Periodic Reporting for period 2 - NanED (Electron Nanocrystallography)
Période du rapport: 2023-03-01 au 2025-10-31
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 proposed 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 has been 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 has set up is going to impact in the society in terms of understanding new material functions and properties. We have been able to understand which are the products of chemical syntheses that were neglected for their polycrystalline yield. We have discovered new polytypes of pharmaceutical compounds that were previously ignored because they were elusive to all the available characterization methods. We demonstrated to be capable of determining the structure of unknown proteins bound to a drug, that up to now were studied in this form since they could 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 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. Data collection was made automatic increasing dramatically the number of crystals that could be studied in one session.
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. In case of extremely beam sensitive samples, a serial electron diffraction strategy was set up, allowing the microscope to collect thousands of single shot patterns over operator unsupervised sessions. In this way we successfully determined the structures of proteins and beam sensitive MOFs.
Studying 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. We set up a specific procedure for getting protein crystals of a proper size for 3D ED analysis and for collecting serialED data. With serialED data we demonstrated that it is possible to identify the binding of drugs to proteins.
Another goal of the project was accurate electron crystallography. In this area we demonstrated that with 3D ED we can obtain on nanocrystals the same accuracy and structure details that x-ray diffraction gets on microcrystals. We have shown that 3D ED is more sensitive to the absolute structure than x-ray diffraction and that it can also detect charge density effect and determine the ionization state of an atom in the crystal structure.
Finally we approached the problem of when a crystal is so small or so disorder to lose its periodicity, becoming a nanomaterial or an amorphous. Analysis of nanoparticles with 3D ED testified that the minimum crystal size that can be studied with 3D ED can go down to 5nm and that the crystals, even if larger, at this scale can exhibit significant differences from one point to another. If the sample is amorphous electron diffraction can still deliver important structure information using pair distribution function analysis. With respect to x-ray diffraction electron diffraction has a higher spatial resolution and we demonstrated how it can be used to distinguish different amorphous phases intergrown at the nanoscale.
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 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. Data collection was made automatic increasing dramatically the number of crystals that could be studied in one session.
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. In case of extremely beam sensitive samples, a serial electron diffraction strategy was set up, allowing the microscope to collect thousands of single shot patterns over operator unsupervised sessions. In this way we successfully determined the structures of proteins and beam sensitive MOFs.
Studying 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. We set up a specific procedure for getting protein crystals of a proper size for 3D ED analysis and for collecting serialED data. With serialED data we demonstrated that it is possible to identify the binding of drugs to proteins.
Another goal of the project was accurate electron crystallography. In this area we demonstrated that with 3D ED we can obtain on nanocrystals the same accuracy and structure details that x-ray diffraction gets on microcrystals. We have shown that 3D ED is more sensitive to the absolute structure than x-ray diffraction and that it can also detect charge density effect and determine the ionization state of an atom in the crystal structure.
Finally we approached the problem of when a crystal is so small or so disorder to lose its periodicity, becoming a nanomaterial or an amorphous. Analysis of nanoparticles with 3D ED testified that the minimum crystal size that can be studied with 3D ED can go down to 5nm and that the crystals, even if larger, at this scale can exhibit significant differences from one point to another. If the sample is amorphous electron diffraction can still deliver important structure information using pair distribution function analysis. With respect to x-ray diffraction electron diffraction has a higher spatial resolution and we demonstrated how it can be used to distinguish different amorphous phases intergrown at the nanoscale.
The results of our project allow to foresee a bright future for electron diffraction in material science. For sure the most important step we did was automation. This has demonstrated that the method has a great potential to enter production control pipelines in chemistry and pharmaceutical industries. The unexpected entrance of AI into the project as a way to distinguish crystals in unsupervised crystal search or for phasing diffraction and high resolution imaging of proteins testifies how its wide application to electron diffraction and crystallography in general will be the next development. Another area of strong expansion will be in-situ experiments. During the project we have been able to observe in-situ activation of nanocrystalline MOFs with direct detection of the emptying of the channels, however effective experimental procedures for collecting 3D ED in liquid or in other in-situ environments are still lacking.