Igor Nederlof

A Medipix quantum area detector allows rotation electron diffraction data collection from submicrometre three-dimensional protein crystals.

An ultrasensitive Medipix2 detector allowed the collection of rotation electron-diffraction data from single three-dimensional protein nanocrystals for the first time. The data could be analysed using the standard X-ray crystallography programs MOSFLM and SCALA.

Ab initio structure determination of nanocrystals of organic pharmaceutical compounds by electron diffraction at room temperature using a Timepix quantum area direct electron detector

A specialized quantum area detector for electron diffraction studies makes it possible to solve the structure of small organic compound nanocrystals in non-cryo conditions by direct methods.

Imaging protein three‐dimensional nanocrystals with cryo‐EM

Flash-cooled three-dimensional crystals of the small protein lysozyme with a thickness of the order of 100 nm were imaged by 300 kV cryo-EM on a Falcon direct electron detector. The images were taken close to focus and to the eye appeared devoid of contrast. Fourier transforms of the images revealed the reciprocal lattice up to 3 Å resolution in favourable cases and up to 4 Å resolution for about half the crystals. The reciprocal-lattice spots showed structure, indicating that the ordering of the crystals was not uniform. Data processing revealed details at higher than 2 Å resolution and indicated the presence of multiple mosaic blocks within the crystal which could be separately processed. The prospects for full three-dimensional structure determination by electron imaging of protein three-dimensional nanocrystals are discussed.

Image processing and lattice determination for three-dimensional nanocrystals.

Three-dimensional nanocrystals can be studied by electron diffraction using transmission cryo-electron microscopy. For molecular structure determination of proteins, such nanosized crystalline samples are out of reach for traditional single-crystal X-ray crystallography. For the study of materials that are not sensitive to the electron beam, software has been developed for determining the crystal lattice and orientation parameters. These methods require radiation-hard materials that survive careful orienting of the crystals and measuring diffraction of one and the same crystal from different, but known directions. However, as such methods can only deal with well-oriented crystalline samples, a problem exists for three-dimensional (3D) crystals of proteins and other radiation sensitive materials that do not survive careful rotational alignment in the electron microscope. Here, we discuss our newly released software AMP that can deal with nonoriented diffraction patterns, and we discuss the progress of our new preprocessing program that uses autocorrelation patterns of diffraction images for lattice determination and indexing of 3D nanocrystals.

Protein Crystal Growth

The biological activity of most proteins is determined by their 3D structure. For instance, a substantial number of molecular diseases are caused by protein structural alterations, which are genetically encoded. Drugs operate by binding to proteins, inducing alteration of their functional structure and thereby affecting their biological activity. Hence the design and improvement of drugs is greatly facilitated by knowledge of the 3D structures of their macromolecular targets. In the light of these considerations, it is clear that elucidation of the 3D structure of proteins is of prime importance for understanding the underlying mechanisms of molecular diseases. It was initially believed that any protein that could be made soluble and could be purified would be relatively easy to crystallize. However, the results have indicated that solubility and purity of proteins, although being important factors, do not secure a yield of useful crystals. The crystallization behavior of proteins turns out to be very complex. In an effort to identify the naturally occurring protein folds, large structural genomics consortia were set up. The somewhat disappointing outcome of these efforts is that only about 3% of all proteins that were targeted by these consortia yielded a crystal structure (http://targetdb.pdb.org/statistics/TargetStatistics.html), despite massive investments in high-throughput, automated protein production, purification and crystallization. It is clear that in order to improve the current situation, better strategies for protein crystallization are required, combined with techniques that allow the use of smaller nano-crystalline material.

Lattice filter for processing image data of three-dimensional protein nanocrystals

A specialized filter for finding lattices in images of three-dimensional nanocrystals devoid of any contrast is described.

Electron crystallography of protein nano-crystals

Sub-micron protein crystals are beyond the range of standard X-ray diffraction experiments. Many proteins however fail to grow large, diffraction grade, crystals due to stacking faults. Using electron diffraction the structure can be solved of crystals that are 1-2 orders of magnitude smaller than what is standard using X-rays. In this talk I will show 3D electron crystallography data and the solved structure of lysozyme. I will touch on the possibilities and limitation of this new method and show recent advances in detector hardware that have enabled this exciting new field.

Electron Diffraction of Protein 3D Nanocrystals

Protein crystallography, being one of the most established methods for structure determination of biomacromolecules, relies on the diffraction analysis using X-rays for 3D (micro-)crystals. Electron diffraction is only employed for single layer 2D nano-crystals. However, there is no established method for analysing multi-layered 3D nano-sized protein crystals. Electron diffraction may fill this important niche, but several problems have to be surmounted for this method to become mainstream. Our group aims tackling some of the bottlenecks, and although our work is still very much in progress, we can report advances in some important areas. Here we summarise improvements in (i) the induction of growth of (nano-)crystals, (ii) electron diffraction data collection using the Medipix quantum area detector, (iii) unit cell determination using single, non- oriented diffraction patterns, (iv) integration of diffraction data and (v) phasing of electron diffraction data.