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1.  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
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.  Hydrogen atoms in protein structures: high-resolution X-ray diffraction structure of the DFPase 
BMC Research Notes  2013;6:308.
Background
Hydrogen atoms represent about half of the total number of atoms in proteins and are often involved in substrate recognition and catalysis. Unfortunately, X-ray protein crystallography at usual resolution fails to access directly their positioning, mainly because light atoms display weak contributions to diffraction. However, sub-Ångstrom diffraction data, careful modeling and a proper refinement strategy can allow the positioning of a significant part of hydrogen atoms.
Results
A comprehensive study on the X-ray structure of the diisopropyl-fluorophosphatase (DFPase) was performed, and the hydrogen atoms were modeled, including those of solvent molecules. This model was compared to the available neutron structure of DFPase, and differences in the protein and the active site solvation were noticed.
Conclusions
A further examination of the DFPase X-ray structure provides substantial evidence about the presence of an activated water molecule that may constitute an interesting piece of information as regard to the enzymatic hydrolysis mechanism.
doi:10.1186/1756-0500-6-308
PMCID: PMC3737025  PMID: 23915572
Sub-Ångstrom X-ray crystallography; Hydrogen atoms; DFPase; MoPro software
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.  Structural insights and ab initio sequencing within the DING proteins family 
Journal of Synchrotron Radiation  2010;18(Pt 1):45-49.
DING proteins constitute a recently discovered protein family that is ubiquitous in eukaryotes. The structural insights and the physiological involvements of these intriguing proteins are hereby deciphered.
DING proteins constitute an intriguing family of phosphate-binding proteins that was identified in a wide range of organisms, from prokaryotes and archae to eukaryotes. Despite their seemingly ubiquitous occurrence in eukaryotes, their encoding genes are missing from sequenced genomes. Such a lack has considerably hampered functional studies. In humans, these proteins have been related to several diseases, like atherosclerosis, kidney stones, inflammation processes and HIV inhibition. The human phosphate binding protein is a human representative of the DING family that was serendipitously discovered from human plasma. An original approach was developed to determine ab initio the complete and exact sequence of this 38 kDa protein by utilizing mass spectrometry and X-ray data in tandem. Taking advantage of this first complete eukaryotic DING sequence, a immunohistochemistry study was undertaken to check the presence of DING proteins in various mice tissues, revealing that these proteins are widely expressed. Finally, the structure of a bacterial representative from Pseudomonas fluorescens was solved at sub-angstrom resolution, allowing the molecular mechanism of the phosphate binding in these high-affinity proteins to be elucidated.
doi:10.1107/S0909049510036009
PMCID: PMC3004253  PMID: 21169690
serendipity; DING protein; ab initio sequencing; sub-angstrom crystallography; HIV inhibition

Results 1-5 (5)