Biology: an autonomous discipline

Dame Ottoline Leyser, Professor of Plant Development at the University of Cambridge, UK, was quoted recently as saying “The defining feature of biology during the past few decades has been figuring out details of the parts. But biological systems don’t think they have parts” [1]. Scepticism of the role of reductionism in understanding biology is a theme in Ernst Mayr’s book What Makes Biology Unique? Considerations on the Autonomy of a Scientific Discipline [2]. Mayr even argues against the relevance of the discovery of the DNA double helix to understanding biology. Mayr, now 100 years old, retired from Harvard University in 1975, where he is now Emeritus Professor of Zoology. So, where do I think these strong views stand today against reductionist research, namely structural biology within which crystallography is a key player, as are the microscopies and spectroscopies? These latter two areas include cryoelectron microscopy (cryoEM) and nuclear magnetic resonance (NMR) spectrosocopy. CryoEM in particular in recent years has taken the fight to these leading ‘whole biologists’ by opening up studies of large complexes that won’t crystallize yielding atomic details.

A thorny issue at its peak during the years of my graduate studies (1974 to 1977) was the relevance of the protein crystalline state to the solution state of a protein inside the biological cell. NMR provided atomically detailed results in solution and of course protein crystallography provided atomic details for a protein in the solid state. Studies of the structure and function of an enzyme in the crystalline state were to my mind greatly facilitated by the invention of the flow cell [3]. An example executing this to excellent effect was in the crystallographic studies of glycogen phosphorylase [4]. A pioneer in enzyme crystallography was David Blow and in his late-career overview [5] he lamented that a prediction of an enzyme’s reaction rate was still not possible, in effect defining it as one of today’s continuing Grand Challenges for science. This seems a harsh assessment by David Blow to my view in that qualitatively one can now see directly for example that large substrate enzymes are much slower than small substrate enzymes. Furthermore, an enzyme’s reaction rate can be deliberately slowed down, even stopped, by working with a designed mutant of the enzyme, guided by its 3D structure, again illustrating that, if not exactly a prediction of a specific reaction rate, this is a deliberate and successful alteration of enzyme reaction rate based on solid-state studies. Overall then crystallography has provided a powerful approach to this issue of the relevance of solid-state results with a resounding yes that these results are relevant to function; this important field has been reviewed by Keith Moffat [6]. These results overcame then the objections of the NMR solution-state spectroscopists to the crystallographer’s results in the solid state. Weaknesses in the armoury of crystallography remain such as crystallization conditions, to a greater or lesser degree, taking one’s results away from biological functioning conditions. Since those combative times in studies of protein structure, crystallography and NMR have worked in tandem to great effect in understanding structure and dynamics.

As protein crystallography has delivered ever more 3D structures, getting closer to complexity has proved a new challenge and focus. The accomplishments of virus crystallography and the crystal structure studies of the ribosome are testimony to the big steps forward to complexity yielding atomically detailed models of these molecular machines. The ribosome studies in particular required the perfecting of freezing conditions for the crystal so it would yield adequate amounts of X-ray diffraction data on the intense synchrotron beamlines needed to measure these data in a workable period of time.

A theme has steadily emerged in structural biology these last 20 years where there is a question about the strict relevance to biology of crystallography results now predominantly based on X-ray diffraction data measured at cryotemperature. This has been compounded by observations of specific X-ray damage to the crystallized protein. Conducting crystallography at physiological temperatures has then become an objective. Neutron macromolecular crystallography (nMX), whilst pursuing protein structures with protonation states experimentally determinable, has also automatically yielded room-temperature structures. Given also that projects succeed in getting time only where all other methods have failed, X-ray, electron or NMR based, it is clear that in structural biology there is a strategic importance of this method. nMX has seen a sustained growth of the instruments, the software and methods at leading neutron sources [7]. Furthermore, in an unconnected development, the new X-ray lasers yield X-ray diffraction data at room temperature and before radiation damage can kick in, the ‘diffract before the sample is destroyed’ approach [8]. Synchrotron facilities are now also adopting the X-ray laser methods for delivery of streams of micron-sized samples and thereby are also yielding results at physiological temperatures albeit not free of radiation damage like the X-ray lasers. To my mind an amazing accomplishment is the room-temperature crystal structure of the 30S ribosome using the Stanford LCLS [9]. The use of streams of micron-sized crystals raises the question of variations in those samples of the biological molecules being studied.

In making comparisons between a structure at cryotemperature and the same one at room temperature one has to be sure that each model refinement is at least at finality. The importance of refereeing of the underpinning data of a submitted article about a new crystal structure is to my mind paramount [10]. Even more, where comparisons between structures at two temperatures are concerned, then both should be refined to convergence. Validation of the structures of biological macromolecules is still developing [11].

In summary, the physical methods of crystallography, microscopy and spectroscopy continue to strive for, and do clearly deliver, biologically relevant results. Prediction of some aspects of biological function from atomic structure is possible, and that are also physiologically relevant. A founding father of quantum mechanics, the physicist Erwin Schrödinger posed the question What is Life? in his influential book [12]. It certainly influenced me as a young physicist looking towards what to pick as a research career. The fields of structural biology and crystallography have to my mind great contributions to make in the future. Shall we ever convince ‘whole biology biologists’? Time will tell but it is unlikely to be in my lifetime. In one of my own research themes studying the basis of the coloration of the lobster shell our explanations cover the molecular basis of the coloration [13, 14] but of course not why the lobster has that colour for its shell. One could say that our research on that is only ‘skin deep’.

Lobsters caught by fisherman at the Isle of Whithorn, Scotland. [Author's own photograph.]

References

[1] Turney, J. (2019). The puzzle of life. Times Higher Education, 21 February 2019, pp. 44–45.

[2] Mayr, E. (2004). What Makes Biology Unique? Considerations on the Autonomy of a Scientific Discipline. Cambridge: Cambridge University Press.

[3] Wyckoff, H. W., Doscher, M., Tsernoglou, D., Inagami, T., Johnson, L. N., Hardman, K. D., Allewell, N. M., Kelly, D. M. & Richards, F. M. (1967). Design of a diffractometer and flow cell system for X-ray analysis of crystalline proteins with applications to the crystal chemistry of ribonuclease-S. J. Mol. Biol. 27, 563–578.

[4] Hajdu, J., Acharya, K. R., Stuart, D. I., McLaughlin, P. J., Barford, D., Oikonomakos, N. G., Klein, H. & Johnson, L. N. (1987). Catalysis in the crystal: synchrotron radiation studies with glycogenphosphorylase b. EMBO J. 6, 539–546.

[5] Blow, D. M. (2000). So do we understand how enzymes work? Structure, 8, R77–R81.

[6] Moffat, K. (2001). Time-Resolved Biochemical Crystallography: A Mechanistic Perspective. Chem. Rev. 101, 1569–1581.

[7] Blakeley, M. P. & Podjarny, A. D. (2018). Neutron macromolecular crystallography. Emerg. Top. Life Sci. 2, 39–55.

[8] Neutze, R., Wouts, R., van der Spoel, D., Weckert, E. & Hajdu, J. (2000). Potential for biomolecular imaging with femtosecond X-ray pulses. Nature, 406, 752–757.

[9] Dao, E. H., Poitevin, F., Sierra, R. G., Gati, C., Rao, Y., Ciftci, H. I., Akşit, F., McGurk, A., Obrinski, T., Mgbam, P., Hayes, B., De Lichtenberg, C., Pardo-Avila, F., Corsepius, N., Zhang, L., Seaberg, M. H., Hunter, M. S., Liang, M., Koglin, J. E., Wakatsuki, S. & Demirci, H. (2018). Structure of the 30S ribosomal decoding complex at ambient temperature. RNA, 24, 1667–1676.

[10] Helliwell, J. R. (2018). Data science skills for referees: I biological X-ray crystallography. Cryst. Rev. 24, 263–272.

[11] Helliwell, J. R. (2017). New developments in crystallography: exploring its methods, technology and scope in the molecular biosciences. Biosci. Rep. 37, BSR20170204.

[12] Schrödinger, E. (1943). What is Life? The Physical Aspect of the Living Cell. Based on lectures delivered under the auspices of the Dublin Institute for Advanced Studies at Trinity College, Dublin, in February 1943. Reprinted by Cambridge University Press, 2012.

[13] Cianci, M., Rizkallah, P. J., Olczak, A., Raftery, J., Chayen, N. E., Zagalsky, P. F. & Helliwell, J. R. (2002). The molecular basis of the coloration mechanism in lobster shell: β-crustacyanin at 3.2 Å resolution. Proc. Natl Acad. Sci. USA, 99, 9795–9800.

[14] Rhys, N. H., Wang, M.-C., Jowitt, T. A., Helliwell, J. R., Grossmann, J. G. & Baldock, C. (2011). Deriving the ultrastructure of α-crustacyanin using lower-resolution structural and biophysical methods. J. Synchrotron Rad. 18, 79–83.