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1.  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
2.  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
3.  On the reproducibility of protein crystal structures: five atomic resolution structures of trypsin 
Details of five very high-resolution accurate structures of bovine trypsin are compared in the context of the reproducibility of models obtained from crystals grown under identical conditions.
Structural studies of proteins usually rely on a model obtained from one crystal. By investigating the details of this model, crystallographers seek to obtain insight into the function of the macromolecule. It is therefore important to know which details of a protein structure are reproducible or to what extent they might differ. To address this question, the high-resolution structures of five crystals of bovine trypsin obtained under analogous conditions were compared. Global parameters and structural details were investigated. All of the models were of similar quality and the pairwise merged intensities had large correlation coefficients. The Cα and backbone atoms of the structures superposed very well. The occupancy of ligands in regions of low thermal motion was reproducible, whereas solvent molecules containing heavier atoms (such as sulfur) or those located on the surface could differ significantly. The coordination lengths of the calcium ion were conserved. A large proportion of the multiple conformations refined to similar occupancies and the residues adopted similar orientations. More than three quarters of the water-molecule sites were conserved within 0.5 Å and more than one third were conserved within 0.1 Å. An investigation of the protonation states of histidine residues and carboxylate moieties was consistent for all of the models. Radiation-damage effects to disulfide bridges were observed for the same residues and to similar extents. Main-chain bond lengths and angles averaged to similar values and were in agreement with the Engh and Huber targets. Other features, such as peptide flips and the double conformation of the inhibitor molecule, were also reproducible in all of the trypsin structures. Therefore, many details are similar in models obtained from different crystals. However, several features of residues or ligands located in flexible parts of the macromolecule may vary significantly, such as side-chain orientations and the occupancies of certain fragments.
doi:10.1107/S0907444913009050
PMCID: PMC3727327  PMID: 23897468
atomic resolution; structure comparison; trypsin; structural reproducibility
4.  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
5.  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
6.  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

Results 1-6 (6)