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Assembly and symmetry of the fungal E3BP-containing core of the Pyruvate Dehydrogenase Complex
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
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(English)Manuscript (preprint) (Other academic)
Abstract [en]

The pyruvate dehydrogenase complex (PDC) is a central component of all aerobic respiration, connecting glycolysis to mitochondrial oxidation of pyruvate. Despite its central metabolic role, its precise composition and means of regulation remain unknown. To explain the variation in stoichiometry reported for the E3-recruiting protein X (PX) in the fungal PDC, we established cryo-EM reconstructions of the native and recombinant PDC from the filamentous fungus and model organism Neurospora crassa. We find that the PX C-terminal domain localizes interior to the E2 core. Critically, we show that two distinct arrangements of a trimeric oligomer exists, which both result in strict tetrahedral symmetry of the PDC core interior. Both oligomerization and volume occlusion of the PDC interior by PX appears to limit its binding stoichiometry, which explains the variety of stoichiometries found previously for S. cerevisiae. This also suggests that the PX oligomer stability and size are potential mechanisms to dynamically adjust PDC compostion in response to external cues. Moreover, we find that the site where PX binds is conserved within fungi but not mammals, suggesting that it could be therapeutically targeted. To this end, we also show that a PX knockout results in loss of activity through dysfunctional E3 recruitment, leading to severely impaired N. crassa growth on sucrose. The fungal PDC is thus shown to be fundamentally similar to the mammalian PDC in function but subject to other conditions of possible regulation, conditioned by a steric restrictions imposed by the symmetry of the PDC and its components.

National Category
Biochemistry and Molecular Biology
Research subject
Biochemistry towards Bioinformatics
Identifiers
URN: urn:nbn:se:su:diva-179830OAI: oai:DiVA.org:su-179830DiVA, id: diva2:1413308
Available from: 2020-03-10 Created: 2020-03-10 Last updated: 2020-03-13Bibliographically approved
In thesis
1. Fast and reliable alignment and classification of biological macromolecules in electron microscopy images
Open this publication in new window or tab >>Fast and reliable alignment and classification of biological macromolecules in electron microscopy images
2020 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

In the last century, immense progress has been made to charter and understand a wide range of biological phenomena. The origin of genetic inheritance was determined, showing that DNA holds genes that determine the architecture of proteins, utilized by the cell for most functions. Mapping of the human genome eventually revealed around 20000 genes, showing a vast complexity of biology at its most fundamental level.

To study the molecular structure, function and regulation of proteins, spectroscopic techniques and microscopy are employed. Until just over a decade ago, the determination of atomic detail of biomolecules like proteins was limited to those that were small or possible to crystallize. However recent technological advances in cryogenic electron microscopy (cryo-EM) now allows it to routinely reach resolutions where it can provide a wealth of new information on molecular biological phenomena by permitting new targets to be structurally characterized.

In cryo-EM, biological molecules are suspended in thin vitreous sheet of ice and imaged in projection. Collecting millions of such images permits the reconstruction of the original molecular structure, by appropriate alignment and averaging of the particle images. This however requires immense computational effort, which just a few years ago was prohibitive to full use of the image data.

In this thesis, I describe the development of fast algorithms for processing of cryo-EM data, utilizing GPUs by exposing the inherent parallelism of its alignment and classification. The acceleration of this processing has changed how biological research can utilize cryo-EM data. The drastically reduced processing time now allows more extensive processing, development of new and more demanding processing tools, and broader access to cryo-EM as a method for biological investigation. As an example of what is now possible, I show the processing of the fungal pyruvate dehydrogenase complex (PDC), which poses unique processing challenges. Through extensive processing, new biological information can be inferred, reconciling numerous previous findings from biochemical research. The processing of PDC also exemplifies current limitations to established.

Place, publisher, year, edition, pages
Stockholm: Institutionen för biokemi och biofysik, Stockholms Universitet, 2020. p. 100
Keywords
cryo-EM, electron microscopy, GPU, parallel processing, protein structure
National Category
Biochemistry and Molecular Biology
Research subject
Biochemistry towards Bioinformatics
Identifiers
urn:nbn:se:su:diva-179802 (URN)978-91-7911-050-5 (ISBN)978-91-7911-051-2 (ISBN)
Public defence
2020-04-24, Magnélisalen, Kemiska övningslaboratoriet, Svante Arrhenius väg 16 B, Stockholm, 13:00 (English)
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Supervisors
Note

At the time of the doctoral defense, the following paper was unpublished and had a status as follows: Paper 5: Manuscript.

Available from: 2020-04-01 Created: 2020-03-10 Last updated: 2020-03-17Bibliographically approved

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