Diffraction cartography: applying microbeams to macromolecular crystallography sample evaluation and data collection

2010 ◽  
Vol 66 (8) ◽  
pp. 855-864 ◽  
Author(s):  
Matthew W. Bowler ◽  
Matias Guijarro ◽  
Sebastien Petitdemange ◽  
Isabel Baker ◽  
Olof Svensson ◽  
...  
Keyword(s):  
Data Collection ◽  
Structural Biology ◽  
Graphical User Interface ◽  
Screening Methods ◽  
Biological Macromolecules ◽  
Macromolecular Crystallography ◽  
Large Crystal ◽  
X Ray ◽  
Automated Methods

Crystals of biological macromolecules often exhibit considerable inter-crystal and intra-crystal variation in diffraction quality. This requires the evaluation of many samples prior to data collection, a practice that is already widespread in macromolecular crystallography. As structural biologists move towards tackling ever more ambitious projects, new automated methods of sample evaluation will become crucial to the success of many projects, as will the availability of synchrotron-based facilities optimized for high-throughput evaluation of the diffraction characteristics of samples. Here, two examples of the types of advanced sample evaluation that will be required are presented: searching within a sample-containing loop for microcrystals using an X-ray beam of 5 µm diameter and selecting the most ordered regions of relatively large crystals using X-ray beams of 5–50 µm in diameter. A graphical user interface developed to assist with these screening methods is also presented. For the case in which the diffraction quality of a relatively large crystal is probed using a microbeam, the usefulness and implications of mapping diffraction-quality heterogeneity (diffraction cartography) are discussed. The implementation of these techniques in the context of planned upgrades to the ESRF's structural biology beamlines is also presented.

scholarly journals Energy optimization of a regular macromolecular crystallography beamline for ultra-high-resolution crystallography

2015 ◽  
Vol 22 (1) ◽  
pp. 172-174 ◽  
Author(s):  
Gerd Rosenbaum ◽  
Stephan L. Ginell ◽  
Julian C.-H. Chen
Keyword(s):  
High Resolution ◽  
Data Collection ◽  
Structural Biology ◽  
Practical Method ◽  
Accurate Data ◽  
Macromolecular Crystallography ◽  
X Ray ◽  
Standard Operating ◽  
Photon Source ◽  
Synchrotron Beamlines

A practical method for operating existing undulator synchrotron beamlines at photon energies considerably higher than their standard operating range is described and applied at beamline 19-ID of the Structural Biology Center at the Advanced Photon Source enabling operation at 30 keV. Adjustments to the undulator spectrum were critical to enhance the 30 keV flux while reducing the lower- and higher-energy harmonic contamination. A Pd-coated mirror and Al attenuators acted as effective low- and high-bandpass filters. The resulting flux at 30 keV, although significantly lower than with X-ray optics designed and optimized for this energy, allowed for accurate data collection on crystals of the small protein crambin to 0.38 Å resolution.

scholarly journals Cool data: quantity AND quality

1999 ◽  
Vol 55 (10) ◽  
pp. 1641-1653 ◽  
Author(s):  
Elspeth Garman
Keyword(s):  
Experimental Data ◽  
Data Collection ◽  
Radiation Damage ◽  
Macromolecular Crystallography ◽  
X Ray ◽  
Vital Part ◽  
Data Collection Methods ◽  
Collection Methods

The use of cryo-techniques in macromolecular crystallography has increased enormously over the last eight years and has become a vital part of modern X-ray data-collection methods. This paper presents some reasons for the rise in popularity of cryo-techniques and a brief outline of the basic methods, followed by a detailed discussion of factors to be considered when trying to optimize both the quantity and quality of the data collected. As more experimenters at synchrotrons observe significant radiation damage to crystals held near 100 K, the available options for further prolonging crystal lifetime and extending the techniques become worth investigating. Some possibilities and parameters to be considered are presented, although these must remain speculative until more experimental data are available.

ID14 `Quadriga', a Beamline for Protein Crystallography at the ESRF

1998 ◽  
Vol 5 (3) ◽  
pp. 215-221 ◽  
Author(s):  
S. Wakatsuki ◽  
H. Belrhali ◽  
E. P. Mitchell ◽  
W. P. Burmeister ◽  
S. M. McSweeney ◽  
...  
Keyword(s):  
User Interface ◽  
Data Collection ◽  
Graphical User Interface ◽  
Macromolecular Crystallography ◽  
Double Crystal ◽  
X Ray ◽  
Double Crystal Monochromator ◽  
Diamond Crystals ◽  
Ccd Detector ◽  
First Results

The ESRF undulator beamline ID14 `Quadriga' is dedicated to monochromatic macromolecular crystallography. Using two undulators with 23 mm and 42 mm periods and a minimum gap of 16 mm installed on a high-β section, it will provide high-brilliance X-ray beams at around 13.5 keV, as well as a wide tuneability between 6.8 and 40 keV. Based on the Troika concept, this beamline has four simultaneously operating experimental stations: three side stations, EH1, EH2 and EH3, using thin diamond crystals, and an end station, EH4, with a fast-scan double-crystal monochromator. Station EH3 has a κ-diffractometer, and an off-line Weissenberg camera with a large 80 × 80 cm active area combined with a 2048 × 2048 CCD detector. During data collection the image plates are placed and removed by a robot located inside the hutch using a cassette system. After data collection the image plates are scanned with an off-line drum scanner. Station EH4 is designed for MAD applications, including Xe K-edge anomalous experiments, and is equipped with a 2048 × 2048 CCD detector on a pseudo 2θ arm. A common graphical user interface and a database will be available to cover all aspects of data collection, including strategy optimization. First results on the performance of the optics elements and initial crystallographic results are presented.

scholarly journals Protein microcrystallography using synchrotron radiation

IUCrJ ◽  
2017 ◽  
Vol 4 (5) ◽  
pp. 529-539 ◽  
Author(s):  
Masaki Yamamoto ◽  
Kunio Hirata ◽  
Keitaro Yamashita ◽  
Kazuya Hasegawa ◽  
Go Ueno ◽  
...  
Keyword(s):  
Synchrotron Radiation ◽  
Data Collection ◽  
Diffraction Data ◽  
Structure Determination ◽  
Structural Features ◽  
X Ray ◽  
Data Collection Strategy ◽  
Technological Developments ◽  
Novel Method

The progress in X-ray microbeam applications using synchrotron radiation is beneficial to structure determination from macromolecular microcrystals such as smallin mesocrystals. However, the high intensity of microbeams causes severe radiation damage, which worsens both the statistical quality of diffraction data and their resolution, and in the worst cases results in the failure of structure determination. Even in the event of successful structure determination, site-specific damage can lead to the misinterpretation of structural features. In order to overcome this issue, technological developments in sample handling and delivery, data-collection strategy and data processing have been made. For a few crystals with dimensions of the order of 10 µm, an elegant two-step scanning strategy works well. For smaller samples, the development of a novel method to analyze multiple isomorphous microcrystals was motivated by the success of serial femtosecond crystallography with X-ray free-electron lasers. This method overcame the radiation-dose limit in diffraction data collection by using a sufficient number of crystals. Here, important technologies and the future prospects for microcrystallography are discussed.

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.

scholarly journals Time-Resolved Macromolecular Crystallography at Pulsed X-ray Sources

2019 ◽  
Vol 20 (6) ◽  
pp. 1401 ◽  
Author(s):  
Marius Schmidt
Keyword(s):  
Structural Biology ◽  
Free Electron ◽  
Reaction Dynamics ◽  
X Rays ◽  
Free Electron Lasers ◽  
Macromolecular Crystallography ◽  
Time Resolved ◽  
X Ray ◽  
Methodological Aspects

The focus of structural biology is shifting from the determination of static structures to the investigation of dynamical aspects of macromolecular function. With time-resolved macromolecular crystallography (TRX), intermediates that form and decay during the macromolecular reaction can be investigated, as well as their reaction dynamics. Time-resolved crystallographic methods were initially developed at synchrotrons. However, about a decade ago, extremely brilliant, femtosecond-pulsed X-ray sources, the free electron lasers for hard X-rays, became available to a wider community. TRX is now possible with femtosecond temporal resolution. This review provides an overview of methodological aspects of TRX, and at the same time, aims to outline the frontiers of this method at modern pulsed X-ray sources.

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.

Small-Angle X-Ray Scattering for the Discerning Macromolecular Crystallographer

2014 ◽  
Vol 67 (12) ◽  
pp. 1786 ◽  
Author(s):  
Lachlan W. Casey ◽  
Alan E. Mark ◽  
Bostjan Kobe
Keyword(s):  
Structural Biology ◽  
Small Angle ◽  
Significant Risk ◽  
Macromolecular Crystallography ◽  
Saxs Data ◽  
X Ray ◽  
X Ray Scattering ◽  
Ray Scattering

The role of small-angle X-ray scattering (SAXS) in structural biology is now well established, and its usefulness in combination with macromolecular crystallography is clear. However, the highly averaged SAXS data present a significant risk of over-interpretation to the unwary practitioner, and it can be challenging to frame SAXS results in a manner that maximises the reliability of the conclusions drawn. In this review, a series of recent examples are used to illustrate both the challenges for interpretation and approaches through which these can be overcome.

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.

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