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Structural characterisation of mitochondrial macromolecular complexes using cryo-EM: Mitoribosome biogenesis and respiratory chain supercomplex
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.ORCID iD: 0000-0001-9178-1006
2020 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Mitochondria, popularly known as powerhouse of the cell, contain specialised mitoribosomes that synthesise essential membrane proteins. These essential proteins are required to form enzyme complexes, which carry out the process of oxidative phosphorylation (OXPHOS). OXPHOS is carried out by five enzyme complexes (Complex I-V), out of which complex I, III and IV pump protons during electron transfer from NADH to Oand complex V uses the generated proton gradient to synthesise ATP. Cryo-EM, as a revolutionary technique in structural biology made it possible to determine the structures of mitoribosome assembly intermediates and respiratory chain supercomplexes. These structures have allowed us to investigate the mitoribosome biogenesis pathway in human and yeast and to gain deeper insights into the architecture of supercomplexes. In the first area of research, using cryo-EM we were for the first time able to capture mitoribosomes in different late stages of assembly and to determine their high-resolution structures with novel factors bound. Investigation of this process was previously unreachable due to lack of techniques to trap these mitoribosome complexes in different states of assembly. The structures of these assembly intermediates establish the role of assembly factors such as MALSU1, LOR8F8, mt-ACP, MTG1 and mitoribosomal proteins (MRPs) in mitoribosome biogenesis and to ensure proper maturation of each subunit, reflecting their role in regulating translation. Furthermore, genetic deletion studies of MTG1 and uL16m in yeast show the importance of transiently acting factors and MRPs in the mitoribosome assembly process and their effects on translation. The assembly pathway of mitoribosomes is critical for protein synthesis since defects in the translation process causes inherited human pathologies. Therefore, elucidation of mitoribosomal biogenesis pathways may also contribute to the development of potential new therapeutic opportunities. In the second research area, structures of the respiratory chain supercomplex from yeast were determined. These are the first near-atomic resolution structures that show organization of complex III and complex IV into two distinct classes that form higher order assemblies (III2IV1and III2IV2). Moreover, the architecture of the supercomplex structures differs from the previously determined respirasomes (I1III2IV1) structures in mammals. We obtained a near-atomic resolution structure of the yeast complex IV, revealed core protein-protein and protein-lipid interactions that hold the supercomplex together. Moreover we found novel subunits required for supercomplex formation in S. cerevisiae. The last part of my study focuses on cryo-EM sample method development where we could successfully demonstrate the usefulness of a simple pressure-assisted sample preparation method for microcrystals, proteins and mitochondria. Our findings show great resolution improvements of selected area electron diffraction patterns of microcrystals, a significant reduction in needed sample concentration for single particle studies and an enrichment of gold nano-particles for tomographic studies.

Place, publisher, year, edition, pages
Stockholm: Department of Biochemistry and Biophysics, Stockholm University , 2020. , p. 50
Keywords [en]
Mitochondria, mitoribosome biogenesis, mitoribosome assembly factors, yeast respiratory supercomplexes, single particle electron cryo-microscopy, cryo-EM sample preparation
National Category
Structural Biology Biochemistry and Molecular Biology
Research subject
Biochemistry
Identifiers
URN: urn:nbn:se:su:diva-180776ISBN: 978-91-7911-156-4 (print)ISBN: 978-91-7911-157-1 (electronic)OAI: oai:DiVA.org:su-180776DiVA, id: diva2:1423978
Public defence
2020-06-08, Magnélisalen, Kemiska övningslaboratoriet, Svante Arrhenius väg 16 B, Stockholm, 13:00 (English)
Opponent
Supervisors
Note

At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 2: Manuscript. Paper 4: Manuscript.

Available from: 2020-05-14 Created: 2020-04-16 Last updated: 2020-05-26Bibliographically approved
List of papers
1. Structures of the human mitochondrial ribosome in native states of assembly
Open this publication in new window or tab >>Structures of the human mitochondrial ribosome in native states of assembly
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2017 (English)In: Nature Structural & Molecular Biology, ISSN 1545-9993, E-ISSN 1545-9985, Vol. 24, no 10, p. 866-869Article in journal (Refereed) Published
Abstract [en]

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 similar to 3-angstrom 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.

Keywords
Cryoelectron microscopy, Ribosome
National Category
Biological Sciences
Research subject
Biochemistry towards Bioinformatics; Biochemistry
Identifiers
urn:nbn:se:su:diva-148876 (URN)10.1038/nsmb.3464 (DOI)000412278000014 ()28892042 (PubMedID)
Available from: 2017-11-22 Created: 2017-11-22 Last updated: 2020-04-19Bibliographically approved
2. Cryo-EM reveals different mitoribosome assembly intermediates in yeast knockout strains of Mtg1 and uL16m
Open this publication in new window or tab >>Cryo-EM reveals different mitoribosome assembly intermediates in yeast knockout strains of Mtg1 and uL16m
(English)Manuscript (preprint) (Other academic)
National Category
Structural Biology
Research subject
Biochemistry
Identifiers
urn:nbn:se:su:diva-180734 (URN)
Available from: 2020-04-07 Created: 2020-04-07 Last updated: 2020-04-19Bibliographically approved
3. Cryo-EM structure of the yeast respiratory supercomplex
Open this publication in new window or tab >>Cryo-EM structure of the yeast respiratory supercomplex
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2019 (English)In: Nature Structural & Molecular Biology, ISSN 1545-9993, E-ISSN 1545-9985, Vol. 26, no 1, p. 50-57Article in journal (Refereed) Published
Abstract [en]

Respiratory chain complexes execute energy conversion by connecting electron transport with proton translocation over the inner mitochondrial membrane to fuel ATP synthesis. Notably, these complexes form multi-enzyme assemblies known as respiratory supercomplexes. Here we used single-particle cryo-EM to determine the structures of the yeast mitochondria! respiratory supercomplexes III2IV and III2IV2, at 3.2-angstrom and 3.5-angstrom resolutions, respectively. We revealed the overall architecture of the supercomplex, which deviates from the previously determined assemblies in mammals; obtained a near-atomic structure of the yeast complex IV; and identified the protein-protein and protein-lipid interactions implicated in supercomplex formation. Take together, our results demonstrate convergent evolution of supercomplexes in mitochondria that, while building similar assemblies, results in substantially different arrangements and structural solutions to support energy conversion.

National Category
Biological Sciences
Research subject
Biochemistry
Identifiers
urn:nbn:se:su:diva-165785 (URN)10.1038/s41594-018-0169-7 (DOI)000454902900007 ()30598556 (PubMedID)
Available from: 2019-02-26 Created: 2019-02-26 Last updated: 2020-04-19Bibliographically approved
4. A simple pressure-assisted method for cryo-EM specimen preparation
Open this publication in new window or tab >>A simple pressure-assisted method for cryo-EM specimen preparation
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(English)Manuscript (preprint) (Other academic)
National Category
Structural Biology
Research subject
Biochemistry
Identifiers
urn:nbn:se:su:diva-180736 (URN)
Available from: 2020-04-07 Created: 2020-04-07 Last updated: 2020-04-19Bibliographically approved

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