Alternative phasing method in macromolecular crystallography

Selenium is the most widely used heavy atom for experimental phasing, either by single anomalous scattering (SAD) or multiple-wavelength anomalous diffraction (MAD) procedures. The use of the single isomorphous replacement (SIR) or single isomorphous replacement with anomalous scattering (SIRAS) phasing procedure with selenomethionine (Mse) containing proteins is not so commonly used, as it requires isomorphous native data. Several non-redundant X-ray diffraction data sets from various Mse derivatised protein crystals were collected at energies far below the absorption edge before and after exposing the crystal to ultraviolet (UV) radiation with 266 nm lasers. A detailed analysis revealed that significant changes in diffracted intensities were induced by ultraviolet irradiation whilst retaining crystal isomorphism. These intensity changes allowed the crystal structures to be solved by the radiation damage-induced phasing (RIP) technique [1]. These can be coupled with the anomalous signal from the dataset collected at the selenium absorption edge to obtain SIRAS phases in a UV-RIPAS phasing experiment [2]. Inspection of the crystal structures and electron-density maps demonstrated that covalent bonds between selenium and carbon at all sites located in the core of the proteins or in a hydrophobic environment were much more susceptible to UV radiation-induced cleavage than other bonds typically present in Mse proteins. The rapid UV radiation-induced bond cleavage opens a reliable new paradigm for phasing at synchrotron [1,2] and at in-house X-ray source [3].

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

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1602531113 ◽

2016 ◽

Vol 113 (46) ◽

pp. 13039-13044 ◽

Cited By ~ 28

Author(s):

Takanori Nakane ◽

Shinya Hanashima ◽

Mamoru Suzuki ◽

Haruka Saiki ◽

Taichi Hayashi ◽

...

Keyword(s):

Membrane Protein ◽

Structure Determination ◽

Free Electron ◽

Heavy Atom ◽

Anomalous Scattering ◽

X Ray ◽

Isomorphous Replacement ◽

Experimental Phasing ◽

Serial Femtosecond Crystallography

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.

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CHOOCH: a program for deriving anomalous-scattering factors from X-ray fluorescence spectra

Journal of Applied Crystallography ◽

10.1107/s0021889800014655 ◽

2001 ◽

Vol 34 (1) ◽

pp. 82-86 ◽

Cited By ~ 150

Author(s):

Gwyndaf Evans ◽

Robert F. Pettifer

Keyword(s):

Absorption Edge ◽

Fluorescence Spectra ◽

Heavy Atom ◽

Fortran Program ◽

Anomalous Scattering ◽

Tabular Data ◽

X Ray ◽

Scattering Factor ◽

Experimental Values ◽

Kronig Relation

A Fortran programCHOOCH, which derives experimental values of the anomalous-scattering factorsf′′ andf′ from X-ray fluorescence spectra, is described. The program assumes knowledge of theoretical values for the imaginary term,f′′, of the anomalous-scattering factor away from the absorption edge to scale the experimental fluorescence spectrum and thus derive values off′′ near the absorption edge, where tabular data are inappropriate. The Kramers–Kronig relation is used to calculate the real part,f′, of the anomalous-scattering factor. The program aids the decision-making process in macromolecular crystallographic experiments where optimal wavelength selection is required. Magnitudes off′ andf′′ at selected wavelengths can later be used as starting values for heavy-atom refinement with crystallographic phasing programs.

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The information available from anomalous scattering on fibres at different wavelengths

Journal of Applied Crystallography ◽

10.1107/s0021889887086643 ◽

1987 ◽

Vol 20 (4) ◽

pp. 295-299 ◽

Cited By ~ 1

Author(s):

C. Nave

Keyword(s):

Absorption Edge ◽

Heavy Atom ◽

Imaginary Component ◽

Anomalous Scattering ◽

Phase Problem ◽

Phase Information ◽

Helical Symmetry ◽

X Ray ◽

The Real ◽

X Ray Scattering

The contribution of a particular atom in a molecule to the total X-ray scattering can be altered by varying the wavelength in the region of the absorption edge of the atom. It is shown that only the changes in the real part of the anomalous scattering of the atom provide significant changes in a pattern from a fibre containing molecules with helical symmetry. Changes due to the imaginary component are small and Friedel differences cannot be observed, owing to the fibre disorder. The information which can be obtained is equivalent to that given by a truly isomorphous heavy-atom derivative. For the general case this is not sufficient to provide unambiguous phase information. If a twofold axis is present at right angles to the fibre axis then the amplitudes are real and the phase problem can, in favourable cases, be solved.

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Preliminary X-ray diffraction analysis of CfaA, a molecular chaperone essential for the assembly of CFA/I fimbriae of human enterotoxigenicEscherichia coli

Acta Crystallographica Section F Structural Biology Communications ◽

10.1107/s2053230x13033967 ◽

2014 ◽

Vol 70 (2) ◽

pp. 196-199

Author(s):

Rui Bao ◽

Lothar Esser ◽

Steven Poole ◽

Annette McVeigh ◽

Yu-xing Chen ◽

...

Keyword(s):

Tertiary Structure ◽

Heavy Atom ◽

Diffusion Method ◽

Anomalous Scattering ◽

X Ray Diffraction ◽

X Ray ◽

Cell Parameters ◽

Colonization Factor ◽

Isomorphous Replacement ◽

Periplasmic Chaperone

Understanding of pilus bioassembly in Gram-negative bacteria stems mainly from studies of P pili and type 1 fimbriae of uropathogenicEscherichia coli, which are mediated by the classic chaperone–usher pathway (CUP). However, CFA/I fimbriae, a class 5 fimbria and intestinal colonization factor for enterotoxigenicE. coli(ETEC), are proposed to assembleviathe alternate chaperone pathway (ACP). Both CUP and ACP fimbrial bioassembly pathways require the function of a periplasmic chaperone, but their corresponding proteins share very low similarity in primary sequence. Here, the crystallization of the CFA/I periplasmic chaperone CfaA by the hanging-drop vapor-diffusion method is reported. X-ray diffraction data sets were collected from a native CfaA crystal to 2 Å resolution and to 1.8 and 2.8 Å resolution, respectively, from a lead and a platinum derivative. These crystals displayed the symmetry of space groupC2, with unit-cell parametersa= 103.6,b= 28.68,c= 90.60 Å, β = 119.7°. Initial phases were derived from multiple isomorphous replacement with anomalous scattering experiments using the data from the platinum and lead derivatives. This resulted in an interpretable electron-density map showing one CfaA molecule in an asymmetric unit. Sequence assignments were aided by anomalous signals from the heavy-atom derivatives. Refinement of the atomic model of CfaA is ongoing, which is expected to further understanding of the essential aspects and allowable variations in tertiary structure of the greater family of chaperones involved in chaperone–usher mediated bioassembly.

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Introduction to phasing

Acta Crystallographica Section D Biological Crystallography ◽

10.1107/s0907444910006694 ◽

2010 ◽

Vol 66 (4) ◽

pp. 325-338 ◽

Cited By ~ 44

Author(s):

Garry L. Taylor

Keyword(s):

Small Molecule ◽

Heavy Atom ◽

Protein Crystallography ◽

Anomalous Scattering ◽

Phase Information ◽

X Ray ◽

Heavy Atoms ◽

Isomorphous Replacement ◽

Diffracted Waves ◽

Complete Protein

When collecting X-ray diffraction data from a crystal, we measure the intensities of the diffracted waves scattered from a series of planes that we can imagine slicing through the crystal in all directions. From these intensities we derive the amplitudes of the scattered waves, but in the experiment we lose the phase information; that is, how we offset these waves when we add them together to reconstruct an image of our molecule. This is generally known as the `phase problem'. We can only derive the phases from some knowledge of the molecular structure. In small-molecule crystallography, some basic assumptions about atomicity give rise to relationships between the amplitudes from which phase information can be extracted. In protein crystallography, theseab initiomethods can only be used in the rare cases in which there are data to at least 1.2 Å resolution. For the majority of cases in protein crystallography phases are derived either by using the atomic coordinates of a structurally similar protein (molecular replacement) or by finding the positions of heavy atoms that are intrinsic to the protein or that have been added (methods such as MIR, MIRAS, SIR, SIRAS, MAD, SAD or combinations of these). The pioneering work of Perutz, Kendrew, Blow, Crick and others developed the methods of isomorphous replacement: adding electron-dense atoms to the protein without disturbing the protein structure. Nowadays, methods from small-molecule crystallography can be used to find the heavy-atom substructure and the phases for the whole protein can be bootstrapped from this prior knowledge. More recently, improved X-ray sources, detectors and software have led to the routine use of anomalous scattering to obtain phase information from either incorporated selenium or intrinsic sulfurs. In the best cases, only a single set of X-ray data (SAD) is required to provide the positions of the anomalous scatters, which together with density-modification procedures can reveal the structure of the complete protein.

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Isomorphous replacement in electron diffraction of wet crystals of rat hemoglobin

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100075968 ◽

1983 ◽

Vol 41 ◽

pp. 446-447

Author(s):

William F. Tivol ◽

Murray Vernon King ◽

D. F. Parsons

Keyword(s):

Electron Diffraction ◽

Heavy Atom ◽

Isomorphous Substitution ◽

X Ray Diffraction ◽

Salt Solutions ◽

X Ray ◽

Isomorphous Replacement ◽

Structure Factors ◽

Diffraction Structure ◽

The Mean

Feasibility of isomorphous substitution in electron diffraction is supported by a calculation of the mean alteration of the electron-diffraction structure factors for hemoglobin crystals caused by substituting two mercury atoms per molecule, following Green, Ingram & Perutz, but with allowance for the proportionality of f to Z3/4 for electron diffraction. This yields a mean net change in F of 12.5%, as contrasted with 22.8% for x-ray diffraction.Use of the hydration chamber in electron diffraction opens prospects for examining many proteins that yield only very thin crystals not suitable for x-ray diffraction. Examination in the wet state avoids treatments that could cause translocation of the heavy-atom labels or distortion of the crystal. Combined with low-fluence techniques, it enables study of the protein in a state as close to native as possible.We have undertaken a study of crystals of rat hemoglobin by electron diffraction in the wet state. Rat hemoglobin offers a certain advantage for hydration-chamber work over other hemoglobins in that it can be crystallized from distilled water instead of salt solutions.

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Using High-Resolution Anomalous-Scattering X-Ray Diffraction to Observe Off-Stoichiometric CU2ZnSnS4 Crystal Structures

2017 IEEE 44th Photovoltaic Specialist Conference (PVSC) ◽

10.1109/pvsc.2017.8366136 ◽

2017 ◽

Author(s):

Christopher J. Bosson ◽

Max T. Birch ◽

Douglas P. Halliday ◽

Chiu C. Tang ◽

Peter D. Hatton

Keyword(s):

High Resolution ◽

Crystal Structures ◽

Anomalous Scattering ◽

X Ray Diffraction ◽

X Ray

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

Acta Crystallographica Section D Biological Crystallography ◽

10.1107/s1399004714008943 ◽

2014 ◽

Vol 70 (7) ◽

pp. 1873-1883 ◽

Cited By ~ 2

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.

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