Long-wavelength Macromolecular Crystallography Research Articles

Location and identification of light element co-factors by long-wavelength macromolecular crystallography

Location and identification of light element co-factors by long-wavelength macromolecular crystallography

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, chlorine, or calcium, that are often natively present in macromolecules. This enables the direct structure solution of proteins and nucleic acids via experimental phasing without the need of additional labelling. To eliminate the significant air absorption of X-rays in this wavelength regime, these experiments are performed in a vacuum environment. Beamline I23 at Diamond Light Source, UK, is the first synchrotron instrument of its kind, designed and optimized for MX experiments in the long wavelength range towards 5 Å. To make this possible, a large vacuum vessel encloses all endstation components of the sample environment. The necessity to maintain samples at cryogenic temperatures during storage and data collection in vacuum requires the use of thermally conductive sample holders. This facilitates efficient heat removal to ensure sample cooling to approximately 50 K. The current protocol describes the procedures used for sample preparation and transfer of samples into vacuum on beamline I23. Ensuring uniformity in practices and methods already established within the macromolecular crystallography community, sample cooling to liquid nitrogen temperature can be performed in any laboratory setting equipped with standard MX tools. Cryogenic storage and transport of samples only require standard commercially available equipment. Specialized equipment is required for the transfer of cryogenically cooled crystals from liquid nitrogen into the vacuum endstation. Bespoke sample handling tools and a dedicated Cryogenic Transfer System (CTS) have been developed in house. Diffraction data collected on samples prepared using this protocol show excellent merging statistics, indicating that the quality of samples is unaltered during the procedure. This opens unique opportunities for in-vacuum MX in a wavelength range beyond standard synchrotron beamlines.

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, chlorine, or calcium, that are often natively present in macromolecules. This enables the direct structure solution of proteins and nucleic acids via experimental phasing without the need of additional labelling. To eliminate the significant air absorption of X-rays in this wavelength regime, these experiments are performed in a vacuum environment. Beamline I23 at Diamond Light Source, UK, is the first synchrotron instrument of its kind, designed and optimized for MX experiments in the long wavelength range towards 5 Å. To make this possible, a large vacuum vessel encloses all endstation components of the sample environment. The necessity to maintain samples at cryogenic temperatures during storage and data collection in vacuum requires the use of thermally conductive sample holders. This facilitates efficient heat removal to ensure sample cooling to approximately 50 K. The current protocol describes the procedures used for sample preparation and transfer of samples into vacuum on beamline I23. Ensuring uniformity in practices and methods already established within the macromolecular crystallography community, sample cooling to liquid nitrogen temperature can be performed in any laboratory setting equipped with standard MX tools. Cryogenic storage and transport of samples only require standard commercially available equipment. Specialized equipment is required for the transfer of cryogenically cooled crystals from liquid nitrogen into the vacuum endstation. Bespoke sample handling tools and a dedicated Cryogenic Transfer System (CTS) have been developed in house. Diffraction data collected on samples prepared using this protocol show excellent merging statistics, indicating that the quality of samples is unaltered during the procedure. This opens unique opportunities for in-vacuum MX in a wavelength range beyond standard synchrotron beamlines.

X-ray tomographic reconstruction and segmentation pipeline for the long-wavelength macromolecular crystallography beamline at Diamond Light Source.

In this paper a practical solution for the reconstruction and segmentation of low-contrast X-ray tomographic data of protein crystals from the long-wavelength macromolecular crystallography beamline I23 at Diamond Light Source is provided. The resulting segmented data will provide the path lengths through both diffracting and non-diffracting materials as basis for analytical absorption corrections for X-ray diffraction data taken in the same sample environment ahead of the tomography experiment. X-ray tomography data from protein crystals can be difficult to analyse due to very low or absent contrast between the different materials: the crystal, the sample holder and the surrounding mother liquor. The proposed data processing pipeline consists of two major sequential operations: model-based iterative reconstruction to improve contrast and minimize the influence of noise and artefacts, followed by segmentation. The segmentation aims to partition the reconstructed data into four phases: the crystal, mother liquor, loop and vacuum. In this study three different semi-automated segmentation methods are experimented with by using Gaussian mixture models, geodesic distance thresholding and a novel morphological method, RegionGrow, implemented specifically for the task. The complete reconstruction-segmentation pipeline is integrated into the MPI-based data analysis and reconstruction framework Savu, which is used to reduce computation time through parallelization across a computing cluster and makes the developed methods easily accessible.

The Long-wavelength Macromolecular Crystallography I23 at Diamond Light Source

The Long-wavelength Macromolecular Crystallography I23 at Diamond Light Source

The long-wavelength macromolecular crystallography beamline I23 at Diamond Light Source

The long-wavelength macromolecular crystallography beamline I23 at Diamond Light Source

First results from the long-wavelength macromolecular crystallography beamline I23 at Diamond Light Source

First results from the long-wavelength macromolecular crystallography beamline I23 at Diamond Light Source

Long-wavelength macromolecular crystallography - First successful native SAD experiment close to the sulfur edge.

Phasing of novel macromolecular crystal structures has been challenging since the start of structural biology. Making use of anomalous diffraction of natively present elements, such as sulfur and phosphorus, for phasing has been possible for some systems, but hindered by the necessity to access longer X-ray wavelengths in order to make most use of the anomalous scattering contributions of these elements. Presented here are the results from a first successful experimental phasing study of a macromolecular crystal structure at a wavelength close to the sulfur K edge. This has been made possible by the in-vacuum setup and the long-wavelength optimised experimental setup at the I23 beamline at Diamond Light Source. In these early commissioning experiments only standard data collection and processing procedures have been applied, in particular no dedicated absorption correction has been used. Nevertheless the success of the experiment demonstrates that the capability to extract phase information can be even further improved once data collection protocols and data processing have been optimised.

In-vacuum long-wavelength macromolecular crystallography.

Structure solution based on the weak anomalous signal from native (protein and DNA) crystals is increasingly being attempted as part of synchrotron experiments. Maximizing the measurable anomalous signal by collecting diffraction data at longer wavelengths presents a series of technical challenges caused by the increased absorption of X-rays and larger diffraction angles. A new beamline at Diamond Light Source has been built specifically for collecting data at wavelengths beyond the capability of other synchrotron macromolecular crystallography beamlines. Here, the theoretical considerations in support of the long-wavelength beamline are outlined and the in-vacuum design of the endstation is discussed, as well as other hardware features aimed at enhancing the accuracy of the diffraction data. The first commissioning results, representing the first in-vacuum protein structure solution, demonstrate the promising potential of the beamline.

The long-wavelength macromolecular crystallography beamline I23 at Diamond Light Source

The long-wavelength macromolecular crystallography beamline I23 at Diamond Light Source

The Long-Wavelength MX Beamline I23 at Diamond Light Source

The long-wavelength macromolecular crystallography (MX) beamline I23 at Diamond Light Source will be the first dedicated beamline for long-wavelength phasing experiments from macromolecular crystals. By exploiting the weak anomalous differences from sulfur or phosphorous present in proteins or RNA/DNA molecules the crystallographic phase problem can be experimentally solved by anomalous diffraction methods based on their intrinsic signal without labeling the crystals with additional anomalous scatterers. Additionally, anomalous contrast can be used to unambiguously identify biologically important ions such as Ca2+, K+ or Cl-. The beamline will operate in a core wavelength range from 1.5 to 4 Å, offering a complementary setup to the suite of already five existing MX beamlines at Diamond. To minimize absorption effects, the complete beamline including sample, goniometer and detector will be operated in vacuum. An X-ray tomography setup will be integrated into the experimental end station to determine the crystal shape and size as a basis for an analytical absorption correction. A large curved Pilatus 12M detector will allow access to diffraction data up to 2theta =1100° (figure 1). Sample cooling is realized by a conductive path from a pulse tube cryo-cooler through the kappa goniometer. The beamline is currently in its construction phase with first users being expected in summer 2014. The challenges of in-vacuum long-wavelength macromolecular crystallography and the opportunities by extending the wavelength range towards the sulfur and phosphorous K-absorption edges will be discussed and an overview on the current status of the project will be given.

The long-wavelength macromolecular crystallography beamline at Diamond Light Source

The long-wavelength macromolecular crystallography beamline at Diamond Light Source

The long-wavelength macromolecular crystallography beamline at Diamond Light Source

The long-wavelength macromolecular crystallography beamline at Diamond Light Source

Low-energy X-ray detection with an in-vacuum PILATUS detector

The feasibility of using PILATUS single-X-ray-photon counting detectors for long-wavelength macromolecular crystallography was investigated by carrying out a series of experiments at Diamond Light Source. A water-cooled PILATUS 100k detector was tested in vacuum with monochromatic 3 keV X-rays on the Diamond test beamline B16. Effects of detector cooling on noise performance, energy calibration and threshold trimming were investigated. When detecting 3 keV X-rays, the electronic noise of the analogue output of pixel preamplifiers forces the threshold to be set at a higher level than the 50% energy level recommended to minimize charge-sharing effects. The influence of threshold settings at low X-ray energy was studied by characterizing the detector response to a collimated beam of 3 keV X-rays scanned across several pixels. The relationship between maximum count rate and minimum energy threshold was investigated separately for various detector gain settings.

Performance of PILATUS detector technology for long-wavelength macromolecular crystallography

The long-wavelength MX beamline I23 currently under design at Diamond Light Source will be optimized in the X-ray energy range between 3 and 5 keV. At the moment no commercial off-the-shelf detector with high quantum efficiency and dynamic range is available to cover the large area required for diffraction experiments in this energy range. The hybrid pixel detector technology used in PILATUS detectors could overcome these limitations as the modular design could allow a large coverage in reciprocal space and high detection efficiency. Experiments were carried out on the Microfocus Spectroscopy beamline I18 at Diamond Light Source to test the performance of a 100K PILATUS module in the low-energy range from 2.3 to 3.7 keV.