scholarly journals Can I solve my structure by SAD phasing? Anomalous signal in SAD phasing

2016 ◽  
Vol 72 (3) ◽  
pp. 346-358 ◽  
Author(s):  
Thomas C. Terwilliger ◽  
Gábor Bunkóczi ◽  
Li-Wei Hung ◽  
Peter H. Zwart ◽  
Janet L. Smith ◽  
...  
Keyword(s):  
Signal To Noise Ratio ◽  
Atomic Displacement ◽  
Peak Height ◽  
High Ratio ◽  
Data Set ◽  
High Data ◽  
Sad Phasing ◽  
Anomalous Signal ◽  
Displacement Factor ◽  
Difference Fourier

A key challenge in the SAD phasing method is solving a structure when the anomalous signal-to-noise ratio is low. A simple theoretical framework for describing measurements of anomalous differences and the resulting useful anomalous correlation and anomalous signal in a SAD experiment is presented. Here, the useful anomalous correlation is defined as the correlation of anomalous differences with ideal anomalous differences from the anomalous substructure. The useful anomalous correlation reflects the accuracy of the data and the absence of minor sites. The useful anomalous correlation also reflects the information available for estimating crystallographic phases once the substructure has been determined. In contrast, the anomalous signal (the peak height in a model-phased anomalous difference Fourier at the coordinates of atoms in the anomalous substructure) reflects the information available about each site in the substructure and is related to the ability to find the substructure. A theoretical analysis shows that the expected value of the anomalous signal is the product of the useful anomalous correlation, the square root of the ratio of the number of unique reflections in the data set to the number of sites in the substructure, and a function that decreases with increasing values of the atomic displacement factor for the atoms in the substructure. This means that the ability to find the substructure in a SAD experiment is increased by high data quality and by a high ratio of reflections to sites in the substructure, and is decreased by high atomic displacement factors for the substructure.

scholarly journals Can I solve my structure by SAD phasing? Planning an experiment, scaling data and evaluating the useful anomalous correlation and anomalous signal

2016 ◽  
Vol 72 (3) ◽  
pp. 359-374 ◽  
Author(s):  
Thomas C. Terwilliger ◽  
Gábor Bunkóczi ◽  
Li-Wei Hung ◽  
Peter H. Zwart ◽  
Janet L. Smith ◽  
...  
Keyword(s):  
Figure Of Merit ◽  
Signal To Noise Ratio ◽  
Theoretical Framework ◽  
Data Sets ◽  
Signal To Noise ◽  
Acta Cryst ◽  
Data Set ◽  
Local Scaling ◽  
Sad Phasing ◽  
Anomalous Signal

A key challenge in the SAD phasing method is solving a structure when the anomalous signal-to-noise ratio is low. Here, algorithms and tools for evaluating and optimizing the useful anomalous correlation and the anomalous signal in a SAD experiment are described. A simple theoretical framework [Terwilligeret al.(2016),Acta Cryst.D72, 346–358] is used to develop methods for planning a SAD experiment, scaling SAD data sets and estimating the useful anomalous correlation and anomalous signal in a SAD data set. Thephenix.plan_sad_experimenttool uses a database of solved and unsolved SAD data sets and the expected characteristics of a SAD data set to estimate the probability that the anomalous substructure will be found in the SAD experiment and the expected map quality that would be obtained if the substructure were found. Thephenix.scale_and_mergetool scales unmerged SAD data from one or more crystals using local scaling and optimizes the anomalous signal by identifying the systematic differences among data sets, and thephenix.anomalous_signaltool estimates the useful anomalous correlation and anomalous signal after collecting SAD data and estimates the probability that the data set can be solved and the likely figure of merit of phasing.

Determination of the site of incorporation of cobalt in CoZnPO-CZP by multiple-wavelength anomalous-dispersion crystallography

1999 ◽  
Vol 55 (3) ◽  
pp. 327-332 ◽  
Author(s):  
M. Helliwell ◽  
J. R. Helliwell ◽  
V. Kaucic ◽  
N. Zabukovec Logar ◽  
L. Barba ◽  
...  
Keyword(s):  
Metal Atom ◽  
Peak Height ◽  
Data Sets ◽  
White Line ◽  
Data Set ◽  
Small Peak ◽  
Multiple Wavelength ◽  
Subsequent Calculation ◽  
Difference Fourier ◽  
Two Peaks

Data were collected from a crystal of CoZnPO-CZP {sodium cobalt–zinc phosphate hydrate, Na6[Co0.2Zn0.8PO4]6.6H2O} using synchrotron radiation at ELETTRA at the inflection point and `white line' for both the cobalt and zinc K edges, and at 1.45 Å, a wavelength remote from the K edges of both metals. The data were processed using the programs DENZO and SCALEPACK. The CCP4 program suite was used for the scaling of data sets and the subsequent calculation of dispersive difference Fourier maps. Optimal scaling was achieved by using a subset of reflections with little or no contribution from the metal atoms (i.e. which were essentially wavelength independent in their intensities) and using weights based on the σ's to obtain an overall scale factor in each case. Phases were calculated with SHELXL97 based on the refined structure using a much higher resolution and complete Cu Kα data set. An occupancy of 100% by zinc at the two metal-atom sites was assumed. The dispersive difference Fourier map calculated for zinc gave two peaks above the background of similar heights at the expected metal-atom sites. The peak height at the Zn1 site was a little higher than at the Zn2 site. The dispersive difference Fourier map calculated for cobalt gave just one peak above the background, at the Zn1 site, and only a small peak at the Zn2 site, thus indicating that incorporation of cobalt takes place mainly at one site. Refinement of the zinc occupancies using MLPHARE reinforces this conclusion. The chemical environment of each site is discussed.

Combining cross-crystal averaging and MRSAD to phase a 4354-amino-acid structure

2016 ◽  
Vol 72 (2) ◽  
pp. 182-191
Author(s):  
Jason Nicholas Busby ◽  
J. Shaun Lott ◽  
Santosh Panjikar
Keyword(s):  
Radiation Damage ◽  
Model Building ◽  
Data Sets ◽  
Low Resolution ◽  
Data Set ◽  
C Protein ◽  
Large Size ◽  
Anomalous Signal ◽  
Difference Fourier ◽  
Combined Data

The B and C proteins from the ABC toxin complex ofYersinia entomophagaform a large heterodimer that cleaves and encapsulates the C-terminal toxin domain of the C protein. Determining the structure of the complex formed by B and the N-terminal region of C was challenging owing to its large size, the non-isomorphism of different crystals and their sensitivity to radiation damage. A native data set was collected to 2.5 Å resolution and a non-isomorphous Ta6Br12-derivative data set was collected that showed strong anomalous signal at low resolution. The tantalum-cluster sites could be found, but the anomalous signal did not extend to a high enough resolution to allow model building. Selenomethionine (SeMet)-derivatized protein crystals were produced, but the high number (60) of SeMet sites and the sensitivity of the crystals to radiation damage made phasing using the SAD or MAD methods difficult. Multiple SeMet data sets were combined to provide 30-fold multiplicity, and the low-resolution phase information from the Ta6Br12data set was transferred to this combined data set by cross-crystal averaging. This allowed the Se atoms to be located in an anomalous difference Fourier map; they were then used inAuto-Rickshawfor multiple rounds of autobuilding and MRSAD.

scholarly journals Development of a dose-limiting data collection strategy for serial synchrotron rotation crystallography

2017 ◽  
Vol 24 (1) ◽  
pp. 29-41 ◽  
Author(s):  
Kazuya Hasegawa ◽  
Keitaro Yamashita ◽  
Tomohiro Murai ◽  
Nipawan Nuemket ◽  
Kunio Hirata ◽  
...  
Keyword(s):  
Data Collection ◽  
Exposure Time ◽  
Signal To Noise Ratio ◽  
Single Shot ◽  
Resolution Limit ◽  
Sample Rotation ◽  
Data Collection Strategy ◽  
Sad Phasing ◽  
Anomalous Signal ◽  
Serial Femtosecond Crystallography

Serial crystallography, in which single-shot diffraction images are collected, has great potential for protein microcrystallography. Although serial femtosecond crystallography (SFX) has been successfully demonstrated, limited beam time prevents its routine use. Inspired by SFX, serial synchrotron crystallography (SSX) has been investigated at synchrotron macromolecular crystallography beamlines. Unlike SFX, the longer exposure time of milliseconds to seconds commonly used in SSX causes radiation damage. However, in SSX, crystals can be rotated during the exposure, which can achieve efficient coverage of the reciprocal space. In this study, mercury single-wavelength anomalous diffraction (Hg-SAD) phasing of the luciferin regenerating enzyme (LRE) was performed using serial synchrotron rotation crystallography. The advantages of rotation and influence of dose on the data collected were evaluated. The results showed that sample rotation was effective for accurate data collection, and the optimum helical rotation step depended on multiple factors such as multiplicity and partiality of reflections, exposure time per rotation angle and the contribution from background scattering. For the LRE microcrystals, 0.25° was the best rotation step for the achievable resolution limit, whereas a rotation step larger than or equal to 1° was favorable for Hg-SAD phasing. Although an accumulated dose beyond 1.1 MGy caused specific damage at the Hg site, increases in resolution and anomalous signal were observed up to 3.4 MGy because of a higher signal-to-noise ratio.

scholarly journals Rapid cadmium SAD phasing at the standard wavelength (1 Å)

2017 ◽  
Vol 73 (7) ◽  
pp. 581-590 ◽  
Author(s):  
Saravanan Panneerselvam ◽  
Esa-Pekka Kumpula ◽  
Inari Kursula ◽  
Anja Burkhardt ◽  
Alke Meents
Keyword(s):  
Crystal Growth ◽  
Data Set ◽  
Cadmium Ions ◽  
Experimental Phasing ◽  
Wide Range ◽  
Sad Phasing ◽  
Anomalous Signal ◽  
Single Data ◽  
Nucleotide Binding Proteins ◽  
Cation Complexes

Cadmium ions can be effectively used to promote crystal growth and for experimental phasing. Here, the use of cadmium ions as a suitable anomalous scatterer at the standard wavelength of 1 Å is demonstrated. The structures of three different proteins were determined using cadmium single-wavelength anomalous dispersion (SAD) phasing. Owing to the strong anomalous signal, the structure of lysozyme could be automatically phased and built using a very low anomalous multiplicity (1.1) and low-completeness (77%) data set. Additionally, it is shown that cadmium ions can easily substitute divalent ions in ATP–divalent cation complexes. This property could be generally applied for phasing experiments of a wide range of nucleotide-binding proteins. Improvements in crystal growth and quality, good anomalous signal at standard wavelengths (i.e.no need to change photon energy) and rapid phasing and refinement using a single data set are benefits that should allow cadmium ions to be widely used for experimental phasing.

Difference Fourier Analysis of Glucose Embedded and Frozen Hydrated Purple Membrane

1982 ◽  
Vol 40 ◽  
pp. 74-75
Author(s):  
Jules S. Jaffe ◽  
Robert M. Glaeser
Keyword(s):  
Purple Membrane ◽  
Data Set ◽  
High Resolution Data ◽  
X Ray ◽  
X Ray Crystallography ◽  
Fourier Techniques ◽  
Versus Protein ◽  
The Difference ◽  
Difference Fourier ◽  
Ideal Method

Although difference Fourier techniques are standard in X-ray crystallography it has only been very recently that electron crystallographers have been able to take advantage of this method. We have combined a high resolution data set for frozen glucose embedded Purple Membrane (PM) with a data set collected from PM prepared in the frozen hydrated state in order to visualize any differences in structure due to the different methods of preparation. The increased contrast between protein-ice versus protein-glucose may prove to be an advantage of the frozen hydrated technique for visualizing those parts of bacteriorhodopsin that are embedded in glucose. In addition, surface groups of the protein may be disordered in glucose and ordered in the frozen state. The sensitivity of the difference Fourier technique to small changes in structure provides an ideal method for testing this hypothesis.

scholarly journals Purification, crystallization and preliminary X-ray diffraction analysis of the effector protein PevD1 fromVerticillium dahliae

2012 ◽  
Vol 68 (7) ◽  
pp. 802-805 ◽  
Author(s):  
Lei Han ◽  
Zheng Liu ◽  
Xinqi Liu ◽  
Dewen Qiu
Keyword(s):  
Pathogenic Fungus ◽  
Sodium Formate ◽  
Diffusion Method ◽  
Effector Protein ◽  
X Ray Diffraction ◽  
Hanging Drop ◽  
Dispersion Method ◽  
Data Set ◽  
X Ray ◽  
Anomalous Signal

The effector protein PevD1 from the pathogenic fungusVerticillium dahliaewas purified and crystallized using the hanging-drop vapour-diffusion method. Native crystals appeared in a solution consisting of 4.0 Msodium formate. A native data set was collected at 1.9 Å resolution at 100 K using an in-house X-ray source. Because of the absence of useful methinione in the protein sequence, derivative crystals that contained iodine were obtained by soaking in 1.25 Mpotassium iodide, and a data set that contained anomalous signal was collected using the same X-ray facility at a wavelength of 1.54 Å. The single-wavelength anomalous dispersion method was used to successfully solve the structure based on the anomalous signal generated from iodine.

Combined Near-Infrared Raman Microprobe and Laser Trapping System: Application to the Analysis of a Single Organic Microdroplet in Water

1998 ◽  
Vol 52 (3) ◽  
pp. 339-342 ◽  
Author(s):  
Katsuhiro Ajito
Keyword(s):  
Raman Spectrum ◽  
Near Infrared ◽  
Laser Light ◽  
Signal To Noise Ratio ◽  
Peak Height ◽  
Laser Spot ◽  
Laser Trapping ◽  
Raman Microprobe ◽  
A Charge ◽  
Nir Laser

A combined Raman microprobe and laser trapping system using near-infrared (NIR) laser light was developed for the investigation of single organic microdroplets. The NIR laser light is noninvasive and reduces fluorescence interference in the Raman spectrum for organic molecules. The focused laser beam used for the laser trapping of a microdroplet serves simultaneously as the laser microprobe for Raman measurement. With this system, the focused laser spot is about 1 μm in diameter, which is small enough for the laser trapping of a single toluene microdroplet in water. The system also makes it possible to visualize a focused laser spot together with a laser-trapped microdroplet by using holographic notch filters. The Raman spectrum for a single laser-trapped toluene microdroplet can be obtained from below 100 cm−1 to above 3000 cm−1 with a charge-coupled device (CCD) detector. Fluorescence interference in the Raman spectrum is completely removed by using NIR laser light. The signal-to-noise ratio (SNR), defined as the ratio of the peak height to the standard deviation of the baseline noise in the spectrum, exceeded 250 for the 1003 cm−1 band of a toluene microdroplet at 1 s, which is sufficient to allow identification of the molecular species of a microdroplet.

scholarly journals A search for transiting planets in the β Pictoris system

2018 ◽  
Vol 615 ◽  
pp. A145 ◽  
Author(s):  
M. Mol Lous ◽  
E. Weenk ◽  
M. A. Kenworthy ◽  
K. Zwintz ◽  
R. Kuschnig
Keyword(s):  
Signal To Noise Ratio ◽  
Large Fraction ◽  
Young Star ◽  
High Signal ◽  
Data Set ◽  
Transiting Planets ◽  
Using Data ◽  
Exoplanet Systems ◽  
Transit Signals

Context. Transiting exoplanets provide an opportunity for the characterization of their atmospheres, and finding the brightest star in the sky with a transiting planet enables high signal-to-noise ratio observations. The Kepler satellite has detected over 365 multiple transiting exoplanet systems, a large fraction of which have nearly coplanar orbits. If one planet is seen to transit the star, then it is likely that other planets in the system will transit the star too. The bright (V = 3.86) star β Pictoris is a nearby young star with a debris disk and gas giant exoplanet, β Pictoris b, in a multi-decade orbit around it. Both the planet’s orbit and disk are almost edge-on to our line of sight. Aims. We carry out a search for any transiting planets in the β Pictoris system with orbits of less than 30 days that are coplanar with the planet β Pictoris b. Methods. We search for a planetary transit using data from the BRITE-Constellation nanosatellite BRITE-Heweliusz, analyzing the photometry using the Box-Fitting Least Squares Algorithm (BLS). The sensitivity of the method is verified by injection of artificial planetary transit signals using the Bad-Ass Transit Model cAlculatioN (BATMAN) code. Results. No planet was found in the BRITE-Constellation data set. We rule out planets larger than 0.6 RJ for periods of less than 5 days, larger than 0.75 RJ for periods of less than 10 days, and larger than 1.05 RJ for periods of less than 20 days.

scholarly journals S-SAD phasing on SOLEIL Beamline PROXIMA 1

2014 ◽  
Vol 70 (a1) ◽  
pp. C609-C609
Author(s):  
Patrick Gourhant ◽  
Beatriz Guimaraes ◽  
Tatiana Isabet ◽  
Sebastian Klinke ◽  
Pierre Legrand ◽  
...  
Keyword(s):  
Model Building ◽  
De Novo ◽  
Signal To Noise Ratio ◽  
Beam Line ◽  
Synchrotron Source ◽  
Combining Data ◽  
Low Energies ◽  
Sad Phasing ◽  
Multiple Sample ◽  
Data Collections

"PROXIMA 1, a beamline for macro-molecular crystallography at the 3rd generation synchrotron source SOLEIL, is equipped with a multi-circle goniometer (alpha 50 degrees) as well as a PILATUS 6M detector. These features, along with the extended energy range of the beam line towards the low energies (down to 5.5 keV) and the possibility to adapt the source size to the sample in order to optimize signal to noise ratio, have made the beam line very attractive for S-SAD phasing with more than seven examples of successful de novo phasing achieved over the last two years. The use of low energies has also proved a significant aid in assisting with MODEL building. The technical capabilities of the beam line for low energy data collections will be presented, along with a number of examples of the successful use of low wavelengths on the beam line. The importance of combining data from multiple sample orientations in order to achieve ""true multiplicity"" will be highlighted, as well as the importance of combining data from multiple crystals in order to achieve high multiplicity."

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