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1.  Likelihood-based molecular-replacement solution for a highly pathological crystal with tetartohedral twinning and sevenfold translational noncrystallographic symmetry 
With the implementation of a molecular-replacement likelihood target that accounts for translational noncrystallographic symmetry, it became possible to solve the crystal structure of a protein with seven tetrameric assemblies arrayed translationally along the c axis. The new algorithm found 56 protein molecules in reduced symmetry (P1), which was used to resolve space-group ambiguity caused by severe twinning.
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 in Phaser to 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 the c axis. To resolve space-group ambiguities caused by tetartohedral twinning, the structure was initially solved by placing 56 copies of the monomer in space group P1 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.
doi:10.1107/S1399004713030319
PMCID: PMC3940205  PMID: 24531481
maximum likelihood; translational noncrystallographic symmetry; molecular replacement; commensurate modulation; pseudo-symmetry
2.  Dimeric structure of the N-terminal domain of PriB protein from Thermoanaerobacter tengcongensis solved ab initio  
The N-terminal domain of the PriB protein from the thermophilic bacterium T. tengcongensis (TtePriB) was expressed and its crystal structure has been solved at the atomic resolution of 1.09 Å by direct methods.
PriB is one of the components of the bacterial primosome, which catalyzes the reactivation of stalled replication forks at sites of DNA damage. The N-terminal domain of the PriB protein from the thermophilic bacterium Thermoanaerobacter tengcongensis (TtePriB) was expressed and its crystal structure was solved at the atomic resolution of 1.09 Å by direct methods. The protein chain, which encompasses the first 104 residues of the full 220-residue protein, adopts the characteristic oligonucleotide/oligosaccharide-binding (OB) structure consisting of a five-stranded β-barrel filled with hydrophobic residues and equipped with four loops extending from the barrel. In the crystal two protomers dimerize, forming a six-stranded antiparallel β-sheet. The structure of the N-terminal OB domain of T. tengcongensis shows significant differences compared with mesophile PriBs. While in all other known structures of PriB a dimer is formed by two identical OB domains in separate chains, TtePriB contains two consecutive OB domains in one chain. However, sequence comparison of both the N-terminal and the C-terminal domains of TtePriB suggests that they have analogous structures and that the natural protein possesses a structure similar to a dimer of two N-terminal domains.
doi:10.1107/S0907444912041637
PMCID: PMC3498933  PMID: 23151633
PriB protein; OB domains; atomic resolution; direct methods
3.  Structural basis for bathochromic shift of fluorescence in far-red fluorescent proteins eqFP650 and eqFP670 
The crystal structures of the far-red fluorescent proteins eqFP650 and eqFP670 have been solved at 1.8 and 1.6 Å resolution, respectively. This permitted identification of the structural elements responsible for the bathochromic shift in both considered far-red fluorescent proteins.
The crystal structures of the far-red fluorescent proteins (FPs) eqFP650 (λex max/λem max 592/650 nm) and eqFP670 (λex max/λem max 605/670 nm), the successors of the far-red FP Katushka (λex max/λem max 588/635 nm), have been determined at 1.8 and 1.6 Å resolution, respectively. An examination of the structures demonstrated that there are two groups of changes responsible for the bathochromic shift of excitation/emission bands of these proteins relative to their predecessor. The first group of changes resulted in an increase of hydrophilicity at the acylimine site of the chromophore due to the presence of one and three water molecules in eqFP650 and eqFP670, respectively. These water molecules provide connection of the chromophore with the protein scaffold via hydrogen bonds causing an ∼15 nm bathochromic shift of the eqFP650 and eqFP670 emission bands. The second group of changes observed in eqFP670 arises from substitution of both Ser143 and Ser158 by asparagines. Asn143 and Asn158 of eqFP670 are hydrogen bonded with each other, as well as with the protein scaffold and with the p-hydroxyphenyl group of the chromophore, resulting in an additional ∼20 nm bathochromic shift of the eqFP670 emission band as compared to eqFP650. The role of the observed structural changes was verified by mutagenesis.
doi:10.1107/S0907444912020598
PMCID: PMC3489099  PMID: 22948909
far-red fluorescent proteins; cell imaging; tissue visualization; Katushka
4.  How good can our beamlines be? 
A repetitive measurement of the same diffraction image allows to judge the performance of a data collection facility.
The accuracy of X-ray diffraction data depends on the properties of the crystalline sample and on the performance of the data-collection facility (synchrotron beamline elements, goniostat, detector etc.). However, it is difficult to evaluate the level of performance of the experimental setup from the quality of data sets collected in rotation mode, as various crystal properties such as mosaicity, non-uniformity and radiation damage affect the measured intensities. A multiple-image experiment, in which several analogous diffraction frames are recorded consecutively at the same crystal orientation, allows minimization of the influence of the sample properties. A series of 100 diffraction images of a thaumatin crystal were measured on the SBC beamline 19BM at the APS (Argonne National Laboratory). The obtained data were analyzed in the context of the performance of the data-collection facility. An objective way to estimate the uncertainties of individual reflections was achieved by analyzing the behavior of reflection intensities in the series of analogous diffraction images. The multiple-image experiment is found to be a simple and adequate method to decompose the random errors from the systematic errors in the data, which helps in judging the performance of a data-collection facility. In particular, displaying the intensity as a function of the frame number allows evaluation of the stability of the beam, the beamline elements and the detector with minimal influence of the crystal properties. Such an experiment permits evaluation of the highest possible data quality potentially achievable at the particular beamline.
doi:10.1107/S0907444912034658
PMCID: PMC3447404  PMID: 22993097
diffraction data precision; signal-to-noise ratio; measurement uncertainty; beamline performance
5.  High-resolution structures of complexes of plant S-­adenosyl-l-homocysteine hydrolase (Lupinus luteus) 
Crystal structures of S-adenosyl-l-homocysteine hydrolase from L. luteus in complex with adenosine, cordycepin and adenine are presented.
S-Adenosyl-l-homocysteine hydrolase (SAHase) catalyzes the reversible breakdown of S-adenosyl-l-homocysteine (SAH) to adenosine and homocysteine. SAH is formed in methylation reactions that utilize S-adenosyl-l-methionine (SAM) as a methyl donor. By removing the SAH byproduct, SAHase serves as a major regulator of SAM-dependent biological methylation reactions. Here, the first crystal structure of SAHase of plant origin, that from the legume yellow lupin (LlSAHase), is presented. Structures have been determined at high resolution for three complexes of the enzyme: those with a reaction byproduct/substrate (adenosine), with its nonoxidizable analog (cordycepin) and with a product of inhibitor cleavage (adenine). In all three cases the enzyme has a closed conformation. A sodium cation is found near the active site, coordinated by residues from a conserved loop that hinges domain movement upon reactant binding. An insertion segment that is present in all plant SAHases is located near a substrate-pocket access channel and participates in its formation. In contrast to mammalian and bacterial SAHases, the channel is open when adenosine or cordycepin is bound and is closed in the adenine complex. In contrast to SAHases from other organisms, which are active as tetramers, the plant enzyme functions as a homodimer in solution.
doi:10.1107/S0907444911055090
PMCID: PMC3282620  PMID: 22349223
S-Adenosyl-l-homocysteine hydrolase; Lupinus luteus
6.  Structures of NodZ α1,6-fucosyltransferase in complex with GDP and GDP-fucose 
Crystal structures of the bacterial α1,6-fucosyltransferase NodZ in complex with GDP and GDP-fucose are presented.
Rhizobial NodZ α1,6-fucosyltransferase (α1,6-FucT) catalyzes the transfer of the fucose (Fuc) moiety from guanosine 5′-­diphosphate-β-l-fucose to the reducing end of the chitin oligosaccharide core during Nod-factor (NF) biosynthesis. NF is a key signalling molecule required for successful symbiosis with a legume host for atmospheric nitrogen fixation. To date, only two α1,6-FucT structures have been determined, both without any donor or acceptor molecule that could highlight the structural background of the catalytic mechanism. Here, the first crystal structures of α1,6-FucT in complex with its substrate GDP-Fuc and with GDP, which is a byproduct of the enzymatic reaction, are presented. The crystal of the complex with GDP-Fuc was obtained through soaking of native NodZ crystals with the ligand and its structure has been determined at 2.35 Å resolution. The fucose residue is exposed to solvent and is disordered. The enzyme–product complex crystal was obtained by cocrystallization with GDP and an acceptor molecule, penta-N-acetyl-l-­glucosamine (penta-NAG). The structure has been determined at 1.98 Å resolution, showing that only the GDP molecule is present in the complex. In both structures the ligands are located in a cleft formed between the two domains of NodZ and extend towards the C-terminal domain, but their conformations differ significantly. The structures revealed that residues in three regions of the C-­terminal domain, which are conserved among α1,2-, α1,6- and protein O-fucosyltransferases, are involved in interactions with the sugar-donor molecule. There is also an interaction with the side chain of Tyr45 in the N-terminal domain, which is very unusual for a GT-B-type glycosyltransferase. Only minor conformational changes of the protein backbone are observed upon ligand binding. The only exception is a movement of the loop located between strand βC2 and helix αC3. In addition, there is a shift of the αC3 helix itself upon GDP-Fuc binding.
doi:10.1107/S0907444911053157
PMCID: PMC3266854  PMID: 22281745
glycosyltransferases; fucosyltransferases; family GT-23 glycosyltransferases; chitooligosaccharide fucosylation; Nod-factor biosynthesis; nodulation; Nod factors; legume–rhizobium symbiosis; nitrogen fixation
7.  Deprotonated imidodiphosphate in AMPPNP-­containing protein structures 
In certain AMPPNP-containing protein structures, the nitrogen bridging the two terminal phosphate groups can be deprotonated.
Many different proteins utilize the chemical energy provided by the cofactor adenosine triphosphate (ATP) for their proper function. A number of structures in the Protein Data Bank (PDB) contain adenosine 5′-(β,γ-imido)triphosphate (AMPPNP), a nonhydrolysable analog of ATP in which the bridging O atom between the two terminal phosphate groups is substituted by the imido function. Under mild conditions imides do not have acidic properties and thus the imide nitrogen should be protonated. However, an analysis of protein structures containing AMPPNP reveals that the imide group is deprotonated in certain complexes if the negative charges of the phosphate moieties in AMPPNP are in part neutralized by coordinating divalent metals or a guanidinium group of an arginine.
doi:10.1107/S0907444911046105
PMCID: PMC3225179  PMID: 22120745
imidodiphosphate; adenosine 5′-(β,γ-methylene)triphosphate; AMPPNP
8.  Human Suv3 protein reveals unique features among SF2 helicases 
Crystal structures of the human mitochondrial helicase hSuv3 in complex with AMPPNP and with a short strand of RNA are presented.
Suv3 is a helicase that is involved in efficient turnover and surveillance of RNA in eukaryotes. In vitro studies show that human Suv3 (hSuv3) in complex with human polynucleotide phosphorylase has RNA degradosome activity. The enzyme is mainly localized in mitochondria, but small fractions are found in cell nuclei. Here, two X-ray crystallographic structures of human Suv3 in complex with AMPPNP, a nonhydrolysable analog of ATP, and with a short five-nucleotide strand of RNA are presented at resolutions of 2.08 and 2.9 Å, respectively. The structure of the enzyme is very similar in the two complexes and consists of four domains. Two RecA-like domains form the tandem typical of all helicases from the SF2 superfamily which together with the C-terminal all-helical domain makes a ring structure through which the nucleotide strand threads. The mostly helical N-terminal domain is positioned externally with respect to the core of the enzyme. Most of the typical helicase motifs are present in hSuv3, but the protein shows certain unique characteristics, suggesting that Suv3 enzymes may constitute a separate subfamily of helicases.
doi:10.1107/S0907444911040248
PMCID: PMC3211972  PMID: 22101826
mitochondrial helicases; human Suv3; SF2 helicases
9.  Structure of the N-terminal fragment of Escherichia coli Lon protease 
The medium-resolution structure of the N-terminal fragment of E. coli Lon protease shows that this part of the enzyme consists of two compact domains and a very long α-helix.
The structure of a recombinant construct consisting of residues 1–245 of Escherichia coli Lon protease, the prototypical member of the A-type Lon family, is reported. This construct encompasses all or most of the N-terminal domain of the enzyme. The structure was solved by SeMet SAD to 2.6 Å resolution utilizing trigonal crystals that contained one molecule in the asymmetric unit. The molecule consists of two compact subdomains and a very long C-terminal α-helix. The structure of the first subdomain (residues 1–117), which consists mostly of β-strands, is similar to that of the shorter fragment previously expressed and crystallized, whereas the second subdomain is almost entirely helical. The fold and spatial relationship of the two subdomains, with the exception of the C-terminal helix, closely resemble the structure of BPP1347, a 203-amino-acid protein of unknown function from Bordetella parapertussis, and more distantly several other proteins. It was not possible to refine the structure to satisfactory convergence; however, since almost all of the Se atoms could be located on the basis of their anomalous scattering the correctness of the overall structure is not in question. The structure reported here was also compared with the structures of the putative substrate-binding domains of several proteins, showing topological similarities that should help in defining the binding sites used by Lon substrates.
doi:10.1107/S0907444910019554
PMCID: PMC2917273  PMID: 20693685
anomalous diffraction; ATP-dependent proteases; protein domains; structure quality; Lon protease
10.  Structure of the Taz2 domain of p300: insights into ligand binding 
The crystal structure of the Taz2 zinc-finger domain of the human p300 transcriptional coactivator was determined using the anomalous diffraction signal of the bound Zn ions. Crystal contacts suggested a possible novel mode of Taz2–peptide ligand interactions.
CBP and its paralog p300 are histone acetyl transferases that regulate gene expression by interacting with multiple transcription factors via specialized domains. The structure of a segment of human p300 protein (residues 1723–1836) corresponding to the extended zinc-binding Taz2 domain has been investigated. The crystal structure was solved by the SAD approach utilizing the anomalous diffraction signal of the bound Zn ions. The structure comprises an atypical helical bundle stabilized by three Zn ions and closely resembles the solution structures determined previously for shorter peptides. Residues 1813–1834 from the current construct form a helical extension of the C-terminal helix and make extensive crystal-contact interactions with the peptide-binding site of Taz2, providing additional insights into the mechanism of the recognition of diverse transactivation domains (TADs) by Taz2. On the basis of these results and molecular modeling, a hypothetical model of the binding of phosphorylated p53 TAD1 to Taz2 has been proposed.
doi:10.1107/S0907444909040153
PMCID: PMC2789004  PMID: 19966416
zinc-finger proteins; anomalous diffraction; protein recognition; transcription regulation
11.  Rotational order–disorder structure of fluorescent protein FP480 
An analysis of the rotational order–disorder structure of fluorescent protein FP480 is presented.
In the last decade, advances in instrumentation and software development have made crystallography a powerful tool in structural biology. Using this method, structural information can now be acquired from pathological crystals that would have been abandoned in earlier times. In this paper, the order–disorder (OD) structure of fluorescent protein FP480 is discussed. The structure is composed of tetramers with 222 symmetry incorporated into the lattice in two different ways, namely rotated 90° with respect to each other around the crystal c axis, with tetramer axes coincident with crystallo­graphic twofold axes. The random distribution of alternatively oriented tetramers in the crystal creates a rotational OD structure with statistically averaged I422 symmetry, although the presence of very weak and diffuse additional reflections suggests that the randomness is only approximate.
doi:10.1107/S0907444909020927
PMCID: PMC2733879  PMID: 19690368
order–disorder structures; rotational order–disorder; fluorescent proteins
12.  To scavenge or not to scavenge: that is the question 
Analysis of a series of diffraction data sets measured from several native as well as nicotinic acid-soaked crystals of trypsin suggests that this potential scavenger does not have any statistically significant effect on the amount of radiation damage incurred in the crystals on X-ray irradiation at 100 K.
Analysis of a series of diffraction data sets measured from four native as well as four nicotinic acid-soaked crystals of trypsin at 100 K shows a high variability in radiation-sensitivity among individual crystals for both nicotinic acid-soaked and native crystals. The level of radiation-sensitivity and the extent of its variability is statistically indistinguishable between the two conditions. This suggests that this potential scavenger does not have any statistically significant effect on the amount of radiation damage incurred in the crystals on X-ray irradiation. This is in contrast to previous results [Kauffmann et al. (2006 ▶), Structure, 14, 1099–1105] where only one crystal specimen was used for each condition (native and nicotinic acid-soaked).
doi:10.1107/S0907444909026821
PMCID: PMC2733885  PMID: 19690379
protein crystallography; radiation damage; scavengers; nicotinic acid
13.  Carrying out an optimal experiment 
Diffraction data collection parameters leading to optimal data quality are discussed in the context of different applications of these data.
Diffraction data collection is the last experimental stage in structural crystallography. It has several technical and theor­etical aspects and a compromise usually has to be found between various parameters in order to achieve optimal data quality. The influence and importance of various experimental parameters and their consequences are discussed in the context of different data applications, such as molecular replacement, anomalous phasing, high-resolution refinement or searching for ligands.
doi:10.1107/S0907444909038578
PMCID: PMC2852303  PMID: 20382992
diffraction data collection; data-collection strategies; diffraction experiments

Results 1-13 (13)