Browse
Submit Data
Databases
API
Help

Dataset Information

22 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Fast iodide-SAD phasing for high-throughput membrane protein structure determination.

ABSTRACT: We describe a fast, easy, and potentially universal method for the de novo solution of the crystal structures of membrane proteins via iodide-single-wavelength anomalous diffraction (I-SAD). The potential universality of the method is based on a common feature of membrane proteins-the availability at the hydrophobic-hydrophilic interface of positively charged amino acid residues with which iodide strongly interacts. We demonstrate the solution using I-SAD of four crystal structures representing different classes of membrane proteins, including a human G protein-coupled receptor (GPCR), and we show that I-SAD can be applied using data collection strategies based on either standard or serial x-ray crystallography techniques.

SUBMITTER: Melnikov I

PROVIDER: S-EPMC5429034 | biostudies-literature | 2017 May

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Publications

Fast iodide-SAD phasing for high-throughput membrane protein structure determination.

Melnikov Igor I Polovinkin Vitaly V Kovalev Kirill K Kovalev Kirill K Gushchin Ivan I Shevtsov Mikhail M Shevchenko Vitaly V Mishin Alexey A Alekseev Alexey A Rodriguez-Valera Francisco F Borshchevskiy Valentin V Cherezov Vadim V Leonard Gordon A GA Gordeliy Valentin V Popov Alexander A

Science advances 20170512 5

We describe a fast, easy, and potentially universal method for the de novo solution of the crystal structures of membrane proteins via iodide-single-wavelength anomalous diffraction (I-SAD). The potential universality of the method is based on a common feature of membrane proteins-the availability at the hydrophobic-hydrophilic interface of positively charged amino acid residues with which iodide strongly interacts. We demonstrate the solution using I-SAD of four crystal structures representing ...[more]

PMID: 28508075

Similar Datasets

SAD phasing using iodide ions in a high-throughput structural genomics environment.

Project description:The Seattle Structural Genomics Center for Infectious Disease (SSGCID) focuses on the structure elucidation of potential drug targets from class A, B, and C infectious disease organisms. Many SSGCID targets are selected because they have homologs in other organisms that are validated drug targets with known structures. Thus, many SSGCID targets are expected to be solved by molecular replacement (MR), and reflective of this, all proteins are expressed in native form. However, many community request targets do not have homologs with known structures and not all internally selected targets readily solve by MR, necessitating experimental phase determination. We have adopted the use of iodide ion soaks and single wavelength anomalous dispersion (SAD) experiments as our primary method for de novo phasing. This method uses existing native crystals and in house data collection, resulting in rapid, low cost structure determination. Iodide ions are non-toxic and soluble at molar concentrations, facilitating binding at numerous hydrophobic or positively charged sites. We have used this technique across a wide range of crystallization conditions with successful structure determination in 16 of 17 cases within the first year of use (94% success rate). Here we present a general overview of this method as well as several examples including SAD phasing of proteins with novel folds and the combined use of SAD and MR for targets with weak MR solutions. These cases highlight the straightforward and powerful method of iodide ion SAD phasing in a high-throughput structural genomics environment.

| S-EPMC3123459 | biostudies-literature

Membrane protein structure determination by SAD, SIR, or SIRAS phasing in serial femtosecond crystallography using an iododetergent.

Project description:The 3D structure determination of biological macromolecules by X-ray crystallography suffers from a phase problem: to perform Fourier transformation to calculate real space density maps, both intensities and phases of structure factors are necessary; however, measured diffraction patterns give only intensities. Although serial femtosecond crystallography (SFX) using X-ray free electron lasers (XFELs) has been steadily developed since 2009, experimental phasing still remains challenging. Here, using 7.0-keV (1.771 Å) X-ray pulses from the SPring-8 Angstrom Compact Free Electron Laser (SACLA), iodine single-wavelength anomalous diffraction (SAD), single isomorphous replacement (SIR), and single isomorphous replacement with anomalous scattering (SIRAS) phasing were performed in an SFX regime for a model membrane protein bacteriorhodopsin (bR). The crystals grown in bicelles were derivatized with an iodine-labeled detergent heavy-atom additive 13a (HAD13a), which contains the magic triangle, I3C head group with three iodine atoms. The alkyl tail was essential for binding of the detergent to the surface of bR. Strong anomalous and isomorphous difference signals from HAD13a enabled successful phasing using reflections up to 2.1-Å resolution from only 3,000 and 4,000 indexed images from native and derivative crystals, respectively. When more images were merged, structure solution was possible with data truncated at 3.3-Å resolution, which is the lowest resolution among the reported cases of SFX phasing. Moreover, preliminary SFX experiment showed that HAD13a successfully derivatized the G protein-coupled A2a adenosine receptor crystallized in lipidic cubic phases. These results pave the way for de novo structure determination of membrane proteins, which often diffract poorly, even with the brightest XFEL beams.

| S-EPMC5135358 | biostudies-literature

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

Project description: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.

| S-EPMC4784666 | biostudies-literature

SAD phasing towards structure determination of a thermostable Rieske ferredoxin with a novel stabilizing disulfide bridge.

Project description:Rieske proteins and Rieske ferredoxins are ubiquitous electron-transfer metalloproteins that are characterized by a [2Fe-2S] cluster coordinated by pairs of cysteine and histidine residues. The thermoacidophilic archaeon Acidianus ambivalens contains a Rieske ferredoxin termed RFd2, which has an hitherto unknown additional region of 40-44 residues at the C-terminus with a Cx3C motif that introduces a novel disulfide bond within the Rieske fold. RFd2 was crystallized with the aim of determining its three-dimensional structure in order to understand the contribution of this as yet unique disulfide bridge to the function and stability of RFd2. RFd2 crystals were successively improved, increasing their diffraction to 1.9?Å resolution. Molecular replacement did not solve the RFd2 structure, but a highly multiple in-house diffraction data set collected at the Cu?K? edge led to solution of the phase problem.

| S-EPMC3660900 | biostudies-literature

Combinatorial use of disulfide bridges and native sulfur-SAD phasing for rapid structure determination of coiled-coils.

Project description:Coiled-coils are ubiquitous protein-protein interaction motifs found in many eukaryotic proteins. The elongated, flexible and often irregular nature of coiled-coils together with their tendency to form fibrous arrangements in crystals imposes challenges on solving the phase problem by molecular replacement. Here, we report the successful combinatorial use of native and rational engineered disulfide bridges together with sulfur-SAD phasing as a powerful tool to stabilize and solve the structure of coiled-coil domains in a straightforward manner. Our study is a key example of how modern sulfur SAD combined with mutagenesis can help to advance and simplify the structural study of challenging coiled-coil domains by X-ray crystallography.

| S-EPMC6146289 | biostudies-literature

Experimental phasing for structure determination using membrane-protein crystals grown by the lipid cubic phase method.

Project description:Despite the marked increase in the number of membrane-protein structures solved using crystals grown by the lipid cubic phase or in meso method, only ten have been determined by SAD/MAD. This is likely to be a consequence of the technical difficulties associated with handling proteins and crystals in the sticky and viscous hosting mesophase that is usually incubated in glass sandwich plates for the purposes of crystallization. Here, a four-year campaign aimed at phasing the in meso structure of the integral membrane diacylglycerol kinase (DgkA) from Escherichia coli is reported. Heavy-atom labelling of this small hydrophobic enzyme was attempted by pre-labelling, co-crystallization, soaking, site-specific mercury binding to genetically engineered single-cysteine mutants and selenomethionine incorporation. Strategies and techniques for special handling are reported, as well as the typical results and the lessons learned for each of these approaches. In addition, an assay to assess the accessibility of cysteine residues in membrane proteins for mercury labelling is introduced. The various techniques and strategies described will provide a valuable reference for future experimental phasing of membrane proteins where crystals are grown by the lipid cubic phase method.

| S-EPMC4304691 | biostudies-literature

NMR data collection and analysis protocol for high-throughput protein structure determination.

Project description:A standardized protocol enabling rapid NMR data collection for high-quality protein structure determination is presented that allows one to capitalize on high spectrometer sensitivity: a set of five G-matrix Fourier transform NMR experiments for resonance assignment based on highly resolved 4D and 5D spectral information is acquired in conjunction with a single simultaneous 3D 15N,13C(aliphatic),13C(aromatic)-resolved [1H,1H]-NOESY spectrum providing 1H-1H upper distance limit constraints. The protocol was integrated with methodology for semiautomated data analysis and used to solve eight NMR protein structures of the Northeast Structural Genomics Consortium pipeline. The molecular masses of the hypothetical target proteins ranged from 9 to 20 kDa with an average of approximately 14 kDa. Between 1 and 9 days of instrument time were invested per structure, which is less than approximately 10-25% of the measurement time routinely required to date with conventional approaches. The protocol presented here effectively removes data collection as a bottleneck for high-throughput solution structure determination of proteins up to at least approximately 20 kDa, while concurrently providing spectra that are highly amenable to fast and robust analysis.

| S-EPMC1180791 | biostudies-literature

Crystal structure of bacteriophage T4 Spackle as determined by native SAD phasing.

Project description:The crystal structure of a bacteriophage T4 early gene product, Spackle, was determined by native sulfur single-wavelength anomalous diffraction (SAD) phasing using synchrotron radiation and was refined to 1.52 Å resolution. The structure shows that Spackle consists of a bundle of five α-helices, forming a relatively flat disc-like overall shape. Although Spackle forms a dimer in the crystal, size-exclusion chromatography with multi-angle light scattering shows that it is monomeric in solution. Mass spectrometry confirms that purified mature Spackle lacks the amino-terminal signal peptide and contains an intramolecular disulfide bond, consistent with its proposed role in the periplasm of T4 phage-infected Escherichia coli cells. The surface electrostatic potential of Spackle shows a strikingly bipolar charge distribution, suggesting a possible mode of membrane association and inhibition of the tail lysozyme activity in T4 bacteriophage superinfection exclusion.

| S-EPMC7466748 | biostudies-literature

Cadmium SAD phasing at CuKα wavelength.

Project description: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.

| S-EPMC6446493 | biostudies-literature

High-throughput in situ experimental phasing.

Project description:In this article, a new approach to experimental phasing for macromolecular crystallography (MX) at synchrotrons is introduced and described for the first time. It makes use of automated robotics applied to a multi-crystal framework in which human intervention is reduced to a minimum. Hundreds of samples are automatically soaked in heavy-atom solutions, using a Labcyte Inc. Echo 550 Liquid Handler, in a highly controlled and optimized fashion in order to generate derivatized and isomorphous crystals. Partial data sets obtained on MX beamlines using an in situ setup for data collection are processed with the aim of producing good-quality anomalous signal leading to successful experimental phasing.

| S-EPMC7397491 | biostudies-literature

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data