scholarly journals Getting the Most Out of Your Crystals: Data Collection at the New High-Flux, Microfocus MX Beamlines at NSLS-II

Molecules ◽  
2019 ◽  
Vol 24 (3) ◽  
pp. 496 ◽  
Author(s):  
Michelle Miller ◽  
Sweta Maheshwari ◽  
Wuxian Shi ◽  
Yuan Gao ◽  
Nam Chu ◽  
...  
Keyword(s):  
Data Collection ◽  
State Of The Art ◽  
Crystallographic Data ◽  
High Flux ◽  
Macromolecular Crystallography ◽  
Vector Data ◽  
Linear Trajectory ◽  
X Ray ◽  
Single Loop ◽  
Collection Strategies

Advances in synchrotron technology are changing the landscape of macromolecular crystallography. The two recently opened beamlines at NSLS-II—AMX and FMX—deliver high-flux microfocus beams that open new possibilities for crystallographic data collection. They are equipped with state-of-the-art experimental stations and automation to allow data collection on previously intractable crystals. Optimized data collection strategies allow users to tailor crystal positioning to optimally distribute the X-ray dose over its volume. Vector data collection allows the user to define a linear trajectory along a well diffracting volume of the crystal and perform rotational data collection while moving along the vector. This is particularly well suited to long, thin crystals. We describe vector data collection of three proteins—Akt1, PI3Kα, and CDP-Chase—to demonstrate its application and utility. For smaller crystals, we describe two methods for multicrystal data collection in a single loop, either manually selecting multiple centers (using H108A-PHM as an example), or “raster-collect”, a more automated approach for a larger number of crystals (using CDP-Chase as an example).

scholarly journals Automated harvesting and processing of protein crystals through laser photoablation

2016 ◽  
Vol 72 (4) ◽  
pp. 454-466 ◽  
Author(s):  
Ulrich Zander ◽  
Guillaume Hoffmann ◽  
Irina Cornaciu ◽  
Jean-Pierre Marquette ◽  
Gergely Papp ◽  
...  
Keyword(s):  
Data Collection ◽  
Macromolecular Crystallography ◽  
X Ray Diffraction ◽  
X Ray ◽  
New Methods ◽  
Repeated Sample ◽  
Sample Recovery ◽  
Through Diffusion ◽  
X Ray Background ◽  
Low X

Currently, macromolecular crystallography projects often require the use of highly automated facilities for crystallization and X-ray data collection. However, crystal harvesting and processing largely depend on manual operations. Here, a series of new methods are presented based on the use of a low X-ray-background film as a crystallization support and a photoablation laser that enable the automation of major operations required for the preparation of crystals for X-ray diffraction experiments. In this approach, the controlled removal of the mother liquor before crystal mounting simplifies the cryocooling process, in many cases eliminating the use of cryoprotectant agents, while crystal-soaking experiments are performed through diffusion, precluding the need for repeated sample-recovery and transfer operations. Moreover, the high-precision laser enables new mounting strategies that are not accessible through other methods. This approach bridges an important gap in automation and can contribute to expanding the capabilities of modern macromolecular crystallography facilities.

Optimizing the spatial distribution of dose in X-ray macromolecular crystallography

2012 ◽  
Vol 20 (1) ◽  
pp. 49-57 ◽  
Author(s):  
Oliver B. Zeldin ◽  
Markus Gerstel ◽  
Elspeth F. Garman
Keyword(s):  
Spatial Distribution ◽  
Data Collection ◽  
Rotation Axis ◽  
Macromolecular Crystallography ◽  
X Ray ◽  
Crystal Shapes ◽  
Peak Dose ◽  
Wide Range ◽  
Helical Scan ◽  
Crystal Volume

X-ray data collection for macromolecular crystallography can lead to highly inhomogeneous distributions of dose within the crystal volume for cases when the crystal is larger than the beam or when the beam is non-uniform (Gaussian-like), particularly when crystal rotation is fully taken into account. Here the spatial distribution of dose is quantitatively modelled in order to compare the effectiveness of two dose-spreading data-collection protocols: helical scanning and translational collection. Their effectiveness in reducing the peak dose per unit diffraction is investigatedviasimulations for four common crystal shapes (cube, plate, long and short needles) and beams with a wide range of full width half maximum values. By inspection of the chosen metric, it is concluded that the optimum strategy is always to use as flat (top-hat) a beam as possible and to either match the beam size in both dimensions to the crystal, or to perform a helical scan with a beam which is narrow along the rotation axis and matched to the crystal size along the perpendicular axis. For crystal shapes where this is not possible, the reduction in peak dose per unit diffraction achieved through dose spreading is quantified and tabulated as a reference for experimenters.

scholarly journals Implementation and performance of SIBYLS: a dual endstation small-angle X-ray scattering and macromolecular crystallography beamline at the Advanced Light Source

2013 ◽  
Vol 46 (1) ◽  
pp. 1-13 ◽  
Author(s):  
Scott Classen ◽  
Greg L. Hura ◽  
James M. Holton ◽  
Robert P. Rambo ◽  
Ivan Rodic ◽  
...  
Keyword(s):  
Data Collection ◽  
Light Source ◽  
Small Angle ◽  
National Laboratory ◽  
Department Of Energy ◽  
Macromolecular Crystallography ◽  
X Ray ◽  
X Ray Scattering ◽  
Advanced Light Source ◽  
Ray Scattering

The SIBYLS beamline (12.3.1) of the Advanced Light Source at Lawrence Berkeley National Laboratory, supported by the US Department of Energy and the National Institutes of Health, is optimized for both small-angle X-ray scattering (SAXS) and macromolecular crystallography (MX), making it unique among the world's mostly SAXS or MX dedicated beamlines. Since SIBYLS was commissioned, assessments of the limitations and advantages of a combined SAXS and MX beamline have suggested new strategies for integration and optimal data collection methods and have led to additional hardware and software enhancements. Features described include a dual mode monochromator [containing both Si(111) crystals and Mo/B4C multilayer elements], rapid beamline optics conversion between SAXS and MX modes, active beam stabilization, sample-loading robotics, and mail-in and remote data collection. These features allow users to gain valuable insights from both dynamic solution scattering and high-resolution atomic diffraction experiments performed at a single synchrotron beamline. Key practical issues considered for data collection and analysis include radiation damage, structural ensembles, alternative conformers and flexibility. SIBYLS develops and applies efficient combined MX and SAXS methods that deliver high-impact results by providing robust cost-effective routes to connect structures to biology and by performing experiments that aid beamline designs for next generation light sources.

RADDOSE-3D: time- and space-resolved modelling of dose in macromolecular crystallography

2013 ◽  
Vol 46 (4) ◽  
pp. 1225-1230 ◽  
Author(s):  
Oliver B. Zeldin ◽  
Markus Gerstel ◽  
Elspeth F. Garman
Keyword(s):  
Data Collection ◽  
Radiation Damage ◽  
Diffraction Experiment ◽  
Macromolecular Crystallography ◽  
X Ray Diffraction ◽  
Time Resolved ◽  
X Ray ◽  
Online Methods ◽  
Crystal Volume ◽  
Number Of Iterations

RADDOSE-3D allows the macroscopic modelling of an X-ray diffraction experiment for the purpose of better predicting radiation-damage progression. The distribution of dose within the crystal volume is calculated for a number of iterations in small angular steps across one or more data collection wedges, providing a time-resolved picture of the dose state of the crystal. The code is highly modular so that future contributions from the community can be easily integrated into it, in particular to incorporate online methods for determining the shape of macromolecular crystals and better protocols for imaging real experimental X-ray beam profiles.

RSPACE – a reciprocal-space modelling tool

1989 ◽  
Vol 22 (6) ◽  
pp. 624-627
Author(s):  
M. R. Harris ◽  
M. Fitzgibbon ◽  
F. Hage
Keyword(s):  
Computer Graphics ◽  
Data Collection ◽  
Reciprocal Space ◽  
Crystallographic Data ◽  
Complex Data ◽  
Educational Tool ◽  
Interactive Computer Graphics ◽  
Area Detector ◽  
The Relationship ◽  
Collection Strategies

RSPACE is an interactive computer-graphics program that models the relationship between crystallographic data-collection hardware and reciprocal space. It is designed to help crystallographers plan efficient complex data-collection strategies for area detector systems, and as an educational tool. Because RSPACE models the interaction of crystal, detector and goniostat geometry in a general way, it is of particular interest to users of area detectors interfaced with multi-axis goniostats.

scholarly journals XAS at the Canadian Macromolecular Crystallography Facility

2014 ◽  
Vol 70 (a1) ◽  
pp. C1525-C1525
Author(s):  
Julien Cotelesage ◽  
Pawel Grochulski ◽  
Michel Fodje ◽  
James Gorin ◽  
Kathryn Janzen ◽  
...  
Keyword(s):  
Data Collection ◽  
Diffraction Data ◽  
Three Dimensional ◽  
Minimal Risk ◽  
Macromolecular Crystallography ◽  
X Ray ◽  
Biologically Relevant ◽  
Wide Range ◽  
X Ray Absorption ◽  
Do So

Recent additions to the Canadian Macromolecular Crystallography Facility have expanded the capabilities of its bending magnet beamline. It is now possible to perform x-ray absorption spectroscopy (XAS) on crystals. A wide range of biologically relevant metals can be further studied, supplementing diffraction data. XAS can be used to determine if metalloproteins are photoreducing during diffraction data collection. The geometries of metal complexes can also be inferred with near-edge and EXAFS data, often more accurately than crystallography. CMCF-BM can be employed to do the abovementioned techniques on powder and solution samples that contain a metal of interest. One XAS-based technique that shows promise is single crystal plane polarized EXAFS. This technique combines crystallographic data with the findings from XAS to yield a high resolution three dimensional atomic model. More recently a number of the procedural steps required for the acquisition of XAS-based data have been automated in the MxDC software suite. These changes to data collection make it easier for users new to these disciplines to run the XAS-based experiments. By having the necessary equipment to do XAS at a protein crystallography facility, researchers who may not have had the opportunity delve into the field of XAS now can do so with minimal risk in terms of materials, funds and time.

scholarly journals Low-dose in situ prelocation of protein microcrystals by 2D X-ray phase-contrast imaging for serial crystallography

IUCrJ ◽  
2020 ◽  
Vol 7 (6) ◽  
pp. 1131-1141
Author(s):  
Isabelle Martiel ◽  
Chia-Ying Huang ◽  
Pablo Villanueva-Perez ◽  
Ezequiel Panepucci ◽  
Shibom Basu ◽  
...  
Keyword(s):  
Data Collection ◽  
Phase Contrast ◽  
Low Dose ◽  
Phase Contrast Imaging ◽  
Cubic Phase ◽  
Macromolecular Crystallography ◽  
Contrast Imaging ◽  
X Ray ◽  
Serial Data

Serial protein crystallography has emerged as a powerful method of data collection on small crystals from challenging targets, such as membrane proteins. Multiple microcrystals need to be located on large and often flat mounts while exposing them to an X-ray dose that is as low as possible. A crystal-prelocation method is demonstrated here using low-dose 2D full-field propagation-based X-ray phase-contrast imaging at the X-ray imaging beamline TOMCAT at the Swiss Light Source (SLS). This imaging step provides microcrystal coordinates for automated serial data collection at a microfocus macromolecular crystallography beamline on samples with an essentially flat geometry. This prelocation method was applied to microcrystals of a soluble protein and a membrane protein, grown in a commonly used double-sandwich in situ crystallization plate. The inner sandwiches of thin plastic film enclosing the microcrystals in lipid cubic phase were flash cooled and imaged at TOMCAT. Based on the obtained crystal coordinates, both still and rotation wedge serial data were collected automatically at the SLS PXI beamline, yielding in both cases a high indexing rate. This workflow can be easily implemented at many synchrotron facilities using existing equipment, or potentially integrated as an online technique in the next-generation macromolecular crystallography beamline, and thus benefit a number of dose-sensitive challenging protein targets.

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