A complement to the modern crystallographer's toolbox: caged gadolinium complexes with versatile binding modes

A set of seven caged gadolinium complexes were used as vectors for introducing the chelated Gd3+ion into protein crystals in order to provide strong anomalous scattering forde novophasing. The complexes contained multidentate ligand molecules with different functional groups to provide a panel of possible interactions with the protein. An exhaustive crystallographic analysis showed them to be nondisruptive to the diffraction quality of the prepared derivative crystals, and as many as 50% of the derivatives allowed the determination of accurate phases, leading to high-quality experimental electron-density maps. At least two successful derivatives were identified for all tested proteins. Structure refinement showed that the complexes bind to the protein surface or solvent-accessible cavities, involving hydrogen bonds, electrostatic and CH–π interactions, explaining their versatile binding modes. Their high phasing power, complementary binding modes and ease of use make them highly suitable as a heavy-atom screen for high-throughputde novostructure determination, in combination with the SAD method. They can also provide a reliable tool for the development of new methods such as serial femtosecond crystallography.

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Advances in Structure Modeling Methods for Cryo-Electron Microscopy Maps

Molecules ◽

10.3390/molecules25010082 ◽

2019 ◽

Vol 25 (1) ◽

pp. 82 ◽

Cited By ~ 2

Author(s):

Eman Alnabati ◽

Daisuke Kihara

Keyword(s):

Electron Microscopy ◽

De Novo ◽

Molecular Structures ◽

Structure Modeling ◽

Rapid Progress ◽

Macromolecular Complexes ◽

Modeling Methods ◽

Cryo Electron Microscopy ◽

Density Maps

Cryo-electron microscopy (cryo-EM) has now become a widely used technique for structure determination of macromolecular complexes. For modeling molecular structures from density maps of different resolutions, many algorithms have been developed. These algorithms can be categorized into rigid fitting, flexible fitting, and de novo modeling methods. It is also observed that machine learning (ML) techniques have been increasingly applied following the rapid progress of the ML field. Here, we review these different categories of macromolecule structure modeling methods and discuss their advances over time.

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Improving the accuracy for Photosystem II and other XFEL structures

Acta Crystallographica Section A Foundations and Advances ◽

10.1107/s2053273314094285 ◽

2014 ◽

Vol 70 (a1) ◽

pp. C571-C571

Author(s):

Nicholas Sauter ◽

Aaron Brewster ◽

Johan Hattne ◽

Muhamed Amin ◽

Jan Kern ◽

...

Keyword(s):

Photosystem Ii ◽

De Novo ◽

Anomalous Scattering ◽

Separate Determination ◽

Structure Factors ◽

Detector Geometry ◽

Small Structure ◽

Image Pixels

Femtosecond-scale XFEL pulses can produce diffraction free from radiation damage, under functional physiological conditions where reaction dynamics can be studied for systems such as photosystem II. However, it has been extremely difficult to derive accurate structure factors from the data since every shot is a still exposure from a distinct specimen. Accuracy can be improved by software methods implemented in the program cctbx.xfel, including optimal indexing and retention of data from multiple lattices, and separate determination of the resolution cutoff for individual lattices. Various techniques can produce well-conforming descriptions of the Bragg spot shape and crystal mosaicity, enabled in part by sub-pixel characterization of the detector geometry. By carefully discriminating between image pixels known to contain diffraction signal and the surrounding pixels containing only background noise, and by extending postrefinement techniques that lead to a better crystal orientation, we derive accurate structure factors with substantially fewer crystal specimen exposures. It is hoped that these developments will make it easier to measure small structure factor differences, such as those from anomalous scattering that will enable the de novo determination of macromolecular structure.

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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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Experimental phase determination with selenomethionine or mercury-derivatization in serial femtosecond crystallography

IUCrJ ◽

10.1107/s2052252517008557 ◽

2017 ◽

Vol 4 (5) ◽

pp. 639-647 ◽

Cited By ~ 17

Author(s):

Keitaro Yamashita ◽

Naoyuki Kuwabara ◽

Takanori Nakane ◽

Tomohiro Murai ◽

Eiichi Mizohata ◽

...

Keyword(s):

Structure Determination ◽

Free Electron ◽

De Novo ◽

Heavy Atom ◽

Protein Structure Determination ◽

High Energy ◽

X Rays ◽

Replacement Method ◽

Serial Femtosecond Crystallography

Serial femtosecond crystallography (SFX) using X-ray free-electron lasers (XFELs) holds enormous potential for the structure determination of proteins for which it is difficult to produce large and high-quality crystals. SFX has been applied to various systems, but rarely to proteins that have previously unknown structures. Consequently, the majority of previously obtained SFX structures have been solved by the molecular replacement method. To facilitate protein structure determination by SFX, it is essential to establish phasing methods that work efficiently for SFX. Here, selenomethionine derivatization and mercury soaking have been investigated for SFX experiments using the high-energy XFEL at the SPring-8 Angstrom Compact Free-Electron Laser (SACLA), Hyogo, Japan. Three successful cases are reported of single-wavelength anomalous diffraction (SAD) phasing using X-rays of less than 1 Å wavelength with reasonable numbers of diffraction patterns (13 000, 60 000 and 11 000). It is demonstrated that the combination of high-energy X-rays from an XFEL and commonly used heavy-atom incorporation techniques will enable routinede novostructural determination of biomacromolecules.

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Relative and Absolute Structure Assignments of Alkenes Using Crystalline Osmate Derivatives for X-Ray Analysis

10.26434/chemrxiv.10830143.v1 ◽

2019 ◽

Cited By ~ 1

Author(s):

Alexander S. Burns ◽

Charles dooley ◽

Paul R. Carlson ◽

Joseph W. Ziller ◽

Scott Rychnovsky

Keyword(s):

Absolute Configuration ◽

Osmium Tetroxide ◽

Heavy Atom ◽

Crystallographic Analysis ◽

Enol Ethers ◽

X Ray ◽

Silyl Enol Ethers ◽

Absolute Structure ◽

Structure Of Liquid

<div><div><p>Osmium tetroxide and TMEDA form stable crystalline adducts with alkenes. The structure of liquid alkenes can be determined through X-ray analysis of these derivatives. Osmium, a heavy atom, facilitates the crystallographic analysis and the determination of the absolute configuration using common Mo X-ray sources. The utility of this method for assigning structures and absolute configurations was demonstrated on a number of unsaturated substrates that include simple alkenes, enones, enol ethers, and silyl enol ethers.</p></div></div>

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Facilitating best practices in collecting anomalous scattering data forde novostructure solution at the ESRF Structural Biology Beamlines

Acta Crystallographica Section D Structural Biology ◽

10.1107/s2059798316001042 ◽

2016 ◽

Vol 72 (3) ◽

pp. 413-420 ◽

Cited By ~ 6

Author(s):

Daniele de Sanctis ◽

Marcus Oscarsson ◽

Alexander Popov ◽

Olof Svensson ◽

Gordon Leonard

Keyword(s):

Structural Biology ◽

De Novo ◽

Heavy Atom ◽

Scattering Data ◽

Anomalous Scattering ◽

Automatic Data ◽

Structure Solution ◽

Experimental Phasing ◽

Wide Range ◽

Collection Practice

The constant evolution of synchrotron structural biology beamlines, the viability of screening protein crystals for a wide range of heavy-atom derivatives, the advent of efficient protein labelling and the availability of automatic data-processing and structure-solution pipelines have combined to makede novostructure solution in macromolecular crystallography a less arduous task. Nevertheless, the collection of diffraction data of sufficient quality for experimental phasing is still a difficult and crucial step. Here, some examples of good data-collection practice for projects requiring experimental phasing are presented and recent developments at the ESRF Structural Biology beamlines that have facilitated these are illustrated.

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Protein structure determination by single-wavelength anomalous diffraction phasing of X-ray free-electron laser data

IUCrJ ◽

10.1107/s2052252516002980 ◽

2016 ◽

Vol 3 (3) ◽

pp. 180-191 ◽

Cited By ~ 54

Author(s):

Karol Nass ◽

Anton Meinhart ◽

Thomas R. M. Barends ◽

Lutz Foucar ◽

Alexander Gorel ◽

...

Keyword(s):

Structure Determination ◽

Free Electron ◽

De Novo ◽

Protein Structure Determination ◽

Anomalous Scattering ◽

X Ray ◽

Sad Phasing ◽

Anomalous Signal ◽

Serial Femtosecond Crystallography

Serial femtosecond crystallography (SFX) at X-ray free-electron lasers (XFELs) offers unprecedented possibilities for macromolecular structure determination of systems that are prone to radiation damage. However, phasing XFEL datade novois complicated by the inherent inaccuracy of SFX data, and only a few successful examples, mostly based on exceedingly strong anomalous or isomorphous difference signals, have been reported. Here, it is shown that SFX data from thaumatin microcrystals can be successfully phased using only the weak anomalous scattering from the endogenous S atoms. Moreover, a step-by-step investigation is presented of the particular problems of SAD phasing of SFX data, analysing data from a derivative with a strong anomalous signal as well as the weak signal from endogenous S atoms.

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Challenges of sulfur SAD phasing as a routine method in macromolecular crystallography

Journal of Synchrotron Radiation ◽

10.1107/s0909049511049004 ◽

2011 ◽

Vol 19 (1) ◽

pp. 19-29 ◽

Cited By ~ 7

Author(s):

James Doutch ◽

Michael A. Hough ◽

S. Samar Hasnain ◽

Richard W. Strange

Keyword(s):

Model Building ◽

De Novo ◽

Protein Structures ◽

Anomalous Scattering ◽

X Ray ◽

Long Wavelength ◽

Structure Solution ◽

Average Proportion ◽

Sad Phasing

The sulfur SAD phasing method allows the determination of protein structuresde novowithout reference to derivatives such as Se-methionine. The feasibility for routine automated sulfur SAD phasing using a number of current protein crystallography beamlines at several synchrotrons was examined using crystals of trimericAchromobacter cycloclastesnitrite reductase (AcNiR), which contains a near average proportion of sulfur-containing residues and two Cu atoms per subunit. Experiments using X-ray wavelengths in the range 1.9–2.4 Å show that we are not yet at the level where sulfur SAD is routinely successful forautomatedstructure solution and model building using existing beamlines and current software tools. On the other hand, experiments using the shortest X-ray wavelengths available on existing beamlines could be routinely exploited to solve and produce unbiased structural models using the similarly weak anomalous scattering signals from the intrinsic metal atoms in proteins. The comparison of long-wavelength phasing (the Bijvoet ratio for nine S atoms and two Cu atoms is ∼1.25% at ∼2 Å) and copper phasing (the Bijvoet ratio for two Cu atoms is 0.81% at ∼0.75 Å) forAcNiR suggests that lower data multiplicity than is currently required for success should in general be possible for sulfur phasing if appropriate improvements to beamlines and data collection strategies can be implemented.

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An introduction to experimental phasing of macromolecules illustrated bySHELX; new autotracing features

Acta Crystallographica Section D Structural Biology ◽

10.1107/s2059798317015121 ◽

2018 ◽

Vol 74 (2) ◽

pp. 106-116 ◽

Cited By ~ 50

Author(s):

Isabel Usón ◽

George M. Sheldrick

Keyword(s):

Heavy Atom ◽

Direct Methods ◽

Anomalous Scattering ◽

Structure Factors ◽

Structure Solution ◽

Experimental Phasing ◽

Simplifying Assumptions ◽

Efficient Route ◽

Tracing Algorithm

For the purpose of this article, experimental phasing is understood to mean the determination of macromolecular structures by exploiting small intensity differences of Friedel opposites and possibly of reflections measured at different wavelengths or for heavy-atom derivatives, without the use of specific structural models. TheSHELXprograms provide a robust and efficient route for routine structure solution by the SAD, MAD and related methods, but involve a number of simplifying assumptions that may limit their applicability in borderline cases. The substructure atoms (i.e.those with significant anomalous scattering) are first located by direct methods, and the experimental data are then used to estimate phase shifts that are added to the substructure phases to obtain starting phases for the native reflections. These are then improved by density modification and, if the resolution of the data and the type of structure permit, polyalanine tracing. A number of extensions to the tracing algorithm are discussed; these are designed to improve its performance at low resolution. Given native data to 2.5 Å resolution or better, a correlation coefficient greater than 25% between the structure factors calculated from such a trace and the native data is usually a good indication that the structure has been solved.

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