sample-preparation-transfer-protocol-for-vacuum-long-wavelength

Sample Preparation and Transfer Protocol for In-Vacuum Long-Wavelength Crystallography on Beamline I23 at Diamond Light Source

Long-wavelength macromolecular crystallography (MX) exploits the anomalous scattering properties of elements, such as sulfur, phosphorus, potassium, ...

Diamond Light Source Ltd, Harwell Science & Innovation Campus

Ultralente insulin has been one of the commercially most important insulin preparations in diabetes treatment over the last 50 years. It is a suspension of insulin microcrystals which dissolve slowly following subcutaneous injection. Because of the small crystal size of about 25 x 25 x 5 microm(3) the atomic structure has been elusive until now. Here we present the crystal structures from Ultralente and their precursor microcrystals from the industrial manufacturing process. During this process insulin undergoes a conformational change within the microcrystals. Both structures show canonical folding of the insulin molecules but exhibit a number of new features when compared with other insulin structures. Surprisingly, we found that the Ultralente crystals bind the conservation agent methylparaben, which slows down dissolution of the crystals and thus contributes to the long duration of action.

Crystal structure of ultralente--a microcrystalline insulin suspension.

X-ray crystallography is the method of choice to deduce atomic resolution structural information from macromolecules. In recent years, significant investments in structural genomics initiatives have been undertaken to automate all steps in X-ray crystallography from protein expression to structure solution. Robotic systems are widely used to prepare crystallization screens and change samples on synchrotron beamlines for macromolecular crystallography. The only remaining manual handling step is the transfer of the crystal from the mother liquor onto the crystal holder. Manual mounting is relatively straightforward for crystals with dimensions of >25â€…Âµm; however, this step is nontrivial for smaller crystals. The mounting of microcrystals is becoming increasingly important as advances in microfocus synchrotron beamlines now allow data collection from crystals with dimensions of only a few micrometres. To make optimal usage of these beamlines, new approaches have to be taken to facilitate and automate this last manual handling step. Optical tweezers, which are routinely used for the manipulation of micrometre-sized objects, have successfully been applied to sort and mount macromolecular crystals on newly designed crystal holders. Diffraction data from CPV type 1 polyhedrin microcrystals mounted with laser tweezers are presented.

Microcrystal manipulation with laser tweezers.

Cristallographie aux rayons x est la m&eacute;thode de choix pour d&eacute;duire l&#39;information structurale de r&eacute;solution atomique de macromol&eacute;cules. Ces derni&egrave;res ann&eacute;es, des investissements importants en g&eacute;nomique structurale des initiatives ont &eacute;t&eacute; prises pour automatiser toutes les &eacute;tapes en cristallographie aux rayons x de l&#39;expression de la prot&eacute;ine &agrave; la solution de la structure. Syst&egrave;mes robotis&eacute;s sont largement utilis&eacute;s pour pr&eacute;parer des &eacute;crans de cristallisation et changer des &eacute;chantillons sur des faisceaux de rayonnement synchrotron pour la cristallographie macromol&eacute;culaire. La seule &eacute;tape de manutention manuelle restante est le transfert du cristal de la liqueur m&egrave;re au support de crystal. Manuel de montage est relativement simple pour les cristaux avec des dimensions de &gt; 25 &micro;m ; Toutefois, cette &eacute;tape est non trivial pour les plus petits cristaux. Le montage des microcristaux devient de plus en plus important comme les avances en rayonnement synchrotron microfoyer permettent maintenant de collecte de donn&eacute;es aupr&egrave;s des cristaux avec des dimensions de seulement quelques microm&egrave;tres. Pour faire une utilisation optimale de ces faisceaux de lumi&egrave;re, nouvelles approches doivent &ecirc;tre prises afin de faciliter et d&#39;automatiser cette derni&egrave;re &eacute;tape de la manutention manuelle. Les pinces optiques, qui sont couramment utilis&eacute;s pour la manipulation d&#39;objets microm&egrave;tre, ont &eacute;t&eacute; appliqu&eacute;s avec succ&egrave;s pour trier et monter des cristaux macromol&eacute;culaires sur supports en cristal nouvellement con&ccedil;u. Type de donn&eacute;es de diffraction de CPV 1 poly&eacute;drine microcristaux mont&eacute; avec des pincettes laser est pr&eacute;sent&eacute;s.

Microcristal manipulation avec des pincettes de laser.

Macromolecular crystallography (MX) is the most powerful technique available to structural biologists to visualize in atomic detail the macromolecular machinery of the cell. Since the emergence of structural genomics initiatives, significant advances have been made in all key steps of the structure determination process. In particular, third-generation synchrotron sources and the application of highly automated approaches to data acquisition and analysis at these facilities have been the major factors in the rate of increase of macromolecular structures determined annually. A plethora of tools are now available to users of synchrotron beamlines to enable rapid and efficient evaluation of samples, collection of the best data, and in favorable cases structure solution in near real time. Here, we provide a short overview of the emerging use of collecting X-ray diffraction data directly from the crystallization experiment. These in situ experiments are now routinely available to users at a number of synchrotron MX beamlines. A practical guide to the use of the method on the MX suite of beamlines at Diamond Light Source is given.

Application of in situ diffraction in high-throughput structure determination platforms.

Recent success at X-ray free-electron lasers has led to serial crystallography experiments staging a comeback at synchrotron sources as well. With crystal lifetimes typically in the millisecond range and the latest-generation detector technologies with high framing rates up to 1 kHz, fast sample exchange has become the bottleneck for such experiments. A micro-patterned chip has been developed from single-crystalline silicon, which acts as a sample holder for up to several thousand microcrystals at a very low background level. The crystals can be easily loaded onto the chip and excess mother liquor can be efficiently removed. Dehydration of the crystals is prevented by keeping them in a stream of humidified air during data collection. Further sealing of the sample holder, for example with Kapton, is not required. Room-temperature data collection from insulin crystals loaded onto the chip proves the applicability of the chip for macromolecular crystallography. Subsequent structure refinements reveal no radiation-damage-induced structural changes for insulin crystals up to a dose of 565.6 kGy, even though the total diffraction power of the crystals has on average decreased to 19.1% of its initial value for the same dose. A decay of the diffracting power by half is observed for a dose of  = 147.5 ± 19.1 kGy, which is about 1/300 of the dose before crystals show a similar decay at cryogenic temperatures.

Room-temperature macromolecular crystallography using a micro-patterned silicon chip with minimal background scattering.

Determination of protein crystal structures requires that the phases are derived independently of the observed measurement of diffraction intensities. Many techniques have been developed to obtain phases, including heavy-atom substitution, molecular replacement and substitution during protein expression of the amino acid methionine with selenomethionine. Although the use of selenium-containing methionine has transformed the experimental determination of phases it is not always possible, either because the variant protein cannot be produced or does not crystallize. Phasing of structures by measuring the anomalous diffraction from S atoms could in theory be almost universal since almost all proteins contain methionine or cysteine. Indeed, many structures have been solved by the so-called native sulfur single-wavelength anomalous diffraction (S-SAD) phasing method. However, the anomalous effect is weak at the wavelengths where data are normally recorded (between 1 and 2 Å) and this limits the potential of this method to well diffracting crystals. Longer wavelengths increase the strength of the anomalous signal but at the cost of increasing air absorption and scatter, which degrade the precision of the anomalous measurement, consequently hindering phase determination. A new instrument, the long-wavelength beamline I23 at Diamond Light Source, was designed to work at significantly longer wavelengths compared with standard synchrotron beamlines in order to open up the native S-SAD method to projects of increasing complexity. Here, the first novel structure, that of the oxidase domain involved in the production of the natural product patellamide, solved on this beamline is reported using data collected to a resolution of 3.15 Å at a wavelength of 3.1 Å. The oxidase is an example of a protein that does not crystallize as the selenium variant and for which no suitable homology model for molecular replacement was available. Initial attempts collecting anomalous diffraction data for native sulfur phasing on a standard macromolecular crystallography beamline using a wavelength of 1.77 Å did not yield a structure. The new beamline thus has the potential to facilitate structure determination by native S-SAD phasing for what would previously have been regarded as very challenging cases with modestly diffracting crystals and low sulfur content.

Structure of the cyanobactin oxidase ThcOx from Cyanothece sp. PCC 7425, the first structure to be solved at Diamond Light Source beamline I23 by means of S-SAD.

The ribosome, the largest RNA-containing macromolecular machinery in cells, requires metal ions not only to maintain its three-dimensional fold but also to perform protein synthesis. Despite the vast biochemical data regarding the importance of metal ions for efficient protein synthesis and the increasing number of ribosome structures solved by X-ray crystallography or cryo-electron microscopy, the assignment of metal ions within the ribosome remains elusive due to methodological limitations. Here we present extensive experimental data on the potassium composition and environment in two structures of functional ribosome complexes obtained by measurement of the potassium anomalous signal at the K-edge, derived from long-wavelength X-ray diffraction data. We elucidate the role of potassium ions in protein synthesis at the three-dimensional level, most notably, in the environment of the ribosome functional decoding and peptidyl transferase centers. Our data expand the fundamental knowledge of the mechanism of ribosome function and structural integrity.

Importance of potassium ions for ribosome structure and function revealed by long-wavelength X-ray diffraction.

Although often presented as taking single `snapshots' of the conformation of a protein, X-ray crystallography provides an averaged structure over time and space within the crystal. The important but difficult task of characterizing structural ensembles in crystals is typically limited to small conformational changes, such as multiple side-chain conformations. A crystallographic method was recently introduced that utilizes residual electron and anomalous density (READ) to characterize structural ensembles encompassing large-scale structural changes. Key to this method is an ability to accurately measure anomalous signals and distinguish them from noise or other anomalous scatterers. This report presents an optimized data-collection and analysis strategy for partially occupied iodine anomalous signals. Using the long-wavelength-optimized beamline I23 at Diamond Light Source, the ability to accurately distinguish the positions of anomalous scatterers with occupancies as low as ∼12% is demonstrated. The number and positions of these anomalous scatterers are consistent with previous biophysical, kinetic and structural data that suggest that the protein Im7 binds to the chaperone Spy in multiple partially occupied conformations. Finally, READ selections demonstrate that re-measured data using the new protocols are consistent with the previously characterized structural ensemble of the chaperone Spy with its client Im7. This study shows that a long-wavelength beamline results in easily validated anomalous signals that are strong enough to be used to detect and characterize highly disordered sections of crystal structures.

Article	Year
Crystal structure of ultralente--a microcrystalline insulin suspension. Proteins\| PubMed ID: 18767151	2009
Microcrystal manipulation with laser tweezers. Acta crystallographica. Section D, Biological crystallography\| PubMed ID: 23793156	2013
Application of in situ diffraction in high-throughput structure determination platforms. Methods in molecular biology (Clifton, N.J.)\| PubMed ID: 25502203	2015
Room-temperature macromolecular crystallography using a micro-patterned silicon chip with minimal background scattering. Journal of applied crystallography\| PubMed ID: 27275143	2016
Structure of the cyanobactin oxidase ThcOx from Cyanothece sp. PCC 7425, the first structure to be solved at Diamond Light Source beamline I23 by means of S-SAD. Acta crystallographica. Section D, Structural biology\| PubMed ID: 27841750	2016
Importance of potassium ions for ribosome structure and function revealed by long-wavelength X-ray diffraction. Nature communications\| PubMed ID: 31175275	2019
Identifying dynamic, partially occupied residues using anomalous scattering. Acta crystallographica. Section D, Structural biology\| PubMed ID: 31793902	2019

Armin Wagner

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