Likelihood-based molecular-replacement solution for a highly pathological crystal with tetartohedral twinning and sevenfold translational noncrystallographic symmetry

Translational noncrystallographic symmetry (tNCS) is a pathology of protein crystals in which multiple copies of a molecule or assembly are found in similar orientations. Structure solution is problematic because this breaks the assumptions used in current likelihood-based methods. To cope with such cases, new likelihood approaches have been developed and implemented inPhaserto account for the statistical effects of tNCS in molecular replacement. Using these new approaches, it was possible to solve the crystal structure of a protein exhibiting an extreme form of this pathology with seven tetrameric assemblies arrayed along thecaxis. To resolve space-group ambiguities caused by tetartohedral twinning, the structure was initially solved by placing 56 copies of the monomer in space groupP1 and using the symmetry of the solution to define the true space group,C2. The resulting structure of Hyp-1, a pathogenesis-related class 10 (PR-10) protein from the medicinal herb St John's wort, reveals the binding modes of the fluorescent probe 8-anilino-1-naphthalene sulfonate (ANS), providing insight into the function of the protein in binding or storing hydrophobic ligands.

Download Full-text

Coping with translational non-crystallographic symmetry in molecular replacement

Acta Crystallographica Section A Foundations and Advances ◽

10.1107/s2053273314096806 ◽

2014 ◽

Vol 70 (a1) ◽

pp. C319-C319

Author(s):

Randy Read ◽

Paul Adams ◽

Airlie McCoy

Keyword(s):

Diffraction Pattern ◽

Likelihood Function ◽

Molecular Replacement ◽

Asymmetric Unit ◽

Space Group ◽

New Targets ◽

Likelihood Functions ◽

Crystallographic Symmetry ◽

Modified Likelihood ◽

Statistical Effects

In translational noncrystallographic symmetry (tNCS), two or more copies of a component are present in a similar orientation in the asymmetric unit of the crystal. This causes systematic modulations of the intensities in the diffraction pattern, leading to problems with methods that assume, either implicitly or explicitly, that the distribution of intensities is a function only of resolution. To characterize the statistical effects of tNCS accurately, it is necessary to determine the translation relating the copies, any small rotational differences in their orientations, and the size of random coordinate differences caused by conformational differences. An algorithm has been developed to estimate these parameters and refine their values against a likelihood function. By accounting for the statistical effects of tNCS, it is possible to unmask the competing statistical effects of twinning and tNCS and to more robustly assess the crystal for the presence of twinning. Modified likelihood functions that account for the statistical effects of tNCS have been developed for use in molecular replacement and implemented in Phaser. With the use of these new targets, it is now possible to solve structures that eluded earlier versions of the program. Pseudosymmetry and space group ambiguities often accompany tNCS, but the new version of Phaser is less likely to fall into the traps that these set.

Download Full-text

A crystallographic study of human NONO (p54nrb): overcoming pathological problems with purification, data collection and noncrystallographic symmetry

Acta Crystallographica Section D Structural Biology ◽

10.1107/s2059798316005830 ◽

2016 ◽

Vol 72 (6) ◽

pp. 761-769 ◽

Cited By ~ 7

Author(s):

Gavin J. Knott ◽

Santosh Panjikar ◽

Andrea Thorn ◽

Archa H. Fox ◽

Maria R. Conte ◽

...

Keyword(s):

Data Collection ◽

Nuclear Gene ◽

Beam Width ◽

Molecular Replacement ◽

X Rays ◽

Space Group ◽

Pou Domain ◽

Structure Solution ◽

Oriented Parallel ◽

Nuclear Gene Regulation

Non-POU domain-containing octamer-binding protein (NONO, a.k.a. p54nrb) is a central player in nuclear gene regulation with rapidly emerging medical significance. NONO is a member of the highly conservedDrosophilabehaviour/human splicing (DBHS) protein family, a dynamic family of obligatory dimeric nuclear regulatory mediators. However, work with the NONO homodimer has been limited by rapid irreversible sample aggregation. Here, it is reported that L-proline stabilizes purified NONO homodimers, enabling good-quality solution small-angle X-ray structure determination and crystallization. NONO crystallized in the apparent space groupP21with a unique axis (b) of 408.9 Å and with evidence of twinning, as indicated by the cumulative intensity distributionLstatistic, suggesting the possibility of space groupP1. Structure solution by molecular replacement shows a superhelical arrangement of six NONO homodimers (or 12 inP1) oriented parallel to the long axis, resulting in extensive noncrystallographic symmetry. Further analysis revealed that the crystal was not twinned, but the collected data suffered from highly overlapping reflections that obscured theL-test. Optimized data collection on a new crystal using higher energy X-rays, a smaller beam width and an increased sample-to-detector distance produced non-overlapping reflections to 2.6 Å resolution. The steps taken to analyse and overcome this series of practical difficulties and to produce a biologically informative structure are discussed.

Download Full-text

Crystallization and preliminary X-ray diffraction studies of human catalase

Acta Crystallographica Section D Biological Crystallography ◽

10.1107/s0907444999009695 ◽

1999 ◽

Vol 55 (9) ◽

pp. 1614-1615 ◽

Cited By ~ 3

Author(s):

R. A. P. Nagem ◽

E. A. L. Martins ◽

V. M. Gonçalves ◽

R. Aparício ◽

I. Polikarpov

Keyword(s):

Search Model ◽

Molecular Replacement ◽

X Ray Diffraction ◽

Beef Liver ◽

X Ray ◽

Diffusion Technique ◽

Cell Dimensions ◽

Synchrotron Radiation Diffraction ◽

Replacement Solution ◽

Unit Cell Dimensions

The enzyme catalase (H2O2–H2O2 oxidoreductase; E.C. 11.1.6) was purified from haemolysate of human placenta and crystallized using the vapour-diffusion technique. Synchrotron-radiation diffraction data have been collected to 1.76 Å resolution. The enzyme crystallized in the space group P212121, with unit-cell dimensions a = 83.6, b = 139.4, c = 227.5 Å. A molecular-replacement solution of the structure has been obtained using beef liver catalase (PDB code 4blc) as a search model.

Download Full-text

Structural insight into epothilones antitumor activity based on the conformational preferences and tubulin binding modes of epothilones A and B obtained from molecular dynamics simulations

Journal of Biomolecular Structure and Dynamics ◽

10.1080/07391102.2014.911702 ◽

2014 ◽

Vol 33 (4) ◽

pp. 789-803 ◽

Cited By ~ 5

Author(s):

Verónica A. Jiménez ◽

Joel B. Alderete ◽

Karen R. Navarrete

Keyword(s):

Molecular Dynamics ◽

Antitumor Activity ◽

Molecular Dynamics Simulations ◽

Binding Modes ◽

Conformational Preferences ◽

Tubulin Binding ◽

Structural Insight ◽

Dynamics Simulations ◽

Insight Into

Download Full-text

X-ray crystal structure of a malonate-semialdehyde dehydrogenase fromPseudomonassp. strain AAC

Acta Crystallographica Section F Structural Biology Communications ◽

10.1107/s2053230x16020008 ◽

2017 ◽

Vol 73 (1) ◽

pp. 24-28 ◽

Cited By ~ 1

Author(s):

Matthew Wilding ◽

Colin Scott ◽

Thomas S. Peat ◽

Janet Newman

Keyword(s):

Crystal Structure ◽

Search Model ◽

Molecular Replacement ◽

X Rays ◽

X Ray Diffraction ◽

Acetyl Coa ◽

Space Group ◽

X Ray ◽

Semialdehyde Dehydrogenase

The NAD-dependent malonate-semialdehyde dehydrogenase KES23460 fromPseudomonassp. strain AAC makes up half of a bicistronic operon responsible for β-alanine catabolism to produce acetyl-CoA. The KES23460 protein has been heterologously expressed, purified and used to generate crystals suitable for X-ray diffraction studies. The crystals belonged to space groupP212121and diffracted X-rays to beyond 3 Å resolution using the microfocus beamline of the Australian Synchrotron. The structure was solved using molecular replacement, with a monomer from PDB entry 4zz7 as the search model.

Download Full-text

Mathematical aspects of molecular replacement. IV. Measure-theoretic decompositions of motion spaces

Acta Crystallographica Section A Foundations and Advances ◽

10.1107/s2053273317007227 ◽

2017 ◽

Vol 73 (5) ◽

pp. 387-402 ◽

Cited By ~ 2

Author(s):

Gregory S. Chirikjian ◽

Sajdeh Sajjadi ◽

Bernard Shiffman ◽

Steven M. Zucker

Keyword(s):

General Theory ◽

Rigid Body ◽

Three Dimensional ◽

Molecular Replacement ◽

Translation Group ◽

Common Occurrence ◽

Space Group ◽

Space Groups ◽

Relevant Case ◽

The Subject

In molecular-replacement (MR) searches, spaces of motions are explored for determining the appropriate placement of rigid-body models of macromolecules in crystallographic asymmetric units. The properties of the space of non-redundant motions in an MR search, called a `motion space', are the subject of this series of papers. This paper, the fourth in the series, builds on the others by showing that when the space group of a macromolecular crystal can be decomposed into a product of two space subgroups that share only the lattice translation group, the decomposition of the group provides different decompositions of the corresponding motion spaces. Then an MR search can be implemented by trading off between regions of the translation and rotation subspaces. The results of this paper constrain the allowable shapes and sizes of these subspaces. Special choices result when the space group is decomposed into a product of a normal Bieberbach subgroup and a symmorphic subgroup (which is a common occurrence in the space groups encountered in protein crystallography). Examples of Sohncke space groups are used to illustrate the general theory in the three-dimensional case (which is the relevant case for MR), but the general theory in this paper applies to any dimension.

Download Full-text

A Conserved Domain Is Crucial for Acceptor Substrate Binding in a Family of Glucosyltransferases

Journal of Bacteriology ◽

10.1128/jb.02267-14 ◽

2014 ◽

Vol 197 (3) ◽

pp. 510-517 ◽

Cited By ~ 8

Author(s):

Fan Zhu ◽

Hua Zhang ◽

Hui Wu

Keyword(s):

Bacterial Adhesion ◽

Substrate Binding ◽

Molecular Replacement ◽

Bacterial Sepsis ◽

Content Type ◽

Acceptor Substrate ◽

Loop Region ◽

Conserved Domain ◽

Substrate Binding Domain ◽

Insight Into

Serine-rich repeat glycoproteins (SRRPs) are highly conserved in streptococci and staphylococci. Glycosylation of SRRPs is important for bacterial adhesion and pathogenesis.Streptococcus agalactiaeis the leading cause of bacterial sepsis and meningitis among newborns. Srr2, an SRRP fromS. agalactiaestrain COH1, has been implicated in bacterial virulence. Four genes (gtfA,gtfB,gtfC, and gtfD) located downstream ofsrr2share significant homology with genes involved in glycosylation of other SRRPs. We have shown previously thatgtfAandgtfBencode two glycosyltransferases, GtfA and GtfB, that catalyze the transfer of GlcNAc residues to the Srr2 polypeptide. However, the function of other glycosyltransferases in glycosylation of Srr2 is unknown. In this study, we determined that GtfC catalyzed the direct transfer of glucosyl residues to Srr2-GlcNAc. The GtfC crystal structure was solved at 2.7 Å by molecular replacement. Structural analysis revealed a loop region at the N terminus as a putative acceptor substrate binding domain. Deletion of this domain rendered GtfC unable to bind to its substrate Srr2-GlcNAc, concurrently abolished the glycosyltransferase activity of GtfC, and also altered glycosylation of Srr2. Furthermore, deletion of the corresponding regions from GtfC homologs also abolished their substrate binding and enzymatic activity, indicating that this region is functionally conserved. In summary, we have determined that GtfC is important for the glycosylation of Srr2 and identified a conserved loop region that is crucial for acceptor substrate binding from GtfC homologs in streptococci. These findings shed new mechanistic insight into this family of glycosyltransferases.

Download Full-text

Die Kristallstruktur des Lithium-Benzamidinats {Li3[C6H5 - C(NSiMe3)2]3 · NC - C6H5} / The Crystal Structure of the Lithium Phenylamidinate {Li3[C6H5 - C(NSiMe3)2]3 · NC - C6H5}

Zeitschrift für Naturforschung B ◽

10.1515/znb-1994-1023 ◽

1994 ◽

Vol 49 (10) ◽

pp. 1444-1447 ◽

Cited By ~ 7

Author(s):

Helmut Goesmann ◽

Dieter Fenske

Keyword(s):

Crystal Structure ◽

Nitrogen Atom ◽

Single Crystals ◽

Title Compound ◽

The Other ◽

Space Group ◽

Structure Solution

AbstractSingle crystals of the title compound have been prepared by the reaction of benzonitrile with LiN-(SiMe3)2 in hexane and subsequent evaporation of the solvent. Space group P21/n, Z = 4, structure solution with 7945 observed unique reflections. R = 0.052. Lattice dimensions at -70 °C: a = 1485.2(9); b = 2486.9(11); c = 1568.9(8) pm; β = 91.06(4)°. The compound forms a trimeric ion ensemble in which two of the lithium cations are coordinated by three nitrogen atoms of two phenylamidinate an ions, the other one by four nitrogen atoms of two chelating phenylaminidate anions and in addition by the nitrogen atom of a benzonitrile molecule.

Download Full-text

Thiocarbonyl Complexes of Rhenium. Part I.

Zeitschrift für Naturforschung B ◽

10.1515/znb-1993-0611 ◽

1993 ◽

Vol 48 (6) ◽

pp. 771-777 ◽

Cited By ~ 6

Author(s):

Ulrich Abram ◽

Bernd Lorenz

Keyword(s):

Mass Spectrometry ◽

Nmr Spectroscopy ◽

Coordination Geometry ◽

X Ray Diffraction ◽

Rhenium Atom ◽

Space Group ◽

X Ray ◽

Monomeric Complex ◽

Structure Solution ◽

R Value

Novel rhenium complexes with terminal thiocarbonyl groups have been synthesized from ReCl3(Me2PhP)3 and sodium diethyldithiocarbamate. mer-(Diethyldithiocarbamato)tris-(dimethylphenylphosphine)(thiocarbonyl)rhenium(I), mer-[Re(CS)(Me2PhP)3(Et2dtc)], and tris(diethyldithiocarbamato)(thiocarbonyl)rhenium(III), [Re(CS)(Et2dtc)3] have been studied by infrared and NMR spectroscopy, mass spectrometry and X-ray diffraction.mer-[Re(CS)(Me2PhP)3(Et2dtc)] crystallizes orthorhombic in the space group Pna21 with a = 1516.1(2), b = 2189.8(2) and c = 1035.6(1) pm. Structure solution and refinement converged at R = 0.042. The coordination geometry is a distorted octahedron. The Re—C bond length is found to be 184(2) pm.[Re(CS)(Et2dtc)3] crystallizes monoclinic in the space group P21/c with a = 962.2(6), b = 1744.0(2), c = 1537.4(6) pm and β = 96.21(1)°. The final R value is 0.028. In the monomeric complex the rhenium atom is seven-coordinate with an approximate pentagonal-bipyramidal coordination sphere and a rhenium-carbon distance of 181(1) pm.

Download Full-text