scholarly journals Multi-crystal native SAD analysis at 6 keV

2014 ◽  
Vol 70 (10) ◽  
pp. 2544-2557 ◽  
Author(s):  
Qun Liu ◽  
Youzhong Guo ◽  
Yanqi Chang ◽  
Zheng Cai ◽  
Zahra Assur ◽  
...  
Keyword(s):  
De Novo ◽  
Real Life ◽  
Kinase Domain ◽  
X Rays ◽  
Protein Kinase Domain ◽  
Anomalous Diffraction ◽  
X Ray ◽  
Tyrosine Protein Kinase ◽  
Sad Phasing ◽  
Feasibility Test

Anomalous diffraction signals from typical native macromolecules are very weak, frustrating their use inde novostructure determination. Here, native SAD procedures are described to enhance signal to noise in anomalous diffraction by using multiple crystals in combination with synchrotron X-rays at 6 keV. Increased anomalous signals were obtained at 6 keV compared with 7 keV X-ray energy, which was used for previous native SAD analyses. A feasibility test of multi-crystal-based native SAD phasing was performed at 3.2 Å resolution for a known tyrosine protein kinase domain, and real-life applications were made to two novel membrane proteins at about 3.0 Å resolution. The three applications collectively serve to validate the robust feasibility of native SAD phasing at lower energy.

scholarly journals Native-SAD Phasing at Synchrotrons: A Low-dose and High-redundancy Strategy

2014 ◽  
Vol 70 (a1) ◽  
pp. C600-C600
Author(s):  
Qun Liu ◽  
Wayne Hendrickson
Keyword(s):  
De Novo ◽  
Protein Complexes ◽  
Heavy Atom ◽  
Real Life ◽  
Biological Macromolecules ◽  
X Rays ◽  
Light Elements ◽  
X Ray ◽  
Sad Phasing ◽  
Synchrotron Beamlines

Native biological macromolecules contain intrinsic light elements such as sulfur in proteins and phosphorus in nucleic acids. Native-SAD phasing utilizes the anomalous signals from light elements for de novo structure determination: first the substructure of anomalous scatterers is determined; phase evaluation for the entire structure then follows. Synchrotron beamlines are expected to be ideal instruments for native-SAD phasing due to their brilliant and energy-tunable x-rays. However, anomalous signals from light elements are typically very weak at x-ray energies accessible to most synchrotron beamlines. Efforts have been made to promote the utility of synchrotrons for routine native-SAD phasing with no requirement for heavy-atom incorporation. Our strategy is to limit the x-ray dose per crystal and to enhance the signal-to-noise ratio by increasing data redundancy through use of multiple crystals. We have devised a robust procedure and applied it for routine native-SAD analyses on real-life membrane proteins, protein-protein complexes, and recalcitrant proteins. Here we use these real-life case studies to illustrate our procedures in sample preparation, x-ray energy selection, data collection, data analysis and phasing.

scholarly journals Advancing Native-SAD Phasing at Synchrotron with 13 Real-life Case Studies

2014 ◽  
Vol 70 (a1) ◽  
pp. C601-C601
Author(s):  
Meitian Wang
Keyword(s):  
Data Collection ◽  
Single Crystal ◽  
Measurement Errors ◽  
Model Building ◽  
De Novo ◽  
Real Life ◽  
Data Sets ◽  
X Ray ◽  
Recent Developments ◽  
Sad Phasing

The key step in elucidating de novo 3D X-ray structures relies on the incorporation of heavy elements into proteins or crystals. Selenomethionine incorporation or heavy metal derivatization are however not always possible and require additional efforts. Exploiting anomalous signals from intrinsically present elements like S, P, and Ca2+ from proteins and nucleic acids, as well as Cl-, SO42-, and PO42- from crystallization solutions, is therefore an appealing alternative. Such a method has been shown to be valid by collecting data from several crystals and combining them(1). Recent developments at macromolecular crystallography beamlines are however pushing the limits of what could be obtained out of a single crystal. Here we introduce a novel data collection routine for native-SAD phasing, which distributes tolerable X-ray life-doses to very high multiplicity X-ray diffraction data sets measured at 6 keV energy and at different crystal orientations on a single crystal. This allows the extraction of weak anomalous signals reliably by reducing both systematic and random measurement errors. The data collection method has been applied successfully to thirteen real-life examples including membrane proteins, a protein/DNA complex, and a large protein complex. In addition to de novo structure determination, we advocate such a data collection protocol for molecular replacement solvable structures where unbiased phase information is crucial in objective map interpretation and model building, especially for medium and low-resolution cases.

scholarly journals Cadmium SAD phasing at CuKα wavelength

F1000Research ◽  
2019 ◽  
Vol 8 ◽  
pp. 84
Author(s):  
Igor E. Eliseev ◽  
Anna N. Yudenko ◽  
Valeria M. Ukrainskaya ◽  
Oleg B. Chakchir
Keyword(s):  
Experimental Model ◽  
De Novo ◽  
Common Method ◽  
Single Domain Antibody ◽  
Anomalous Diffraction ◽  
Cadmium Ions ◽  
X Ray ◽  
X Ray Crystallography ◽  
Domain Antibody ◽  
Sad Phasing

Single-wavelength anomalous diffraction (SAD) is the most common method for de novo elucidation of macromolecular structures by X-ray crystallography. It requires an anomalous scatterer in a crystal to calculate phases. A recent study by Panneerselvam et al. emphasized the utility of cadmium ions for SAD phasing at the standard synchrotron wavelength of 1 Å. Here we show that cadmium is also useful for phasing of crystals collected in-house with CuKα radiation. Using a crystal of single-domain antibody as an experimental model, we demonstrate how cadmium SAD can be conveniently employed to solve a CuKα dataset. We then discuss the factors which make this method generally applicable.

scholarly journals Optimization in S-SAD phasing - difference between solved and unsolved structure

2014 ◽  
Vol 70 (a1) ◽  
pp. C613-C613
Author(s):  
Jan Stránský ◽  
Tomáš Kovaľ ◽  
Lars Østergaard ◽  
Jarmila Dušková ◽  
Tereza Skálová ◽  
...  
Keyword(s):  
Data Processing ◽  
De Novo ◽  
Protein Structures ◽  
X Ray Diffraction ◽  
X Ray ◽  
Structure Solution ◽  
Sad Phasing ◽  
Optimal Values ◽  
Grant Agency ◽  
Intensity Reading

Development of X-ray diffraction technologies have made de novo phasing of protein structures by single-wavelength anomalous dispersion by sulphur (S-SAD) more common. As anomalous differences in the sulphur atomic factors are in the order of errors of measurement, careful intensity reading and data processing are crucial. S-SAD was used for de novo phasing of a small 12 kDa protein with 4 sulphur atoms per molecule at 2.3 Å, where the data did not enable a straightforward structure solution. Data processing was performed using XDS [1] and scaling using XSCALE. The sulphur substructure was determined by SHELXD [2] and phases were obtained from SHELXE [2]. Both algorithms strongly depend on input parameters and default values did not lead to the correct phases. Therefore a systematic search of optimal values of several parameters was used to find a solution. This method helped to confirm sulphur substructure and to differentiate the handedness of the solutions. Moreover, a script for comfortable conversion of SHELX outputs to MTZ format was developed, using programmes included in the CCP4 package [3]. The previously unsolvable protein structure was successfully resolved with the described procedure. This work was supported by the Grant Agency of the Czech Technical University in Prague, (SGS13/219/OHK4/3T/14), the Czech Science Foundation (P302/11/0855), project BIOCEV CZ.1.05/1.1.00/02.0109 from the ERDF.

scholarly journals Semi-automated protein crystal mounting device for the sulfur single-wavelength anomalous diffraction method

2010 ◽  
Vol 43 (2) ◽  
pp. 341-346 ◽  
Author(s):  
Yu Kitago ◽  
Nobuhisa Watanabe ◽  
Isao Tanaka
Keyword(s):  
Diffraction Method ◽  
Systematic Errors ◽  
Protein Crystal ◽  
X Rays ◽  
Anomalous Diffraction ◽  
X Ray ◽  
Frozen Solution ◽  
Custom Made ◽  
Protein Molecules ◽  
X Ray Absorption

Use of longer-wavelength X-rays has advantages for the detection of small anomalous signals from light atoms, such as sulfur, in protein molecules. However, the accuracy of the measured diffraction data decreases at longer wavelengths because of the greater X-ray absorption. The capillary-top mounting method (formerly the loopless mounting method) makes it possible to eliminate frozen solution around the protein crystal and reduces systematic errors in the evaluation of small anomalous differences. However, use of this method requires custom-made tools and a large amount of skill. Here, the development of a device that can freeze the protein crystal semi-automatically using the capillary-top mounting method is described. This device can pick up the protein crystal from the crystallization drop using a micro-manipulator, and further procedures, such as withdrawal of the solution around the crystal by suction and subsequent flash freezing of the protein crystal, are carried out automatically. This device makes it easy for structural biologists to use the capillary-top mounting method for sulfur single-wavelength anomalous diffraction phasing using longer-wavelength X-rays.

scholarly journals Selenium single-wavelength anomalous diffraction de novo phasing using an X-ray-free electron laser

2016 ◽  
Vol 7 (1) ◽  
Author(s):  
Mark S. Hunter ◽  
Chun Hong Yoon ◽  
Hasan DeMirci ◽  
Raymond G. Sierra ◽  
E. Han Dao ◽  
...  
Keyword(s):  
Free Electron ◽  
Free Electron Laser ◽  
De Novo ◽  
Anomalous Diffraction ◽  
X Ray

scholarly journals UJI KELAYAKAN PESAWAT SINAR-X TERHADAP PROYEKSI PA (POSTERO-ANTERIOR) DAN LAT (LATERAL) PADA TEKNIK PEMERIKSAAN FOTO THORAX

2017 ◽  
Vol 18 (1) ◽  
pp. 27
Author(s):  
Kadek Miniati ◽  
Gusti Ngurah Sutapa ◽  
I Wayan Balik Sudarsana
Keyword(s):  
Radiation Dose ◽  
X Rays ◽  
Tube Voltage ◽  
Limit Values ◽  
X Ray ◽  
Limit Value ◽  
Irradiation Field ◽  
Interval Distance ◽  
Dosage Guidelines ◽  
Feasibility Test

Research has been conducted to determine feasibility test of the X-ray planePA and LAT projections on chest x-ray techniques. The study using a water phantom object as a substitute for patients with variations in interval distance ofthe  100-180 cm. Measurement of radiation dose X-rays performed five repetitions , measurable doses had be read on the device electrometer. Exposition factors to the PA projection using a tube voltage of 75 kV, current and time of 3,2 mAs, the irradiation field areaof (30 x 30) cm2.For the LAT projection tube voltage of 80 kV, current and time of 6,3 mAs, and the irradiation field area of (20 x 30) cm2. It the study of the radiation dose X-ray plane projection PA and LAT is optimal is below the limit value at the level of dosage guidelines BAPETEN No 08 of 2011. Obtained PA projections are below the value of 0,4 mGy while LAT projection is below the value of 1,5 mGy. The radiation dose X-rays plane using a variation of 100-180 cm distance is still below the dose limit values ??, thus meeting the objectives anssurance quality and quality control.

scholarly journals Low Multiplicity Sulfur SAD Phasing in the Home lab

2014 ◽  
Vol 70 (a1) ◽  
pp. C607-C607 ◽  
Author(s):  
Severine Freisz ◽  
Juergen Graf ◽  
Matthew Benning ◽  
Vernon Smith
Keyword(s):  
De Novo ◽  
Structural Information ◽  
Low Noise ◽  
Intensity Difference ◽  
Anomalous Scattering ◽  
High Quality ◽  
X Ray ◽  
Sad Phasing ◽  
Very High

Advances in crystallographic hardware and software are enabling structural biologists to investigate more challenging projects. Recent developments in hardware and software are greatly increasing the capabilities of in-house diffraction systems making it more routine to obtain de novo structural information in the home lab. Single-wavelength anomalous diffraction (SAD) techniques with Cu Ka or Ga Ka radiation are now widely used for structure solution even in cases involving weak anomalous scatterers, like sulfur. We have now introduced the D8 Venture solution for structural biology with the PHOTON 100 detector featuring the first CMOS active pixel sensor for X-ray crystallography. Our new microfocus source, the METALJET delivers beam intensity exceeding those of typical bending-magnet beamlines. The very high intensity, the small beam focus and the lower air scatter produced by Gallium Kα radiation help to greatly reduce the background scatter. This provides greater signal to noise essential to identify weak anomalous signal. Due to the very weak anomalous scattering of S, data multiplicities in the order of 40 are typically necessary to obtain phases by S-SAD. Collecting high-multiplicity data minimizes systematic experimental errors to measure with very high accuracy the minute intensity difference between Friedel Pairs (1.0 – 1.5 %) [1]. This requires software which optimizes the collection strategy, for example with respect to overall data collection time to minimize radiation damage. The combination of a brighter, more stable X-ray source with a high sensitivity low noise detector have greatly improved the quality of data collected in-house. The high quality allows successful SAD measurements far away from the absorption edge. Here we present a low multiplicity sulfur-SAD phasing experiment on a small Thaumatin crystal showing the high quality of the data collected on the D8 VENTURE with the METALJET.

scholarly journals Cloning, overexpression, purification and preliminary X-ray analysis of the protein kinase domain of enhanced disease resistance 1 (EDR1) fromArabidopsis thaliana

2014 ◽  
Vol 70 (7) ◽  
pp. 959-962
Author(s):  
Heidi Kaljunen ◽  
Saravanan Panneerselvam ◽  
Jochen Mueller-Dieckmann
Keyword(s):  
Disease Resistance ◽  
Protein Kinase ◽  
Kinase Domain ◽  
Mitogen Activated Protein Kinase ◽  
X Rays ◽  
Abiotic And Biotic Stresses ◽  
Protein Kinase Domain ◽  
Biotic Stresses ◽  
Inactive Form ◽  
Mitogen Activated Protein

Enhanced disease resistance 1 is a member of the Raf-like mitogen-activated protein kinase kinase kinase (MAPKKK) family that negatively regulates disease resistance, ethylene-induced senescence and programmed cell death in response to both abiotic and biotic stresses. A catalytically inactive form of the EDR1 kinase domain was successfully cloned, expressed, purified and crystallized. Crystallization was conducted in the presence of the ATP analogue AMP-PNP. The crystals belonged to space groupP3221 and contained two molecules in the asymmetric unit. The crystals diffracted X-rays to 2.55 Å resolution.

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