scholarly journals The magic triangle goes MAD: experimental phasing with a bromine derivative

2010 ◽  
Vol 66 (4) ◽  
pp. 374-380 ◽  
Author(s):  
Tobias Beck ◽  
Tim Gruene ◽  
George M. Sheldrick
Keyword(s):  
Functional Groups ◽  
Heavy Atom ◽  
Anomalous Dispersion ◽  
Proteinase K ◽  
Nonspecific Binding ◽  
Heavy Atoms ◽  
Carboxylate Groups ◽  
Experimental Phasing ◽  
Sad Phasing ◽  
Bromine Derivative

Experimental phasing is an essential technique for the solution of macromolecular structures. Since many heavy-atom ion soaks suffer from nonspecific binding, a novel class of compounds has been developed that combines heavy atoms with functional groups for binding to proteins. The phasing tool 5-amino-2,4,6-tribromoisophthalic acid (B3C) contains three functional groups (two carboxylate groups and one amino group) that interact with proteinsviahydrogen bonds. Three Br atoms suitable for anomalous dispersion phasing are arranged in an equilateral triangle and are thus readily identified in the heavy-atom substructure. B3C was incorporated into proteinase K and a multiwavelength anomalous dispersion (MAD) experiment at the Br Kedge was successfully carried out. Radiation damage to the bromine–carbon bond was investigated. A comparison with the phasing tool I3C that contains three I atoms for single-wavelength anomalous dispersion (SAD) phasing was also carried out.

scholarly journals De novo protein structure determination by heavy-atom soaking in lipidic cubic phase and SIRAS phasing using serial synchrotron crystallography

IUCrJ ◽  
2018 ◽  
Vol 5 (5) ◽  
pp. 524-530 ◽  
Author(s):  
S. Botha ◽  
D. Baitan ◽  
K. E. J. Jungnickel ◽  
D. Oberthür ◽  
C. Schmidt ◽  
...  
Keyword(s):  
Synchrotron Radiation ◽  
Data Collection ◽  
Diffraction Data ◽  
Heavy Atom ◽  
Proteinase K ◽  
Cubic Phase ◽  
Anomalous Scattering ◽  
Experimental Phasing ◽  
Lipidic Cubic Phase ◽  
Serial Crystallography

During the past few years, serial crystallography methods have undergone continuous development and serial data collection has become well established at high-intensity synchrotron-radiation beamlines and XFEL radiation sources. However, the application of experimental phasing to serial crystallography data has remained a challenging task owing to the inherent inaccuracy of the diffraction data. Here, a particularly gentle method for incorporating heavy atoms into micrometre-sized crystals utilizing lipidic cubic phase (LCP) as a carrier medium is reported. Soaking in LCP prior to data collection offers a new, efficient and gentle approach for preparing heavy-atom-derivative crystals directly before diffraction data collection using serial crystallography methods. This approach supports effective phasing by utilizing a reasonably low number of diffraction patterns. Using synchrotron radiation and exploiting the anomalous scattering signal of mercury for single isomorphous replacement with anomalous scattering (SIRAS) phasing resulted in high-quality electron-density maps that were sufficient for building a complete structural model of proteinase K at 1.9 Å resolution using automatic model-building tools.

scholarly journals Phasing and structure of bestrophin-1: a case study in the use of heavy-atom cluster compounds with multi-subunit transmembrane proteins

2016 ◽  
Vol 72 (3) ◽  
pp. 319-325 ◽  
Author(s):  
Veronica Kane Dickson
Keyword(s):  
Heavy Atom ◽  
Three Dimensional ◽  
Transmembrane Proteins ◽  
Antibody Fragments ◽  
Cluster Compounds ◽  
Heavy Atoms ◽  
Moderate Resolution ◽  
Sad Phasing ◽  
Crystal Twinning

The purification and three-dimensional crystallization of membrane proteins are commonly affected by a cumulation of pathologies that are less prevalent in their soluble counterparts. This may include severe anisotropy, poor spot shape, poor to moderate-resolution diffraction, crystal twinning, translational pseudo-symmetry and poor uptake of heavy atoms for derivatization. Such challenges must be circumvented by adaptations in the approach to crystallization and/or phasing. Here, an example of a protein that exhibited all of the above-mentioned complications is presented. Bestrophin-1 is a eukaryotic calcium-activated chloride channel, the structure of which was recently determined in complex with monoclonal antibody fragments using SAD phasing with tantalum bromide clusters (Ta6Br12·Br2). Some of the obstacles to obtaining improved diffraction and phasing for this particular channel are discussed, as well as the approach and adaptations that were key to determining the structure.

A Base Specific Single Heavy Atom Stain for Electron Microscopy

1972 ◽  
Vol 30 ◽  
pp. 184-185
Author(s):  
J. P. Langmore ◽  
N. R. Cozzarelli ◽  
A. V. Crewe
Keyword(s):  
Electron Microscopy ◽  
Electron Microscope ◽  
High Resolution ◽  
Elastic Scattering ◽  
Heavy Atom ◽  
High Vacuum ◽  
Heavy Atoms ◽  
Large Movement ◽  
Base Sequencing ◽  
Scanning Electron

A system has been developed to allow highly specific derivatization of the thymine bases of DNA with mercurial compounds wich should be visible in the high resolution scanning electron microscope. Three problems must be completely solved before this staining system will be useful for base sequencing by electron microscopy: 1) the staining must be shown to be highly specific for one base, 2) the stained DNA must remain intact in a high vacuum on a thin support film suitable for microscopy, 3) the arrangement of heavy atoms on the DNA must be determined by the elastic scattering of electrons in the microscope without loss or large movement of heavy atoms.

The crystal structure of myoglobin. VI. Seal myoglobin

1960 ◽  
Vol 258 (1293) ◽  
pp. 181-201 ◽  
Keyword(s):  
Tertiary Structure ◽  
Heavy Atom ◽  
Three Dimensional ◽  
Sperm Whale ◽  
Fourier Synthesis ◽  
Heavy Atoms ◽  
Isomorphous Replacement ◽  
Unit Cells ◽  
The Common ◽  
Sperm Whale Myoglobin

Myoglobin from the common seal ( Phoca vitulina ) when crystallized from ammonium sulphate forms monoclinic crystals with space group the unit cell, a = 57·9Å, b = 29·6Å, c = 106·4Å, β = 102°15', contains four molecules. The method of isomorphous replacement has been used in an investigation of the centrosymmetric b -axis projection in which it has been possible to determine signs for nearly all the h0l reflexions having spacings greater than 4Å. Three independent heavy-atom derivatives were employed and the signs so determined have been used to compute a map of the electron density projected on the (010) plane. This projection has been interpreted in terms of the molecule of sperm-whale myoglobin, as deduced by Bodo, Dintzis, Kendrew & Wyckoff (1959) from a three-dimensional Fourier synthesis to 6Å resolution. The results of the interpretation show that the two myoglobin molecules are very similar in form (tertiary structure) in spite of the differences in their amino-acid composition. The relative orientation of the two unit cells with respect to the myoglobin molecule is given and a comparison is made of the positions of the heavy atoms in each molecule.

scholarly journals Softer but stronger? Sulfur SAD with low energy X-rays of 2.7 Å

2014 ◽  
Vol 70 (a1) ◽  
pp. C604-C604
Author(s):  
Dorothee Liebschner ◽  
Naohiro Matsugaki ◽  
Miki Senda ◽  
Yusuke Yamada ◽  
Toshiya Senda
Keyword(s):  
Absorption Edge ◽  
Protein Crystal ◽  
X Rays ◽  
Long Wavelength ◽  
Heavy Atoms ◽  
Statistical Validity ◽  
Experimental Phasing ◽  
Recent Developments ◽  
Long Time ◽  
Anomalous Signal

Single wavelength anomalous diffraction (SAD) is a powerful experimental phasing technique used in macromolecular crystallography (MX). SAD is based on the absorption of X-rays by heavy atoms, which can be either incorporated into the protein (crystal) or naturally present in the structure, such as sulfur or metal ions. In particular, sulfur seems to be an attractive candidate for phasing, because most proteins contain a considerable number of S atoms. However, the K-absorption edge of sulfur is around 5.1 Å wavelength (2.4 keV), which is far from the optimal wavelength of most MX-beamlines at synchrotrons. Therefore, phasing experiments have to be performed further away from the absorption edge, which results in weaker anomalous signal. This explains why S-SAD was not commonly used for a long time, although its feasibility was illustrated by the ground-breaking study by Hendrickson and Teeter [1]. Recent developments in instrumentation, software and methodology made it possible to measure intensities more accurately, and, as a consequence, S-SAD has lately obtained more and more attention [2]. The beamline BL-1A at Photon factory (KEK, Japan) is designed to take full advantage of a long wavelength X-ray beam at around 3 Å to further enhance anomalous signals. We performed S-SAD experiments at BL-1A using two different wavelengths (1.9 Å and 2.7 Å) and compared their phasing capabilities. This methodological study was performed with ferredoxin reductase crystals of various sizes. In order to guarantee statistical validity and to exclude the influence of a particular sample, we repeated the comparison with several crystals. The novelty in the approach consists in using very long wavelengths (2.7 Å), not fully exploited in the literature so far. According to our study, the 2.7 Å wavelength shows - despite strong absorption effects of the diffracted X-rays - more successful phasing results than at 1.9 Å.

Completion of crystal structures from powder data: the use of the coordination polyhedra

2000 ◽  
Vol 33 (6) ◽  
pp. 1305-1310 ◽  
Author(s):  
Angela Altomare ◽  
Carmelo Giacovazzo ◽  
Antonietta Guagliardi ◽  
Anna Grazia Giuseppina Moliterni ◽  
Rosanna Rizzi
Keyword(s):  
Structural Model ◽  
Heavy Atom ◽  
Computing Time ◽  
Direct Methods ◽  
Coordination Polyhedra ◽  
Trial Solution ◽  
Final Model ◽  
Heavy Atoms ◽  
Experimental Diffraction Pattern ◽  
User Intervention

Direct methods applied to powder diffraction data often provide well located heavy atoms and unreliable light-atom positions. The completion of the crystal structure is then not always straightforward and may require a considerable amount of user intervention. The heavy-atom connectivity provided by the trial solution may be used to guess the nature of the coordination polyhedra. A Monte Carlo procedure is described which, in the absence of a well defined structural model, is able to locate the light atoms correctly under the restraints of the experimental heavy-atom connectivity model. The correctness of the final model is assessed by criteria based on the agreement between the whole experimental diffraction pattern and the calculated one. The procedure requires little CPU computing time and has been implemented as a routine ofEXPO[Altomareet al.(1999).J. Appl. Cryst.32, 339–340]. The method has proved to be sufficiently robust against the distortion of the coordination polyhedra and has been successfully applied to some test structures.

scholarly journals Exploiting subtle structural differences in heavy-atom derivatives for experimental phasing

2014 ◽  
Vol 70 (7) ◽  
pp. 1873-1883 ◽  
Author(s):  
Jimin Wang ◽  
Yue Li ◽  
Yorgo Modis
Keyword(s):  
Structure Determination ◽  
Heavy Atom ◽  
Data Sets ◽  
Diarrhea Virus ◽  
Isomorphous Replacement ◽  
Experimental Phasing ◽  
Structural Differences ◽  
Structure Determinations ◽  
Weak Derivatives ◽  
Viral Diarrhea Virus

Structure determination using the single isomorphous replacement (SIR) or single-wavelength anomalous diffraction (SAD) methods with weak derivatives remains very challenging. In a recent structure determination of glycoprotein E2 from bovine viral diarrhea virus, three isomorphous uranium-derivative data sets were merged to obtain partially interpretable initial experimental maps. Small differences between them were then exploited by treating them as three independent SAD data sets plus three circular pairwise SIR data sets to improve the experimental maps. Here, how such subtle structural differences were exploited for experimental phasing is described in detail. The basis for why this approach works is also provided: the effective resolution of isomorphous signals between highly isomorphous derivatives is often much higher than the effective resolution of the anomalous signals of individual derivative data sets. Hence, the new phasing approaches outlined here will be generally applicable to structure determinations involving weak derivatives.

scholarly journals Heavy atom effects in the Paternò–Büchi reaction of pyrimidine derivatives with 4,4’-disubstituted benzophenones

2011 ◽  
Vol 7 ◽  
pp. 113-118 ◽  
Author(s):  
Feng-Feng Kong ◽  
Jian-Bo Wang ◽  
Qin-Hua Song
Keyword(s):  
Temperature Effects ◽  
Halogen Atom ◽  
Heavy Atom ◽  
Photochemical Efficiency ◽  
Pyrimidine Derivatives ◽  
Heavy Atoms ◽  
Electron Withdrawing Group ◽  
Photochemical Systems ◽  
Expected Increase ◽  
Atom Effect

The regioselectivity and the photochemical efficiency were investigated in the Paternò–Büchi reaction of 1,3-dimethylthymine (DMT) and 1,3-dimethyluracil (DMU) with benzophenone (1b) and some 4,4’-disubstituted derivatives (dimethoxy (1a), difluoro (1c), dichloro (1d), dibromo (1e) and dicyano benzophenone (1f)) that gives rise to two regioisomeric oxetanes, 2 and 3. The regioselectivity (the ratio of 2/3) decreased gradually for both DMT/DMU photochemical systems from 1a to 1f. That is, a halogen atom as an electron-withdrawing group (EWG) has a pronounced effect on the regioselectivity. However, the photochemical efficiency of the 1e systems did not show the expected increase, but decreased relative to systems with 1b. Temperature effects on the regioselectivity of 1b–e systems showed some interesting features for systems with heavy atoms (including the 1d and 1e systems), such as higher inversion temperatures, and an entropy-controlled regioselectively whereas the regioselectivity for two other systems (1b and 1c) is enthalpy–entropy controlled. A heavy atom effect is suggested to be responsible for these unusual phenomena based on the triplet-diradical mechanism of the Paternò–Büchi reaction.

scholarly journals An overview of heavy-atom derivatization of protein crystals

2016 ◽  
Vol 72 (3) ◽  
pp. 303-318 ◽  
Author(s):  
Ashley C. W. Pike ◽  
Elspeth F. Garman ◽  
Tobias Krojer ◽  
Frank von Delft ◽  
Elisabeth P. Carpenter
Keyword(s):  
Heavy Atom ◽  
Protein Crystals ◽  
Data Sets ◽  
Low Resolution ◽  
Heavy Atoms ◽  
Density Maps ◽  
Resolution Electron ◽  
First Choice ◽  
First Time ◽  
Resolution Data

Heavy-atom derivatization is one of the oldest techniques for obtaining phase information for protein crystals and, although it is no longer the first choice, it remains a useful technique for obtaining phases for unknown structures and for low-resolution data sets. It is also valuable for confirming the chain trace in low-resolution electron-density maps. This overview provides a summary of the technique and is aimed at first-time users of the method. It includes guidelines on when to use it, which heavy atoms are most likely to work, how to prepare heavy-atom solutions, how to derivatize crystals and how to determine whether a crystal is in fact a derivative.

scholarly journals Experimental phasing withSHELXC/D/E: combining chain tracing with density modification

2010 ◽  
Vol 66 (4) ◽  
pp. 479-485 ◽  
Author(s):  
George M. Sheldrick
Keyword(s):  
Main Chain ◽  
Heavy Atoms ◽  
General Account ◽  
Structure Solution ◽  
Experimental Phasing

The programsSHELXC, SHELXDandSHELXEare designed to provide simple, robust and efficient experimental phasing of macromolecules by the SAD, MAD, SIR, SIRAS and RIP methods and are particularly suitable for use in automated structure-solution pipelines. This paper gives a general account of experimental phasing using these programs and describes the extension of iterative density modification in SHELXEby the inclusion of automated protein main-chain tracing. This gives a good indication as to whether the structure has been solved and enables interpretable maps to be obtained from poorer starting phases. The autotracing algorithm starts with the location of possible seven-residue α-helices and common tripeptides. After extension of these fragments in both directions, various criteria are used to decide whether to accept or reject the resulting poly-Ala traces. Noncrystallographic symmetry (NCS) is applied to the traced fragments, not to the density. Further features are the use of a `no-go' map to prevent the traces from passing through heavy atoms or symmetry elements and a splicing technique to combine the best parts of traces (including those generated by NCS) that partly overlap.

Sign in / Sign up

Export Citation Format

Share Document