in situ x-ray crystallographic data collection using the beamline at diamond light source

Automated Protein Crystallization and Real-Time Data Collection

X-ray crystallography remains a key tool for understanding protein structure and function, providing high-resolution structures of proteins or their complexes with, for example, substrates or drug candidates. In many cases, however, obtaining crystals with desirable properties - highly diffracting, crystal form amenable to soaking and without crystal pathologies such as twinning - remains a considerable bottleneck1. As suitable chemical conditions to produce protein crystals cannot, in general, be predicted, crystallization screening exploring thousands of potential chemical mixtures is standard, often aided by automation/robotics in setting screens and crystal hotels for monitoring, often remotely, the crystallization drop images that are recorded.
When crystals appear, typically they must be harvested from the crystallization environment using a nylon or Kapton loop and then, transferred to a droplet containing a cryoprotection agent (the search for which is an additional variable) before plunge-freezing into liquid nitrogen. These additional steps between crystallization and X-ray data collection can involve dehydration of the crystallization drop when its sealed environment is broken, mechanical stresses on the crystal when it is handled, and damage from the cryoprotection agents to the crystal lattice (typically resulting in increased mosaic spread) amongst other factors2. In addition, crystal harvesting is time- and labor-intensive and can lead to inhomogeneity between samples, especially when skin forms on drops during the harvesting process. The VMXi beamline gives access to useable data from crystals that are stuck to the plate, which would otherwise be discarded for data collection.
The vast majority of X-ray crystal structures are determined at 100K using the above approach, enabling straightforward crystal transport and handling and increasing crystal lifetime in the X-ray beam by orders of magnitude. There is increasing interest, however, in determining structures under non-cryogenic conditions, that is, much closer to the physiological conditions relevant to protein function2,3,4. This enables a much better appreciation of the dynamic structure of proteins, avoids amino acid conformations or loops being frozen out in functionally non-relevant states5, and enables ligand binding to be explored under conditions much closer to those in the protein&#39;s natural environment within the cell and organism6.
An alternative approach, implemented at the Versatile Macromolecular Crystallography in situ (VMXi) beamline at the Diamond Light Source synchrotron, UK, is to measure the diffraction data directly from crystals within the environment in which they have grown (i.e., within the crystallization plate), under ambient conditions and without disturbance7,8. This enables very rapid feedback from crystallization screens and optimizations to guide a user to an optimal crystal form for their requirements. It also enables high-quality room-temperature structures to be produced in an automated manner9.
This protocol assumes that a user has a highly pure protein sample ready for crystallization. We describe the user experience accessing the Crystallization Facility at Harwell to produce protein crystals and then use beamline VMXi for data collection (Figure 1).
The Crystallization Facility at Harwell
The Crystallization Facility at Harwell (CF) is located in the Research Complex at Harwell (RCaH) adjacent to the Diamond Light Source. The facility offers users a high-throughput automated laboratory for macromolecular crystallization, using robotics for crystallization screening, crystal optimization, crystal imaging, and characterization. Through close integration with the highly automated VMXi beamline, the pace of determining room temperature structures has greatly accelerated and enables the characterization of novel protein structures, protein-ligand and DNA-ligand complexes, as well as automated fragment screening (Figure 1), all under non-cryogenic conditions.
The CF pipeline is a suite of instrumentation encompassing nanoliter crystallization robots9 for the crystallization of soluble and membrane proteins, liquid handling robots to prepare commercial crystallization screens and complex custom optimization screens, and four imaging instruments (one at 4 &#176;C and three at 20 &#176;C for the imaging of crystallization plates (see the Table of Materials). One imager is capable of imaging lipid cubic phase (LCP) glass plates and one imager is equipped with multi-fluorescence optics (both at 20 &#176;C).
The facility is now used widely by a broad spectrum of academic and industrial users, including the Membrane Protein Laboratory (MPL;https://www.diamond.ac.uk/Instruments/Mx/MPL.html), the XChem fragment screening facility 10, MX beamlines, the XFEL-hub, as well as the Rosalind Franklin Institute (RFI). This well-established and optimized pipeline has enabled crystallization experiments to be performed across a wide spectrum of structural biology projects. This paper describes the pipeline for crystals intended for data collection at VMXi, although crystals may also be harvested and cryo-cooled or directed to the XChem pipeline.
User access is allocated through the Diamond MX proposal system (https://www.diamond.ac.uk/Instruments/Mx/Synchrotron-Access.html) and industrial users are supported through the Diamond Industry Liaison group. All users can come to the site with their sample(s) or plates, which can be transported by hand. It is not recommended to send plates by courier as our experience suggests that drops can move away from the location in which they were dispensed, or drops may be damaged by the crystallization reservoir. Alternatively, by arrangement, users can send their protein samples to the CF, where members of staff set up crystallization experiments on their behalf. The experiments can be monitored remotely by the user by either logging on to Rock Maker Web in the case of CF or via ISPyB in the case of VMXi. Access to the CF can be carried out in an iterative fashion based on the X-ray diffraction results collected at Diamond.
Beamline VMXi at Diamond Light Source
Beamline VMXi (hereafter referred to as &#34;the beamline&#34;) is a unique and recently developed instrument fully dedicated to room-temperature, highly automated X-ray crystallography with a focus on measuring data from crystals within suitable crystallization plates. The beamline offers a micro focus (10 x 10 &#181;m), pink beam (band pass of &#60;5 &#215; 10-2&#916;E/E) with a high flux of ~2 &#215; 1013 photons/s (at 16 KeV)7. This high-flux beam, coupled with a fast detector, enables very high throughput of samples and collection of data from samples upwards of 10 &#181;m in size.
Crystallization plates enter the beamline by being stored in a sample storage system and imaged based on the schedule provided by the user while registering the plates using the ISPyB11 interface SynchWeb12. Typically, users are advised to select a Fibonacci sequence of time points for imaging (0, 12, 24, 36, 60&#8230;7,320 h from the plate being entered into the system). The user is informed by email once a plate has been imaged. Both visible light and UV light imaging are available to users on demand. The images taken by the sample storage system are analyzed by a machine learning algorithm; this automatically locates and defines points of interest of objects that resemble crystals and registers the points of interest ready for the user to add to a queue for data collection. Users may also manually click on the visible light images to register points of interest or can click and drag a region to be analyzed by raster scan. These points are available to users to add to the queue alongside the automatically located points.
Once all samples have appropriate parameters for data collection, the plate enters a queue. When the plate reaches the top of the queue, it is automatically dispensed to the beamline. The crystallization plates are loaded from the crystal hotels into the beamline automatically by a robotic arm, and following image matching, crystallographic data sets of up to 60&#176; rotation are measured from each selected crystal as per user defined instructions. All drops within a plate may be used for these experiments on the beamline. Data are merged from multiple crystals to produce isomorphous, optimally merged data sets in an automated manner7,9. Once all of the queued data sets are collected, the user is sent an email with a link to follow to view the data sets in ISPyB11, as in other Diamond MX beamlines. Users are also directed to the beamline web page (https://www.diamond.ac.uk/Instruments/Mx/VMXi.html).&#160;

Author Spotlight: Advancing Protein Structure Analysis for Drug Development 

Crystallization and In Situ Room Temperature Data Collection Using the Crystallization Facility at Harwell and Beamline VMXi, Diamond Light Source

Understanding the three dimensional structures of proteins underpins drug design, biotechnology, and our understanding of many different diseases. VMXI determines protein structures without cryo cooling, so much closer to their natural temperatures. VMXI also measures diffraction data from protein crystals while still in their crystallization plates, avoiding any damage from mechanical handling.In the future, we really want to exploit the incredible throughput of VMXI to explore large populations of crystals to identify lots of different structural states rather than the single structure people often obtain now. We also want to push towards drug development and carrying out fragment screening and optimization within crystallization plates. A new approach that shows enormous promise.

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Research

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Biochemistry

1.0K visualizações.  Harwell Science and Innovation Campus. Para começar, produza cristais dentro das placas NC2. Em seguida, faça login no sistema I-SPY B usando o ID Diamond Fed fornecido e selecione Propostas. Role a lista ou digite o número da proposta na barra de pesquisa para localizar a proposta desejada.Assim que a proposta for encontrada, escolha a remessa no menu suspenso abaixo do número da proposta. A janela de remessa será aberta exibindo todas as remessas relacionadas à proposta. Para criar uma nova remessa, clique em adicio...