Neutrons, space-grown crystals, and enzymes

An article recently published in Cell Reports Physical Science reveals how researchers have for the first time directly visualised the positions of hydrogen atoms within an enzyme that plays an important role in the metabolism of many microorganisms. Neutron diffraction measurements at ILL of protein crystals grown under microgravity conditions at the International Space Station (ISS) were key ingredients in this work.

Unlike X-rays, which interact with the electrons around the nuclei, neutrons interact directly with the nuclei itself. This means that hydrogen (and its heavier isotope deuterium) scatter neutrons with similar strength to the other common elements of a protein, making them an ideal tool for probing the positions of hydrogen atoms in biological macromolecules. However, as neutron sources are are billions of times less intense than X-ray sources, the challenge is that we need large crystals that are well-ordered. This is where microgravity and the ISS come in.

Deciphering the paper’s title: a bite-sized approach

Neutron diffraction from a microgravity-grown crystal reveals the active site hydrogens of the internal aldimine form of tryptophan synthase”. Interesting as it may be, the title of this publication is not easy to digest. What are they even talking about? Let’s take a scientific approach and split the title into smaller bite-sized pieces. Then we can examine it piece by piece, working our way backwards.


Our first bite-sized piece is “tryptophan synthase", or TS for short. This is a type of protein, an enzyme, which speeds up chemical reactions in living organisms. TS is involved in the metabolic processes of many different microorganisms, including pathogenic bacteria such as Salmonella enterica and Mycobacterium tuberculosis.

Now let’s bite off a second piece: “the internal aldimine form of tryptophan synthase". So we are told that TS is in the “internal aldimine form”. There’s quite a bit of biochemistry behind these three words, but what we need to understand is actually simple. Some enzymes require the addition of a small molecule, called a cofactor, in order to function. The cofactor for TS is pyridoxal 5′-phosphate, or PLP for short. PLP is the active form of vitamin B6, which is present in practically all forms of life and is crucial for countless cellular functions. The internal aldimine is simply a stable enzyme–PLP complex. PLP-dependent enzymes are particularly interesting because they form a large and versatile family of enzymes that are common to many organisms and include many that play a role in human diseases or are of interest in enzyme engineering applications. Despite this obvious interest, important questions remain about how PLP-dependent enzymes really work.

And now for our third bite: “active site hydrogens” in the TS-PLP complex have been revealed. In order to understand and model the structure and functioning of enzymes, scientists need to see them at the atomic level. Around half of the atoms in proteins are hydrogen atoms. Most importantly, the hydrogen atoms in active sites (i.e. the parts of the molecule involved in the functioning of the enzyme) are shuffled around during the reaction. Understanding where the hydrogen atoms are and how they move is therefore essential to understanding how the enzyme works.

Space-grown crystals

A “microgravity-grown crystal” is our next bite.

Proteins are typically amorphous as solids. However, in suitable environments, they can crystallize. Scientists can observe protein crystals at atomic level using both X-ray and neutron diffraction and then create 3D models of the molecules. The larger and more defect-free the crystals, the better the results. However, growing large, well-ordered protein crystals on Earth is challenging, and gravity is part of the problem.

To overcome this, the large (≥ 0.5 mm3) defect-free crystals used in this study were actually grown on board the International Space Station (ISS) over a period of about six months, in which they travelled around 50 million miles.

Neutrons diffraction

This brings us to our biggest chunk of all: “neutron diffraction”. Both X-rays and neutrons allow us to look inside crystals, and their powers are often complementary. X-rays interact with the electron density around atoms, which means that in X-ray diffraction the positions of key functional hydrogen atoms (with only one electron) remain undetermined. Neutrons, on the other hand, interact with atomic nuclei, which means that hydrogen (and its heavier isotope deuterium) scatter neutrons as well as heavier nuclei. Neutrons are therefore ideal for probing hydrogen atom locations in biological macromolecules. With neutrons, however, the challenge is that we need crystals that are large and well-ordered. Hence the use of a microgravity-grown crystal.

Neutron diffraction data were collected on the ILL instrument LADI-III, a Laue diffractometer for biology research at high resolution (1.5 - 2.5 Å). LADI-III uses a large cylindrical area detector composed of neutron-sensitive image-plates that completely surround the crystal and allow large numbers of diffraction spots to be recorded simultaneously. A range of neutron wavelengths from 2.85–3.80 Å was used for data collection, with four different crystal orientations sampled and a total of 57 images collected. The principle is as follows: the neutron beam is diffracted from the atomic nuclei within the crystal producing a pattern of diffraction spots. As there is a specific relationship between the 2D diffraction pattern and the 3D structure within the crystal, scientists can use the information in these patterns to determine the 3D structure, including the all-important hydrogen atoms.

In order to facilitate the location of individual hydrogen atoms, the protein was produced using a technique called perdeuteration: all hydrogen atoms in the molecule were replaced by deuterium in order to become more visible. The ILL offers its user community and in-house researchers not only high-performance instruments but also highly specialized platforms and laboratories to prepare and characterise samples before, during and after neutron experiments, some of them within partnerships with neighbouring institutes. In particular, the ILL Deuteration Laboratory (D-Lab) can produce a variety of partially, or fully deuterated (perdeuterated) biological molecules, including proteins.

And that was the last piece of title. “Neutron diffraction from a microgravity-grown crystal reveals the active site hydrogens of the internal aldimine form of tryptophan synthase” hopefully sounds much easier to decipher after we have examined piece by piece, in bite-sized pieces. Standing out are the importance of understanding the workings of enzymes involved in the spread of infections and the central role of neutron diffraction data in this quest.


Finally, combining the neutron diffraction data with X-ray diffraction data, the team were able to report the 2.1 Å resolution joint X-ray/neutron (XN) structure of TS with PLP in the internal aldimine form where the positions of all atoms have been determined, as well as providing important insights into its functioning. This investigation is part of ongoing biomolecular crystallization studies using microgravity that span decades. It is an example of how fundamental research conducted over a long timescale with cumulative progress can one day enable the rapid development of a much-needed application, as we have seen recently during the pandemic crisis. The work involved ILL’s Life Sciences and Large Scale Structures groups, the Partnership for Structural Biology (PSB), as well as the LINXS Institute for Advanced Neutron and X-ray Science (Lund, Sweden) and the Oak Ridge National Laboratory (Oak Ridge, USA).


ILL Instrument: LADI-III, A quasi-Laue diffractometer for biology

Reference: Neutron diffraction from a microgravity-grown crystal reveals the active site hydrogens of the internal aldimine form of tryptophan synthase,[Open Access]

ILL Contacts: Matthew Blakeley (LADI-III) & Juliette Devos (D-Lab)

Contact: Timothy C. Mueser (University of Toledo, USA)