Periodic Reporting for period 1 - NanED (Electron Nanocrystallography)
Reporting period: 2021-03-01 to 2023-02-28
The properties of each material, organic or inorganic, depend on how the atoms are arranged on its structure. A wide class of materials we use in everyday life is in crystalline form so their structure is ordered, and their atomic arrangement can be described in great detail. However, very often the materials we encounter crystallize in grains with sizes 10 or 100 times smaller than the diameter of a human hair. This happens, either because this is the only way to synthesize them, or because we made them small, since it is their “nano” size that gives them extraordinary properties. When the crystals are so small the determination of their structure changes from a routine characterization to an extremely challenging problem that requires new science to be solved. The standard way in which we determine the crystal structure is based on observing how a crystal deflects an x-ray radiation impinging on it: the so called diffraction phenomena. If the crystal size falls in the ranges discussed above, the scattering of x-ray radiation is too weak, and a new radiation probe must be found. Our project proposes to use electrons as “the radiation” for investigating the crystal structure instead of x-ray. The electrons interact stronger with matter than x-ray, therefore we have detectable diffraction signals from crystals as small as a few hundreds of nanometers. Furthermore, such an experiment can be done in existing instruments: the transmission electron microscopes. The aim of NanED is to train a new generation of young electron crystallographers that will spread this novel technique in Europe and beyond and at the same time to develop and apply electron diffraction to any kind of crystalline material, going from inorganic synthetic products to pharmaceutical compounds, from nanoparticles to proteins. The characterization capability of nanocrystals that NanED is and will be setting up is going to impact in the society in terms of understanding new material functions and properties. We expect to be able to understand which are the products of chemical syntheses that were neglected for their polycrystalline yield. We will discover new polytypes of pharmaceutical compounds that were previously ignored because they were elusive to all the available characterization methods. We want to determine the structure of unknown proteins that up to now remained unknown since they can only crystallize in nanocrystalline forms and are too small for standard cryo-EM imaging.
At the beginning the consortium had to set up the project infrastructure. A web site (https:\\naned.eu) and a twitter account (@NanEd_P) were created and all the ESR were selected through a common recruiting platform. After the ESR were employed, we started their formation through three dedicated workshops one introductory to 3D electron diffraction, one on the treatment and analysis of 3D ED data and one on the complementary use of 3D ED and powder x-ray diffraction.
As proof of concept and part of the training activity all the ESR analyzed a set of three common samples and had to solve their structure. This was the first experiment of this kind in which completely different 3D ED instruments were used to analyze the same structure. The successful structure solution obtained on most of the samples by all ESR demonstrated the portability and the advance development stage of the method. The consortium was able to define a precise protocol for the data collection of 3D ED adapted to any kind of sample, instrument type and detector available.
The extension of the method to any kind of sample passes through the determination of sample preparation routines that are able to protect the samples from the damaging effects of the electron beam and from the high vacuum condition of an electron microscope column. We discovered that freezing the crystals in their crystallization solution is not only a method for protecting the crystals from damage but it avoids the release of molecules trapped inside the crystals. We could in this way study hydrated phases and detect molecules trapped in porous crystal structures.
To study proteins with electron diffraction is one of the most challenging parts of the project. Proteins are huge molecules and the positions of hundreds of atoms should be determined and at the same time they are very sensitive to the harsh environment of the TEM vacuum. However every improvement in this field has a high revenue since the structure of a protein is the key to understanding its functions. In this first part of the project we set up a specific procedure for getting protein crystals of a proper size for 3D ED analysis.
Preliminary analysis of our capability to extract fine structure details from 3D ED analysis stated that we are sensitive to the oxidation state of the atoms in the structure.
As proof of concept and part of the training activity all the ESR analyzed a set of three common samples and had to solve their structure. This was the first experiment of this kind in which completely different 3D ED instruments were used to analyze the same structure. The successful structure solution obtained on most of the samples by all ESR demonstrated the portability and the advance development stage of the method. The consortium was able to define a precise protocol for the data collection of 3D ED adapted to any kind of sample, instrument type and detector available.
The extension of the method to any kind of sample passes through the determination of sample preparation routines that are able to protect the samples from the damaging effects of the electron beam and from the high vacuum condition of an electron microscope column. We discovered that freezing the crystals in their crystallization solution is not only a method for protecting the crystals from damage but it avoids the release of molecules trapped inside the crystals. We could in this way study hydrated phases and detect molecules trapped in porous crystal structures.
To study proteins with electron diffraction is one of the most challenging parts of the project. Proteins are huge molecules and the positions of hundreds of atoms should be determined and at the same time they are very sensitive to the harsh environment of the TEM vacuum. However every improvement in this field has a high revenue since the structure of a protein is the key to understanding its functions. In this first part of the project we set up a specific procedure for getting protein crystals of a proper size for 3D ED analysis.
Preliminary analysis of our capability to extract fine structure details from 3D ED analysis stated that we are sensitive to the oxidation state of the atoms in the structure.
The next step of the project will be to investigate the limit between a crystal and an amorphous and to directly follow phase transitions in-situ on nanocrystals. We want to push 3D ED to its limits, trying to determine the smallest crystal size whose crystal structure can be determined. We have evidence at the moment that we can go down to 20 nm but we want to move beyond.
In situ nanocrystallography is a new open field. We expect to be able to detect phase transitions on nano objects, in liquid or in gas following a chemical reaction in all its steps. This is a powerful tool for understanding the mechanism of a chemical reaction and can have several applications in the development of technological devices like sensors or batteries.
Automation is a necessary step to make any technique more efficient and able to produce a sufficient amount of data for a full description of a phenomenon. Although 3D ED can be implemented in any TEM, its automation is still an open issue. In the remaining half of the project we will push further the automation process trying to find a solution which is able to automatically investigate a large number of crystals in one session and to collect snapshots of single crystals in case they are immediately destroyed by the electron beam. The so called serialED. All this will have an impact on the portability of the technique in the industrial world.
Finally we will work on having a complete protocol for structural analysis of proteins. Working at the same time on the data reduction and on the method of structure solution with 3D ED data, we envisage to be able to solve the structure of unknown proteins completely ab-initio. This can be a breakthrough that can have significant fall out both in medicine and pharmaceutical sciences.
In situ nanocrystallography is a new open field. We expect to be able to detect phase transitions on nano objects, in liquid or in gas following a chemical reaction in all its steps. This is a powerful tool for understanding the mechanism of a chemical reaction and can have several applications in the development of technological devices like sensors or batteries.
Automation is a necessary step to make any technique more efficient and able to produce a sufficient amount of data for a full description of a phenomenon. Although 3D ED can be implemented in any TEM, its automation is still an open issue. In the remaining half of the project we will push further the automation process trying to find a solution which is able to automatically investigate a large number of crystals in one session and to collect snapshots of single crystals in case they are immediately destroyed by the electron beam. The so called serialED. All this will have an impact on the portability of the technique in the industrial world.
Finally we will work on having a complete protocol for structural analysis of proteins. Working at the same time on the data reduction and on the method of structure solution with 3D ED data, we envisage to be able to solve the structure of unknown proteins completely ab-initio. This can be a breakthrough that can have significant fall out both in medicine and pharmaceutical sciences.