondrial translation defect.

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Neha Nirwan, Yuzuru Itoh, Pratima Singh, Sutirtha Bandyopadhyay, Kutti R. Vinothkumar, Alexey Amunts, Kayarat Saikrishnan
Structure-based mechanism for activation of the AAA+ GTPase McrB by the endonuclease McrC
Nature Communications, 10 (1), pp. 1-9
Abstract
The AAA+ GTPase McrB powers DNA cleavage by the endonuclease McrC. The GTPase itself is activated by McrC. The architecture of the GTPase and nuclease complex, and the mechanism of their activation remained unknown. Here, we report a 3.6 Å structure of a GTPase-active and DNA-binding deficient construct of McrBC. Two hexameric rings of McrB are bridged by McrC dimer. McrC interacts asymmetrically with McrB protomers and inserts a stalk into the pore of the ring, reminiscent of the γ subunit complexed to α3β3 of F1-ATPase. Activation of the GTPase involves conformational changes of residues essential for hydrolysis. Three consecutive nucleotide-binding pockets are occupied by the GTP analogue 5’-guanylyl imidodiphosphate and the next three by GDP, which is suggestive of sequential GTP hydrolysis.

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Janna M Bigalke, Shintaro Aibara, Robert Roth, Göran Dahl, Euan Gordon, Sarah Dorbéus, Alexey Amunts, Jenny Sandmark
Cryo-EM structure of the activated RET signaling complex reveals the importance of its cysteine-rich domain
Science Advances, 5 (7), eaau4202
Abstract
Signaling through the receptor tyrosine kinase RET is essential during normal development. Both gain- and loss-of-function mutations are involved in a variety of diseases, yet the molecular details of receptor activation have remained elusive. We have reconstituted the complete extracellular region of the RET signaling complex together with Neurturin (NRTN) and GFRα2 and determined its structure at 5.7-Å resolution by cryo-EM. The proteins form an assembly through RET-GFRα2 and RET-NRTN interfaces. Two key interaction points required for RET extracellular domain binding were observed: (i) the calcium-binding site in RET that contacts GFRα2 domain 3 and (ii) the RET cysteine-rich domain interaction with NRTN. The structure highlights the importance of the RET cysteine-rich domain and allows proposition of a model to explain how complex formation leads to RET receptor dimerization and its activation. This provides a framework for targeting RET activity and for further exploration of mechanisms underlying neurological diseases.

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Victor Tobiasson, Allexa Dow, Sladjana Prisic, Alexey Amunts
Zinc depletion does not necessarily induce ribosome hibernation in mycobacteria
Proceedings of the National Academy of Sciences, 116 (7), pp. 2395-2397
Abstract
In PNAS, Li et al. recently reported analysis of Mycobacterium smegmatis ribosomes formed under zinc-limited conditions. A zinc chelator [N,N,N′,N′-tetrakis(2-pyridylmethyl)-ethylenediamine] was added to growth medium and an extended purification procedure was employed to obtain zinc-depleted (C−) ribosomes for cryo-EM. The structure was solved at 3.5-Å resolution and showed five partially modeled C− ribosomal proteins in place of their zinc-binding (C+) paralogs. Additionally, a hibernation-promoting factor, MSMEG_1878 [designated by Li et al. as a mycobacterial protein Y (MPY)], associated with ribosomes at the coding region of the 30S subunit, resulting in ribosome inactivation and tolerance to aminoglycosides. While this work contributes toward understanding of ribosome hibernation in mycobacteria, we believe that differences in growth conditions used to stimulate production of C+ and C− ribosomes led the authors to erroneously conclude that MPY exclusively binds to C− ribosomes. Here, we present our data and refer to work by others to offer an alternative interpretation of the results.

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Anton S Petrov, Elizabeth C Wood, Chad R Bernier, Ashlyn M Norris, Alan Brown, Alexey Amunts
Structural patching fosters divergence of mitochondrial ribosomes
Molecular biology and evolution, 36 (2), pp. 207-219
Abstract
Mitochondrial ribosomes (mitoribosomes) are essential components of all mitochondria that synthesize proteins encoded by the mitochondrial genome. Unlike other ribosomes, mitoribosomes are highly variable across species. The basis for this diversity is not known. Here, we examine the composition and evolutionary history of mitoribosomes across the phylogenetic tree by combining three-dimensional structural information with a comparative analysis of the secondary structures of mitochondrial rRNAs (mt-rRNAs) and available proteomic data. We generate a map of the acquisition of structural variation and reconstruct the fundamental stages that shaped the evolution of the mitoribosomal large subunit and led to this diversity. Our analysis suggests a critical role for ablation and expansion of rapidly evolving mt-rRNA. These changes cause structural instabilities that are “patched” by the acquisition of pre-existing compensatory elements, thus providing opportunities for rapid evolution. This mechanism underlies the incorporation of mt-tRNA into the central protuberance of the mammalian mitoribosome, and the altered path of the polypeptide exit tunnel of the yeast mitoribosome. We propose that since the toolkits of elements utilized for structural patching differ between mitochondria of different species, it fosters the growing divergence of mitoribosomes.

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2018

Shintaro Aibara, Juni Andréll, Vivek Singh, Alexey Amunts
Rapid isolation of the mitoribosome from HEK cells
JoVE (Journal of Visualized Experiments), 140 , pp. e57877
Abstract
The human mitochondria possess a dedicated set of ribosomes (mitoribosomes) that translate 13 essential protein components of the oxidative phosphorylation complexes encoded by the mitochondrial genome. Since all proteins synthesized by human mitoribosomes are integral membrane proteins, human mitoribosomes are tethered to the mitochondrial inner membrane during translation. Compared to the cytosolic ribosome the mitoribosome has a sedimentation coefficient of 55S, half the rRNA content, no 5S rRNA and 36 additional proteins. Therefore, a higher protein-to-RNA ratio and an atypical structure make the human mitoribosome substantially distinct from its cytosolic counterpart. Despite the central importance of the mitoribosome to life, no protocols were available to purify the intact complex from human cell lines. Traditionally, mitoribosomes were isolated from mitochondria-rich animal tissues that required kilograms of starting material. We reasoned that mitochondria in dividing HEK293-derived human cells grown in nutrient-rich expression medium would have an active mitochondrial translation, and, therefore, could be a suitable source of material for the structural and biochemical studies of the mitoribosome. To investigate its structure, we developed a protocol for large-scale purification of intact mitoribosomes from HEK cells. Herein, we introduce nitrogen cavitation method as a faster, less labor-intensive and more efficient alternative to traditional mechanical shear-based methods for cell lysis. This resulted in preparations of the mitoribosome that allowed for its structural determination to high resolution, revealing the composition of the intact human mitoribosome and its assembly intermediates. Here, we follow up on this work and present an optimized and more cost-effective method requiring only ~1010 cultured HEK cells. The method can be employed to purify human mitoribosomal translating complexes, mutants, quality control assemblies and mitoribosomal subunits intermediates. The purification can be linearly scaled up tenfold if needed, and also applied to other types of cells.

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Annemarie Perez Boerema, Shintaro Aibara, Bijoya Paul, Victor Tobiasson, Dari Kimanius, Björn O Forsberg, Karin Wallden, Erik Lindahl, Alexey Amunts
Structure of the chloroplast ribosome with chl-RRF and hibernation-promoting factor
Nature Plants, 4 (4), pp. 212-217
Abstract
Oxygenic photosynthesis produces oxygen and builds a variety of organic compounds, changing the chemistry of the air, the sea and fuelling the food chain on our planet. The photochemical reactions underpinning this process in plants take place in the chloroplast. Chloroplasts evolved ~1.2 billion years ago from an engulfed primordial diazotrophic cyanobacterium, and chlororibosomes are responsible for synthesis of the core proteins driving photochemical reactions. Chlororibosomal activity is spatiotemporally coupled to the synthesis and incorporation of functionally essential co-factors, implying the presence of chloroplast-specific regulatory mechanisms and structural adaptation of the chlororibosome1,2. Despite recent structural information3,4,5,6, some of these aspects remained elusive. To provide new insights into the structural specialities and evolution, we report a comprehensive analysis of the 2.9–3.1 Å resolution electron cryo-microscopy structure of the spinach chlororibosome in complex with its recycling factor and hibernation-promoting factor. The model reveals a prominent channel extending from the exit tunnel to the chlororibosome exterior, structural re-arrangements that lead to increased surface area for translocon binding, and experimental evidence for parallel and convergent evolution of chloro- and mitoribosomes.

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2017

Joanna Rorbach, Shintaro Aibara, Alexey Amunts
Ribosome origami
Nature Structural & Molecular Biology, 24 (11), pp. 879-881
Abstract
Assembly of the small ribosomal subunit from an RNA strand and 33 proteins is an intricate and dynamic process. Two cryo-EM studies now provide insight into a complicated complex of at least 51 trans-factors that act on the preribosomal small subunit to sequentially fold it into a 3D molecular machine.

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Björn O. Forsberg, Shintaro Aibara, Dari Kimanius, Bijoya Paul, Erik Lindahl, Alexey Amunts
Cryo-EM reconstruction of the chlororibosome to 3.2 Å resolution within 24 h
IUCrJ, 4 (6), pp. 723-727
Abstract
The introduction of direct detectors and the automation of data collection in cryo-EM have led to a surge in data, creating new opportunities for advancing computational processing. In particular, on-the-fly workflows that connect data collection with three-dimensional reconstruction would be valuable for more efficient use of cryo-EM and its application as a sample-screening tool. Here, accelerated on-the-fly analysis is reported with optimized organization of the data-processing tools, image acquisition and particle alignment that make it possible to reconstruct the three-dimensional density of the 70S chlororibosome to 3.2 Å resolution within 24 h of tissue harvesting. It is also shown that it is possible to achieve even faster processing at comparable quality by imposing some limits to data use, as illustrated by a 3.7 Å resolution map that was obtained in only 80 min on a desktop computer. These on-the-fly methods can be employed as an assessment of data quality from small samples and extended to high-throughput approaches.

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Alan Brown, Sorbhi Rathore, Dari Kimanius, Shintaro Aibara, Xiao-chen Bai, Joanna Rorbach, Alexey Amunts, V. Ramakrishnan
Structures of the human mitochondrial ribosome in native states of assembly
Nature Structural & Molecular Biology, 24 (10), pp. 866–869
Abstract
Mammalian mitochondrial ribosomes (mitoribosomes) have less rRNA content and 36 additional proteins compared with the evolutionarily related bacterial ribosome. These differences make the assembly of mitoribosomes more complex than the assembly of bacterial ribosomes, but the molecular details of mitoribosomal biogenesis remain elusive. Here, we report the structures of two late-stage assembly intermediates of the human mitoribosomal large subunit (mt-LSU) isolated from a native pool within a human cell line and solved by cryo-EM to ∼3-Å resolution. Comparison of the structures reveals insights into the timing of rRNA folding and protein incorporation during the final steps of ribosomal maturation and the evolutionary adaptations that are required to preserve biogenesis after the structural diversification of mitoribosomes. Furthermore, the structures redefine the ribosome silencing factor (RsfS) family as multifunctional biogenesis factors and identify two new assembly factors (L0R8F8 and mt-ACP) not previously implicated in mitoribosomal biogenesis.

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Donna Matzov, Shintaro Aibara, Arnab Basu, Ella Zimmerman, Anat Bashan, Mee-Ngan F Yap, Alexey Amunts, Ada E Yonath
The cryo-EM Structure of Hibernating 100S Ribosome Dimer From Pathogenic Staphylococcus Aureus
Nature communications, 8 (1), pp. 1-7
Abstract
Formation of 100S ribosome dimer is generally associated with translation suppression in bacteria. Trans-acting factors ribosome modulation factor (RMF) and hibernating promoting factor (HPF) were shown to directly mediate this process in E. coli. Gram-positive S. aureus lacks an RMF homolog and the structural basis for its 100S formation was not known. Here we report the cryo-electron microscopy structure of the native 100S ribosome from S. aureus, revealing the molecular mechanism of its formation. The structure is distinct from previously reported analogs and relies on the HPF C-terminal extension forming the binding platform for the interactions between both of the small ribosomal subunits. The 100S dimer is formed through interactions between rRNA h26, h40, and protein uS2, involving conformational changes of the head as well as surface regions that could potentially prevent RNA polymerase from docking to the ribosome.

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Nirupa Desai, Alan Brown, Alexey Amunts, V. Ramakrishnan
The structure of the yeast mitochondrial ribosome
Science, 355 (6324), pp. 528-531
Abstract
Mitochondria have specialized ribosomes (mitoribosomes) dedicated to the expression of the genetic information encoded by their genomes. Here, using electron cryomicroscopy, we have determined the structure of the 75-component yeast mitoribosome to an overall resolution of 3.3 angstroms. The mitoribosomal small subunit has been built de novo and includes 15S ribosomal RNA (rRNA) and 34 proteins, including 14 without homologs in the evolutionarily related bacterial ribosome. Yeast-specific rRNA and protein elements, including the acquisition of a putatively active enzyme, give the mitoribosome a distinct architecture compared to the mammalian mitoribosome. At an expanded messenger RNA channel exit, there is a binding platform for translational activators that regulate translation in yeast but not mammalian mitochondria. The structure provides insights into the evolution and species-specific specialization of mitochondrial translation.

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2016

Martin Ott, Alexey Amunts, Alan Brown
Organization and regulation of mitochondrial protein synthesis
Annual Review of Biochemistry, 85
Abstract
Mitochondria are essential organelles of endosymbiotic origin that are responsible for oxidative phosphorylation within eukaryotic cells. Independent evolution between species has generated mitochondrial genomes that are extremely diverse, with the composition of the vestigial genome determining their translational requirements. Typically, translation within mitochondria is restricted to a few key subunits of the oxidative phosphorylation complexes that are synthesized by dedicated ribosomes (mitoribosomes). The dramatically rearranged mitochondrial genomes, the limited set of transcripts, and the need for the synthesized proteins to coassemble with nuclear-encoded subunits have had substantial consequences for the translation machinery. Recent high-resolution cryo-electron microscopy has revealed the effect of coevolution on the mitoribosome with the mitochondrial genome. In this review, we place the new structural information in the context of the molecular mechanisms of mitochondrial translation and focus on the novel ways protein synthesis is organized and regulated in mitochondria.

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2015

Dasmanthie De Silva, Ya-Ting Tu, Alexey Amunts, Flavia Fontanesi, Antoni Barrientos
Mitochondrial ribosome assembly in health and disease
Cell Cycle, 14 (14), pp. 2226-2250
Abstract
The ribosome is a structurally and functionally conserved macromolecular machine universally responsible for catalyzing protein synthesis. Within eukaryotic cells, mitochondria contain their own ribosomes (mitoribosomes), which synthesize a handful of proteins, all essential for the biogenesis of the oxidative phosphorylation system. High-resolution cryo-EM structures of the yeast, porcine and human mitoribosomal subunits and of the entire human mitoribosome have uncovered a wealth of new information to illustrate their evolutionary divergence from their bacterial ancestors and their adaptation to synthesis of highly hydrophobic membrane proteins. With such structural data becoming available, one of the most important remaining questions is that of the mitoribosome assembly pathway and factors involved. The regulation of mitoribosome biogenesis is paramount to mitochondrial respiration, and thus to cell viability, growth and differentiation. Moreover, mutations affecting the rRNA and protein components produce severe human mitochondrial disorders. Despite its biological and biomedical significance, knowledge on mitoribosome biogenesis and its deviations from the much-studied bacterial ribosome assembly processes is scarce, especially the order of rRNA processing and assembly events and the regulatory factors required to achieve fully functional particles. This article focuses on summarizing the current available information on mitoribosome assembly pathway, factors that form the mitoribosome assembly machinery, and the effect of defective mitoribosome assembly on human health.

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Alexey Amunts, Alan Brown, Jaan Toots, Sjors HW Scheres, V. Ramakrishnan
The structure of the human mitochondrial ribosome
Science, 348 (6230), pp. 95-98
Abstract
The highly divergent ribosomes of human mitochondria (mitoribosomes) synthesize 13 essential proteins of oxidative phosphorylation complexes. We have determined the structure of the intact mitoribosome to 3.5 angstrom resolution by means of single-particle electron cryogenic microscopy. It reveals 80 extensively interconnected proteins, 36 of which are specific to mitochondria, and three ribosomal RNA molecules. The head domain of the small subunit, particularly the messenger (mRNA) channel, is highly remodeled. Many intersubunit bridges are specific to the mitoribosome, which adopts conformations involving ratcheting or rolling of the small subunit that are distinct from those seen in bacteria or eukaryotes. An intrinsic guanosine triphosphatase mediates a contact between the head and central protuberance. The structure provides a reference for analysis of mutations that cause severe pathologies and for future drug design.

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Alexey Amunts, Karol Fiedorczuk, Thao T Truong, Josephine Chandler, E Peter Greenberg, V Ramakrishnan
Bactobolin A Binds to a Site on the 70S Ribosome Distinct from Previously Seen Antibiotics
Journal of Molecular Biology, 427 (4), pp. 753-755
Abstract
The ribosome is the target of a large number of antibiotics. Here, we report a 3.4-Å-resolution crystal structure of bactobolin A bound to 70S ribosome-tRNA complex. The antibiotic binds at a previously unseen site in the 50S subunit and displaces tRNA bound at the P-site. It thus likely has a similar mechanism of action as blasticidin S despite binding to a different site. The structure also rationalizes previously identified resistance mutations.

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2014

Alan Brown, Alexey Amunts, Xiao-chen Bai, Yoichiro Sugimoto, Patricia C Edwards, Garib Murshudov, Sjors HW Scheres, V. Ramakrishnan
Structure of the large ribosomal subunit from human mitochondria
Science, 346 (6210), pp. 718-722
Abstract
Human mitochondrial ribosomes are highly divergent from all other known ribosomes and are specialized to exclusively translate membrane proteins. They are linked with hereditary mitochondrial diseases and are often the unintended targets of various clinically useful antibiotics. Using single-particle cryogenic electron microscopy, we have determined the structure of its large subunit to 3.4 angstrom resolution, revealing 48 proteins, 21 of which are specific to mitochondria. The structure unveils an adaptation of the exit tunnel for hydrophobic nascent peptides, extensive remodeling of the central protuberance, including recruitment of mitochondrial valine transfer RNA (tRNAVal) to play an integral structural role, and changes in the tRNA binding sites related to the unusual characteristics of mitochondrial tRNAs.

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Alexey Amunts, Alan Brown, Xiao-chen Bai, Jose L Llácer, Tanweer Hussain, Paul Emsley, Fei Long, Garib Murshudov, Sjors HW Scheres, Venki Ramakrishnan
Structure of the yeast mitochondrial large ribosomal subunit
Science, 343 (6178), pp. 1485-1489
Abstract
Mitochondria have specialized ribosomes that have diverged from their bacterial and cytoplasmic counterparts. We have solved the structure of the yeast mitoribosomal large subunit using single-particle cryo–electron microscopy. The resolution of 3.2 angstroms enabled a nearly complete atomic model to be built de novo and refined, including 39 proteins, 13 of which are unique to mitochondria, as well as expansion segments of mitoribosomal RNA. The structure reveals a new exit tunnel path and architecture, unique elements of the E site, and a putative membrane docking site.

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2011

Nicoleta Herascu, Mehdi Najafi, Alexey Amunts, Jörg Pieper, Klaus-Dieter Irrgang, Rafael Picorel, Michael Seibert, Valter Zazubovich
Parameters of the protein energy landscapes of several light-harvesting complexes probed via spectral hole growth kinetics measurements
The Journal of Physical Chemistry B, 115 (12), pp. 2737-2747
Abstract
The parameters of barrier distributions on the protein energy landscape in the excited electronic state of the pigment/protein system have been determined by means of spectral hole burning for the lowest-energy pigments of CP43 core antenna complex and CP29 minor antenna complex of spinach Photosystem II (PS II) as well as of trimeric and monomeric LHCII complexes transiently associated with the pea Photosystem I (PS I) pool. All of these complexes exhibit sixty to several hundred times lower spectral hole burning yields as compared with molecular glassy solids previously probed by means of the hole growth kinetics measurements. Therefore, the entities (groups of atoms), which participate in conformational changes in protein, appear to be significantly larger and heavier than those in molecular glasses. No evidence of a small (∼1 cm−1) spectral shift tier of the spectral diffusion dynamics has been observed. Therefore, our data most likely reflect the true barrier distributions of the intact protein and not those related to the interface or surrounding host. Possible applications of the barrier distributions as well as the assignments of low-energy states of CP29 and LHCII are discussed in light of the above results.

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2010

Alexey Amunts, Hila Toporik, Anna Borovikova, Nathan Nelson
Structure determination and improved model of plant photosystem I
Journal of Biological Chemistry, 285 (5), pp. 3478-3486
Abstract
Photosystem I functions as a sunlight energy converter, catalyzing one of the initial steps in driving oxygenic photosynthesis in cyanobacteria, algae, and higher plants. Functionally, Photosystem I captures sunlight and transfers the excitation energy through an intricate and precisely organized antenna system, consisting of a pigment network, to the center of the molecule, where it is used in the transmembrane electron transfer reaction. Our current understanding of the sophisticated mechanisms underlying these processes has profited greatly from elucidation of the crystal structures of the Photosystem I complex. In this report, we describe the developments that ultimately led to enhanced structural information of plant Photosystem I. In addition, we report an improved crystallographic model at 3.3-Å resolution, which allows analysis of the structure in more detail. An improved electron density map yielded identification and tracing of subunit PsaK. The location of an additional ten beta-carotenes as well as five chlorophylls and several loop regions, which were previously uninterpretable, are now modeled. This represents the most complete plant Photosystem I structure obtained thus far, revealing the locations of and interactions among 17 protein subunits and 193 non-covalently bound photochemical cofactors. Using the new crystal structure, we examine the network of contacts among the protein subunits from the structural perspective, which provide the basis for elucidating the functional organization of the complex.

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2009

Alexey Amunts, Nathan Nelson
Plant photosystem I design in the light of evolution
Structure, 17 (5), pp. 637-650
Abstract
Photosystem I (PSI) is a membrane protein complex that catalyzes sunlight-driven transmembrane electron transfer as part of the photosynthetic machinery. Photosynthetic organisms appeared on the Earth about 3.5 billion years ago and provided an essential source of potential energy for the development of life. During the course of evolution, these primordial organisms were phagocytosed by more sophisticated eukaryotic cells, resulting in the evolvement of algae and plants. Despite the extended time interval between primordial cyanobacteria and plants, PSI has retained its fundamental mechanism of sunlight conversion. Being probably the most efficient photoelectric apparatus in nature, PSI operates with a quantum efficiency close to 100%. However, adapting to different ecological niches necessitated structural changes in the PSI design. Based on the recently solved structure of plant PSI, which revealed a complex of 17 protein subunits and 178 prosthetic groups, we analyze the evolutionary development of PSI. In addition, some aspects of PSI structure determination are discussed

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2008

Bart Van Oort, Alexey Amunts, Jan Willem Borst, Arie Van Hoek, Nathan Nelson, Herbert Van Amerongen, Roberta Croce
Picosecond fluorescence of intact and dissolved PSI-LHCI crystals
Biophysical Journal, 95 (12), pp. 5851-5861
Abstract
Over the past several years, many crystal structures of photosynthetic pigment-protein complexes have been determined, and these have been used extensively to model spectroscopic results obtained on the same proteins in solution. However, the crystal structure is not necessarily identical to the structure of the protein in solution. Here, we studied picosecond fluorescence of photosystem I light-harvesting complex I (PSI-LHCI), a multisubunit pigment-protein complex that catalyzes the first steps of photosynthesis. The ultrafast fluorescence of PSI-LHCI crystals is identical to that of dissolved crystals, but differs considerably from most kinetics presented in the literature. In contrast to most studies, the data presented here can be modeled quantitatively with only two compartments: PSI core and LHCI. This yields the rate of charge separation from an equilibrated core (22.5 ± 2.5 ps) and rates of excitation energy transfer from LHCI to core (kLC) and vice versa (kCL). The ratio between these rates, R = kCL/kLC, appears to be wavelength-dependent and scales with the ratio of the absorption spectra of LHCI and core, indicating the validity of a detailed balance relation between both compartments. kLC depends slightly but nonsystematically on detection wavelength, averaging (9.4 ± 4.9 ps)−1R ranges from 0.5 (<690 nm) to ∼1.3 above 720 nm.

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Alexey Amunts, Nathan Nelson
Functional organization of a plant Photosystem I: evolution of a highly efficient photochemical machine
Plant Physiology and Biochemistry, 46 (3), pp. 228-237
Abstract
Despite its enormous complexity, a plant Photosystem I (PSI) is arguably the most efficient nano-photochemical machine in Nature. It emerged as a homodimeric structure containing several chlorophyll molecules over 3.5 billion years ago, and has perfected its photoelectric properties ever since. The recently determined structure of plant PSI, which is at the top of the evolutionary tree of this kind of complexes, provided the first relatively high-resolution structural model of the supercomplex containing a reaction center (RC) and a peripheral antenna (LHCI) complexes. The RC is highly homologous to that of the cyanobacterial PSI and maintains the position of most transmembrane helices and chlorophylls during 1.5 years of separate evolution. The LHCI is composed of four nuclear gene products (Lhca1–Lhca4) that are unique among the chlorophyll a/b binding proteins in their pronounced long-wavelength absorbance and their assembly into dimers. In this respect, we describe structural elements, which establish the biological significance of a plant PSI and discuss structural variance from the cyanobacterial version. The present comprehensive structural analysis summarizes our current state of knowledge, providing the first glimpse at the architecture of this highly efficient photochemical machine at the atomic level.

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2007

Alexey Amunts, Omri Drory, Nathan Nelson
The structure of a plant photosystem I supercomplex at 3.4 Å resolution
Nature, 447 (7140), pp. 58-63
Abstract
All higher organisms on Earth receive energy directly or indirectly from oxygenic photosynthesis performed by plants, green algae and cyanobacteria. Photosystem I (PSI) is a supercomplex of a reaction centre and light-harvesting complexes. It generates the most negative redox potential in nature, and thus largely determines the global amount of enthalpy in living systems. We report the structure of plant PSI at 3.4 Å resolution, revealing 17 protein subunits. PsaN was identified in the luminal side of the supercomplex, and most of the amino acids in the reaction centre were traced. The crystal structure of PSI provides a picture at near atomic detail of 11 out of 12 protein subunits of the reaction centre. At this level, 168 chlorophylls (65 assigned with orientations for Qx and Qy transition dipole moments), 2 phylloquinones, 3 Fe4S4 clusters and 5 carotenoids are described. This structural information extends the understanding of the most efficient nano-photochemical machine in nature.

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2005

Alexey Amunts, Adam Ben-Shem, Nathan Nelson
Solving the structure of plant photosystem I—biochemistry is vital
Photochemical & Photobiological Sciences, 4 (12), pp. 1011-1015
Abstract
The recently determined structure of plant photosystem I (PSI) provides the first relatively high-resolution structural model of a supercomplex containing a reaction center and its peripheral antenna. Large amounts of highly purified PSI were required to get enough crystals amenable for structural determination by X-ray crystallography. In addition, a deep biochemical understanding of the large supercomplex was vital for achieving the goal. The stability of PSI was analyzed by sucrose gradient centrifugation and gel electrophoresis. Small amounts of LHCI were detached from PSI following a 12 day incubation under crystallization conditions. The interaction between the reaction center and the peripheral antenna of PSI (LHCI) as well as the interactions among the LHCI monomers are flexible. Nevertheless, the pure and homogeneous preparation of PSI allows for relatively tight crystal packing, which holds promise for obtaining atomic resolution in the future.

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