An analysis of the rotational order–disorder structure of the reversibly photoswitchable red fluorescent protein rsTagRFP is presented.
The rotational order–disorder (OD) structure of the reversibly photoswitchable fluorescent protein rsTagRFP is discussed in detail. The structure is composed of tetramers of 222 symmetry incorporated into the lattice in two different orientations rotated 90° with respect to each other around the crystal c axis and with tetramer axes coinciding with the crystallographic twofold axes. The random distribution of alternatively oriented tetramers in the crystal creates the rotational OD structure with statistically averaged I422 symmetry. Despite order–disorder pathology, the structure of rsTagRFP has electron-density maps of good quality for both non-overlapping and overlapping parts of the model. The crystal contacts, crystal internal architecture and a possible mechanism of rotational OD crystal formation are discussed.
OD structure; rotational order–disorder; fluorescent protein
Quality control of three-dimensional structures of macromolecules is a critical step to ensure the integrity of structural biology data, especially those produced by structural genomics centers. Whereas the Protein Data Bank (PDB) has proven to be a remarkable success overall, the inconsistent quality of structures reveals a lack of universal standards for structure/deposit validation. Here, we review the state-of-the-art methods used in macromolecular structure validation, focusing on validation of structures determined by X-ray crystallography. We describe some general protocols used in the rebuilding and re-refinement of problematic structural models. We also briefly discuss some frontier areas of structure validation, including refinement of protein–ligand complexes, automation of structure redetermination, and the use of NMR structures and computational models to solve X-ray crystal structures by molecular replacement.
Structure quality; Structure validation; Drug discovery; Data mining; Structural genomics
The number of macromolecular structures deposited in the Protein Data Bank now approaches 100 000, with the vast majority of them determined by crystallographic methods. Thousands of papers describing such structures have been published in the scientific literature, and 20 Nobel Prizes in chemistry or medicine have been awarded for discoveries based on macromolecular crystallography. New hardware and software tools have made crystallography appear to be an almost routine (but still far from being analytical) technique and many structures are now being determined by scientists with very limited experience in the practical aspects of the field. However, this apparent ease is sometimes illusory and proper procedures need to be followed to maintain high standards of structure quality. In addition, many noncrystallographers may have problems with the critical evaluation and interpretation of structural results published in the scientific literature. The present review provides an outline of the technical aspects of crystallography for less experienced practitioners, as well as information that might be useful for users of macromolecular structures, aiming to show them how to interpret (but not overinterpret) the information present in the coordinate files and in their description. A discussion of the extent of information that can be gleaned from the atomic coordinates of structures solved at different resolution is provided, as well as problems and pitfalls encountered in structure determination and interpretation.
data collection and processing; electron density maps; protein crystallography; structure refinement; structure solution; structure quality; structure validation
The crystal structure of the novel red emitting fluorescent protein from lancelet Branchiostoma lanceolatum (Chordata) revealed an unusual five residues cyclic unit comprising Gly58-Tyr59-Gly60 chromophore, the following Phe61 and Tyr62 covalently bound to chromophore Tyr59.
A key property of proteins of the green fluorescent protein (GFP) family is their ability to form a chromophore group by post-translational modifications of internal amino acids, e.g. Ser65-Tyr66-Gly67 in GFP from the jellyfish Aequorea victoria (Cnidaria). Numerous structural studies have demonstrated that the green GFP-like chromophore represents the ‘core’ structure, which can be extended in red-shifted proteins owing to modifications of the protein backbone at the first chromophore-forming position. Here, the three-dimensional structures of green laGFP (λex/λem = 502/511 nm) and red laRFP (λex/λem ≃ 521/592 nm), which are fluorescent proteins (FPs) from the lancelet Branchiostoma lanceolatum (Chordata), were determined together with the structure of a red variant laRFP-ΔS83 (deletion of Ser83) with improved folding. Lancelet FPs are evolutionarily distant and share only ∼20% sequence identity with cnidarian FPs, which have been extensively characterized and widely used as genetically encoded probes. The structure of red-emitting laRFP revealed three exceptional features that have not been observed in wild-type fluorescent proteins from Cnidaria reported to date: (i) an unusual chromophore-forming sequence Gly58-Tyr59-Gly60, (ii) the presence of Gln211 at the position of the conserved catalytic Glu (Glu222 in Aequorea GFP), which proved to be crucial for chromophore formation, and (iii) the absence of modifications typical of known red chromophores and the presence of an extremely unusual covalent bond between the Tyr59 Cβ atom and the hydroxyl of the proximal Tyr62. The impact of this covalent bond on the red emission and the large Stokes shift (∼70 nm) of laRFP was verified by extensive structure-based site-directed mutagenesis.
red fluorescent proteins; GYG chromophore; Branchiostoma lanceolatum; lancelets
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.
atomic resolution; structure comparison; trypsin; structural reproducibility
A structural analysis of the recently developed orange fluorescent proteins with novel phenotypes, LSSmOrange (λex/λem at 437/572 nm), PSmOrange (λex/λem at 548/565 nm and for photoconverted form at 636/662 nm) and PSmOrange2 (λex/λem at 546/561 nm and for photoconverted form at 619/651 nm), is presented. The obtained crystallographic structures provide an understanding of how the ensemble of a few key mutations enabled special properties of the orange FPs. While only a single Ile161Asp mutation, enabling excited state proton transfer, is critical for LSSmOrange, other substitutions provide refinement of its special properties and an exceptional 120 nm large Stokes shift. Similarly, a single Gln64Leu mutation was sufficient to cause structural changes resulting in photoswitchability of PSmOrange, and only one additional substitution (Phe65Ile), yielding PSmOrange2, was enough to greatly decrease the energy of photoconversion and increase its efficiency of photoswitching. Fluorescence of photoconverted PSmOrange and PSmOrange2 demonstrated an unexpected bathochromic shift relative to the fluorescence of classic red FPs, such as DsRed, eqFP578 and zFP574. The structural changes associated with this fluorescence shift are of considerable value for the design of advanced far-red FPs. For this reason the chromophore transformations accompanying photoconversion of the orange FPs are discussed.
The yellow fluorescent protein phiYFPv with improved folding has been developed from the spectrally identical wild-type phiYFP found in the marine jellyfish Phialidium.
The yellow fluorescent protein phiYFPv (λem
max ≃ 537 nm) with improved folding has been developed from the spectrally identical wild-type phiYFP found in the marine jellyfish Phialidium. The latter fluorescent protein is one of only two known cases of naturally occurring proteins that exhibit emission spectra in the yellow–orange range (535–555 nm). Here, the crystal structure of phiYFPv has been determined at 2.05 Å resolution. The ‘yellow’ chromophore formed from the sequence triad Thr65-Tyr66-Gly67 adopts the bicyclic structure typical of fluorophores emitting in the green spectral range. It was demonstrated that perfect antiparallel π-stacking of chromophore Tyr66 and the proximal Tyr203, as well as Val205, facing the chromophore phenolic ring are chiefly responsible for the observed yellow emission of phiYFPv at 537 nm. Structure-based site-directed mutagenesis has been used to identify the key functional residues in the chromophore environment. The obtained results have been utilized to improve the properties of phiYFPv and its homologous monomeric biomarker tagYFP.
yellow fluorescent protein; Phialidium; structure–function relationship; chromophores; oligomeric structure; intersubunit surface
Standard ways for the placement of molecules in the unit cell are proposed.
There are currently no rules for a unified, standard way of placing macromolecular structures in the crystal lattice. An analysis of all possible symmetry-equivalent representations of molecular structures in various space groups leads to the concept of the anti-Cheshire symmetry and suggests that the center of a unique structural motif can always be placed within the selected asymmetric unit of the anti-Cheshire cell. The placement of structures according to this suggestion will ensure uniformity of presentation of all structurally equivalent Protein Data Bank models and will therefore diminish the possibility of confusing less crystallographically knowledgeable users of the PDB. The anti-Cheshire cells and their asymmetric units are defined and tabulated for all 65 space groups relevant to macromolecular crystallography that exhibit only rotational symmetry operations.
placement of molecules; Cheshire symmetry; anti-Cheshire symmetry
The dual role of the Protein Data Bank as a repository of all macromolecular structures and as the major source of structural metadata for further analysis is discussed and suggestions are made on how to identify models that contain errors and could potentially degrade the quality of meta analyses.
Whereas the vast majority of the more than 85 000 crystal structures of macromolecules currently deposited in the Protein Data Bank are of high quality, some suffer from a variety of imperfections. Although this fact has been pointed out in the past, it is still worth periodic updates so that the metadata obtained by global analysis of the available crystal structures, as well as the utilization of the individual structures for tasks such as drug design, should be based on only the most reliable data. Here, selected abnormal deposited structures have been analysed based on the Bayesian reasoning that the correctness of a model must be judged against both the primary evidence as well as prior knowledge. These structures, as well as information gained from the corresponding publications (if available), have emphasized some of the most prevalent types of common problems. The errors are often perfect illustrations of the nature of human cognition, which is frequently influenced by preconceptions that may lead to fanciful results in the absence of proper validation. Common errors can be traced to negligence and a lack of rigorous verification of the models against electron density, creation of non-parsimonious models, generation of improbable numbers, application of incorrect symmetry, illogical presentation of the results, or violation of the rules of chemistry and physics. Paying more attention to such problems, not only in the final validation stages but during the structure-determination process as well, is necessary not only in order to maintain the highest possible quality of the structural repositories and databases but most of all to provide a solid basis for subsequent studies, including large-scale data-mining projects. For many scientists PDB deposition is a rather infrequent event, so the need for proper training and supervision is emphasized, as well as the need for constant alertness of reason and critical judgment as absolutely necessary safeguarding measures against such problems. Ways of identifying more problematic structures are suggested so that their users may be properly alerted to their possible shortcomings.
macromolecular crystallography; model validation; Protein Data Bank
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.
maximum likelihood; translational noncrystallographic symmetry; molecular replacement; commensurate modulation; pseudo-symmetry
Singular value decomposition of a matrix is a versatile tool used in multivariate data analysis. Here, its use is presented to test the validity of physical models applied when scaling diffraction data affected by radiation-induced changes.
In an X-ray diffraction experiment, the structure of molecules and the crystal lattice changes owing to chemical reactions and physical processes induced by the absorption of X-ray photons. These structural changes alter structure factors, affecting the scaling and merging of data collected at different absorbed doses. Many crystallographic procedures rely on the analysis of consistency between symmetry-equivalent reflections, so failure to account for the drift of their intensities hinders the structure solution and the interpretation of structural results. The building of a conceptual model of radiation-induced changes in macromolecular crystals is the first step in the process of correcting for radiation-induced inconsistencies in diffraction data. Here the complexity of radiation-induced changes in real and reciprocal space is analysed using matrix singular value decomposition applied to multiple complete datasets obtained from single crystals. The model consists of a resolution-dependent decay correction and a uniform-per-unique-reflection term modelling specific radiation-induced changes. This model is typically sufficient to explain radiation-induced effects observed in diffraction intensities. This analysis will guide the parameterization of the model, enabling its use in subsequent crystallographic calculations.
radiation damage; matrix singular value decomposition; experimental phasing; radiolysis
The importance of presenting macromolecular structures in unified, standard ways is discussed.
To uniquely describe a crystal structure, it is sufficient to specify the crystal unit cell and symmetry, and describe the unique structural motif which is repeated by the space-group symmetry throughout the whole crystal. It is somewhat arbitrary how such a unique motif can be defined and positioned with respect to the unit-cell origin. As a result of such freedom, some isomorphous structures are presented in the Protein Data Bank in different locations and appear as if they have different atomic coordinates, despite being completely equivalent structurally. This may easily confuse those users of the PDB who are less familiar with crystallographic symmetry transformations. It would therefore be beneficial for the community of PDB users to introduce standard rules for locating crystal structures of macromolecules in the unit cells of various space groups.
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.
PriB protein; OB domains; atomic resolution; direct methods
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 592/650 nm) and eqFP670 (λex
max 605/670 nm), the successors of the far-red FP Katushka (λex
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.
far-red fluorescent proteins; cell imaging; tissue visualization; Katushka
The bacterial heat shock protein Hsp33 is a redox-regulated chaperone activated by oxidative stress. In response to oxidation, four cysteines within a Zn2+ binding C-terminal domain form two disulfide bonds with concomitant release of the metal. This leads to the formation of the biologically active Hsp33 dimer. The crystal structure of the N-terminal domain of the E. coli protein has been reported, but neither the structure of the Zn2+ binding motif nor the nature of its regulatory interaction with the rest of the protein are known. Here we report the crystal structure of the full-length B. subtilis Hsp33 in the reduced form. The structure of the N-terminal, dimerization domain is similar to that of the E. coli protein, although there is no domain swapping. The Zn2+ binding domain is clearly resolved showing the details of the tetrahedral coordination of Zn2+ by four thiolates. We propose a structure-based activation pathway for Hsp33.
rsTagRFP the first monomeric red fluorescent protein with reversibly photoswitchable absorbance spectra. The switching is realized by irradiation of rsTagRFP with blue (440 nm) and yellow (567 nm) light, turning the protein fluorescence ON and OFF, respectively. It is perhaps the most useful probe in this color class that has yet been reported. Because of the photoswitchable absorbance, rsTagRFP can be used as an acceptor in photochromic Förster resonance energy transfer (pcFRET). Yellow fluorescent proteins YPet and mVenus have been demonstrated to be excellent pcFRET donors for the rsTagRFP acceptor in its fusion constructs. Analysis of X-ray structures has shown that photoswitching of rsTagRFP is accompanied by cis-trans isomerization and protonation/deprotonation of the chromophore, with the deprotonated cis- and protonated trans- isomers corresponding to its ON and OFF states. Unlike in other photoswitchable fluorescent proteins, both conformers of rsTagRFP chromophore are essentially coplanar. Two other peculiarities of the rsTagRFP chromophore are an essentially hydrophobic environment of its p-hydroxyphenyl site and the absence of direct hydrogen bonding between this moiety and the protein scaffold. The influence of the immediate environment on rsTagRFP chromophore was probed by site-directed mutagenesis. Residues Glu145 and His197 were found to participate in protonation/deprotonation of the chromophore accompanying the photoswitching of rsTagRFP fluorescence, whereas the residues Met160 and Leu174 were shown to spatially restrict chromophore isomerization, favoring its radiative decay.
KFP; Dronpa; TagRFP; PAmCherry; FRET
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.
S-Adenosyl-l-homocysteine hydrolase; Lupinus luteus
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.
glycosyltransferases; fucosyltransferases; family GT-23 glycosyltransferases; chitooligosaccharide fucosylation; Nod-factor biosynthesis; nodulation; Nod factors; legume–rhizobium symbiosis; nitrogen fixation
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.
imidodiphosphate; adenosine 5′-(β,γ-methylene)triphosphate; AMPPNP
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.
mitochondrial helicases; human Suv3; SF2 helicases
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.
diffraction data precision; signal-to-noise ratio; measurement uncertainty; beamline performance
Mutations were introduced to the domain-swapped homodimer of the antiviral lectin griffithsin (GRFT). Whereas several single and double mutants remained dimeric, insertion of either two or four amino acids at the dimerization interface resulted in a monomeric form of the protein (mGRFT). Monomeric character of the modified proteins was confirmed by sedimentation equilibrium ultracentrifugation and by their high resolution X-ray crystal structures, whereas their binding to carbohydrates was assessed by isothermal titration calorimetry. Cell-based antiviral activity assays utilizing different variants of mGRFT indicated that the monomeric form of the lectin had greatly reduced activity against HIV-1, suggesting that the antiviral activity of GRFT stems from crosslinking and aggregation of viral particles via multivalent interactions between GRFT and oligosaccharides present on HIV envelope glycoproteins. Atomic resolution crystal structure of a complex between mGRFT and nonamannoside revealed that a single mGRFT molecule binds to two different nonamannoside molecules through all three carbohydrate-binding sites present on the monomer.
Lectin; Entry Inhibitor; HIV; Man9; High Mannose Oligosaccharide; Branched Carbohydrates; Griffithsin
In the crystal structure of the racemic title compound, C22H25NO2, solved and refined against sychrotron diffraction data, the hydroxy group and the carbonyl O atom participate in the formation of O—H⋯O hydrogen bonds between pairs of enantiomers related by a crystallographic centre of symmetry.