Recent advancements in Cryo-EM Unravels the Secrets of Cellular Respiration

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Overview of Cellular Respiration

Mitochondrion Source: Wikimedia Commons
Mitochondrion Source: Wikimedia Commons

The name of Michaelis in the famous Michaelis-Menton equation in the world of enzyme kinetics is well-connected to the mitochondria, commonly referred to as the powerhouse of living cells. In addition to his role in the formulation of Michaelis-Menton equation, Leonor Michaelis also identified the Janus green B stain that helped in the initial visualisation of the mitochondria, way back in the early 1900s. This paved an interesting way to the deeper understanding of the role played by the mitochondria in cellular respiration.

At a fairly snail’s pace, the next five decades or so led to investigations that identified constituent enzymes and prosthetic groups in the complex that are key role players in the elusive process of cellular respiration. The chemiosmotic theory (1961) indicated that the identified complexes, later termed as complexes I-IV, work in tandem with the ATP synthase (complex V) wherein glucose is broken down in the presence of O2 and this is coupled to the generation of ATP, the energy currency of the cell.

However, these findings only cautioned the deep and elusive molecular mechanisms driving cellular respiration.

Joining forces with Structural biology

The new era opened up gates to studies that focused on the molecular mechanism of these enzymes. This was substantially propelled with the advancements in structural biology, the tool for visualising details at the atomic level. The ambitious approaches in the field of structural biology were successfully fed with the aid of powerful techniques such as X-ray crystallography and complemented by NMR.

To give a fair idea, these enzymes are in the size range of 10-25 nm in length and they are composed of multi-subunit assemblies and are of protein sizes ranging from a few hundred daltons to mega dalton.

In the last 20 years, both X-ray crystallography and NMR have allowed for unveiling atomic details of these nano-sized enzymes. This has helped put together the missing pieces of the jig-saw puzzle of the the entire electron transport chain, with respect to location and events. The limitations stem from the fact that this information is obtained in parts owing to the shortcomings of the so-called high-resolution techniques used.

Graphics credit: Lavanya and Arvind Kumar, Biotechin.Asia
Graphics credit: Lavanya and Arvind Kumar, Biotechin.Asia

Brief idea on Cryo-EM

An answer to these shortcomings is the new technique, widely known as Cryo-Electron Microscopy (Cryo-EM).

As described by Milne et. al 2013, Cryo-EM is a young technique based upon the principle of imaging radiation-sensitive specimens in a transmission electron microscope under cryogenic conditions. Cryo-EM is a malleable tool suited for the analysis of dynamic protein assemblies, resembling their native conformation, that are not amenable to analysis by either NMR or X-ray crystallographic methods.

As the name suggests, these experiments are performed at cryogenic temperatures. This serves as an advantage given that sample damage that occurs in the routinely performed electron microscopy analysis of proteins or cells is averted. The damage is a result of the interaction of the electrons with organic matter leading to chemical bond breakage and generation of free radicals, resulting in secondary damage. This damage can be averted by six-fold using liquid nitrogen temperature (-196°C) during imaging. Lowering the temperature allows for the use of higher electron doses which aides in enhancing the signal to noise ratio.

Advancements using Cryo-EM

The last decade has seen the use of Cryo-EM to obtain high-resolution structure of various protein assemblies like those of ribosomes and photosystems. A soon-to-be-published structure of the complex V, the ATP synthase is believed to reveal certain important structural transitions that were difficult to decipher until recently.

Recent additions include the structures of the respirosome including complexes I, III and IV (~ 6 Å) and also individual structures of the mammalian complex I at 3.9 Å. These high-resolution structures illustrated below, provide information on assembly, maturation and dysfunction and allows detailed molecular analysis of disease-causing mutation. Adding to the exciting advancement in the field of Cryo-EM is a recent report of 3.2 Å resolution structure of haemoglobin using Volta phase plate Cryo-EM.

Credit: Lavanya and Arvind,
Credit: Lavanya and Arvind, Biotechin.Asia

This study is a testimony to the fact that in the years to come, Cryo-EM will be the key technique used by structural biologists. Although many known mutations with pathological implications have been attributed to the core of the complex I, it is only with these structures that a framework has been laid for understanding the molecular basis of mutations and the mechanism of complex I function and regulation.

Until recently, most mechanistic and structural principles for electron transport remained hypothetical. But, the studies on trapped conformations and the transitions between them is made possible only with the use of Cryo-EM.

This young technique is enabling us to probe deeper with high confidence into the complex systems.

Refer to the following research articles for further details:

Zhu, J., Vinothkumar, K. R. & Hirst, J. Structure of mammalian respiratory complex I. Nature 536, 354-358, doi:10.1038/nature19095 (2016).

Fiedorczuk, K. et al. Atomic structure of the entire mammalian mitochondrial complex I. Nature 538, 406-410, doi:10.1038/nature19794 (2016).

Letts, J. A., Fiedorczuk, K. & Sazanov, L. A. The architecture of respiratory supercomplexes. Nature 537, 644-648, doi:10.1038/nature19774 (2016).