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1.  The structure of an authentic spore photoproduct lesion in DNA suggests a basis for recognition. Corrigendum 
A correction is made to the article by Singh et al. [(2014). Acta Cryst. D70, 752–759].
The article by Singh et al. [ (2014). Acta Cryst. D70, 752–759] is corrected.
PMCID: PMC4030664
spore photoproduct; DNA; host–guest approach; corrigendum
2.  Structural basis of sialidase in complex with geranylated flavonoids as potent natural inhibitors. Corrigendum 
The article by Lee et al. [(2014) Acta Cryst. D70, 1357–1365] is corrected.
A correction is made to the article by Lee et al. [(2014) Acta Cryst. D70, 1357–1365].
PMCID: PMC4321492  PMID: 25664753
sialidase; NanI; geranylated flavonoid; diplacone; sialidase inhibitor; corrigendum
3.  Analysis of the HindIII-catalyzed reaction by time-resolved crystallography 
A time-resolved study using the freeze-trap method elucidates the mechanism of the DNA-cleaving reaction of HindIII.
In order to investigate the mechanism of the reaction catalyzed by HindIII, structures of HindIII–DNA complexes with varying durations of soaking time in cryoprotectant buffer containing manganese ions were determined by the freeze-trap method. In the crystal structures of the complexes obtained after soaking for a longer duration, two manganese ions, indicated by relatively higher electron density, are clearly observed at the two metal ion-binding sites in the active site of HindIII. The increase in the electron density of the two metal-ion peaks followed distinct pathways with increasing soaking times, suggesting variation in the binding rate constant for the two metal sites. DNA cleavage is observed when the second manganese ion appears, suggesting that HindIII uses the two-metal-ion mechanism, or alternatively that its reactivity is enhanced by the binding of the second metal ion. In addition, conformational change in a loop near the active site accompanies the catalytic reaction.
PMCID: PMC4321485  PMID: 25664735
HindIII; time-resolved crystallography; freeze-trap method
4.  Nitrogenase MoFe protein from Clostridium pasteurianum at 1.08 Å resolution: comparison with the Azotobacter vinelandii MoFe protein 
Determination of the nitrogenase MoFe protein from C. pasteurianum at 1.08 Å resolution and comparison to its distinct ortholog from A. vinelandii at atomic resolution reveals conserved structural arrangements that are significant to the function of nitrogenase.
The X-ray crystal structure of the nitrogenase MoFe protein from Clostridium pasteurianum (Cp1) has been determined at 1.08 Å resolution by multiwavelength anomalous diffraction phasing. Cp1 and the ortholog from Azotobacter vinelandii (Av1) represent two distinct families of nitrogenases, differing primarily by a long insertion in the α-subunit and a deletion in the β-subunit of Cp1 relative to Av1. Comparison of these two MoFe protein structures at atomic resolution reveals conserved structural arrangements that are significant to the function of nitrogenase. The FeMo cofactors defining the active sites of the MoFe protein are essentially identical between the two proteins. The surrounding environment is also highly conserved, suggesting that this structural arrangement is crucial for nitrogen reduction. The P clusters are likewise similar, although the surrounding protein and solvent environment is less conserved relative to that of the FeMo cofactor. The P cluster and FeMo cofactor in Av1 and Cp1 are connected through a conserved water tunnel surrounded by similar secondary-structure elements. The long α-subunit insertion loop occludes the presumed Fe protein docking surface on Cp1 with few contacts to the remainder of the protein. This makes it plausible that this loop is repositioned to open up the Fe protein docking surface for complex formation.
PMCID: PMC4321486  PMID: 25664737
FeMo cofactor; P cluster; metalloproteins; iron–sulfur clusters
5.  Exploring the speed and performance of molecular replacement with AMPLE using QUARK ab initio protein models 
Two ab initio modelling programs solve complementary sets of targets, enhancing the success of AMPLE with small proteins.
AMPLE clusters and truncates ab initio protein structure predictions, producing search models for molecular replacement. Here, an interesting degree of complementarity is shown between targets solved using the different ab initio modelling programs QUARK and ROSETTA. Search models derived from either program collectively solve almost all of the all-helical targets in the test set. Initial solutions produced by Phaser after only 5 min perform surprisingly well, improving the prospects for in situ structure solution by AMPLE during synchrotron visits. Taken together, the results show the potential for AMPLE to run more quickly and successfully solve more targets than previously suspected.
PMCID: PMC4321487  PMID: 25664744
AMPLE; QUARK; ROSETTA; ab initio modelling; molecular replacement
6.  Data Exploration Toolkit for serial diffraction experiments 
This paper describes a set of tools allowing experimentalists insight into the variation present within large serial data sets.
Ultrafast diffraction at X-ray free-electron lasers (XFELs) has the potential to yield new insights into important biological systems that produce radiation-sensitive crystals. An unavoidable feature of the ‘diffraction before destruction’ nature of these experiments is that images are obtained from many distinct crystals and/or different regions of the same crystal. Combined with other sources of XFEL shot-to-shot variation, this introduces significant heterogeneity into the diffraction data, complicating processing and interpretation. To enable researchers to get the most from their collected data, a toolkit is presented that provides insights into the quality of, and the variation present in, serial crystallography data sets. These tools operate on the unmerged, partial intensity integration results from many individual crystals, and can be used on two levels: firstly to guide the experimental strategy during data collection, and secondly to help users make informed choices during data processing.
PMCID: PMC4321488  PMID: 25664746
Data Exploration Toolkit; ultrafast diffraction; X-ray free-electron lasers
7.  Indexing amyloid peptide diffraction from serial femtosecond crystallography: new algorithms for sparse patterns 
Special methods are required to interpret sparse diffraction patterns collected from peptide crystals at X-ray free-electron lasers. Bragg spots can be indexed from composite-image powder rings, with crystal orientations then deduced from a very limited number of spot positions.
Still diffraction patterns from peptide nanocrystals with small unit cells are challenging to index using conventional methods owing to the limited number of spots and the lack of crystal orientation information for individual images. New indexing algorithms have been developed as part of the Computational Crystallography Toolbox (cctbx) to overcome these challenges. Accurate unit-cell information derived from an aggregate data set from thousands of diffraction patterns can be used to determine a crystal orientation matrix for individual images with as few as five reflections. These algorithms are potentially applicable not only to amyloid peptides but also to any set of diffraction patterns with sparse properties, such as low-resolution virus structures or high-throughput screening of still images captured by raster-scanning at synchrotron sources. As a proof of concept for this technique, successful integration of X-ray free-electron laser (XFEL) data to 2.5 Å resolution for the amyloid segment GNNQQNY from the Sup35 yeast prion is presented.
PMCID: PMC4321489  PMID: 25664747
XFEL; Sup35 yeast prion; indexing methods; crystallography
8.  Structural analysis of DNA binding by C.Csp231I, a member of a novel class of R-M controller proteins regulating gene expression 
The structure of the new class of controller proteins (exemplified by C.Csp231I) in complex with its 21 bp DNA-recognition sequence is presented, and the molecular basis of sequence recognition in this class of proteins is discussed. An unusual extended spacer between the dimer binding sites suggests a novel interaction between the two C-protein dimers.
In a wide variety of bacterial restriction–modification systems, a regulatory ‘controller’ protein (or C-protein) is required for effective transcription of its own gene and for transcription of the endonuclease gene found on the same operon. We have recently turned our attention to a new class of controller proteins (exemplified by C.Csp231I) that have quite novel features, including a much larger DNA-binding site with an 18 bp (∼60 Å) spacer between the two palindromic DNA-binding sequences and a very different recognition sequence from the canonical GACT/AGTC. Using X-ray crystallography, the structure of the protein in complex with its 21 bp DNA-recognition sequence was solved to 1.8 Å resolution, and the molecular basis of sequence recognition in this class of proteins was elucidated. An unusual aspect of the promoter sequence is the extended spacer between the dimer binding sites, suggesting a novel interaction between the two C-protein dimers when bound to both recognition sites correctly spaced on the DNA. A U-bend model is proposed for this tetrameric complex, based on the results of gel-mobility assays, hydrodynamic analysis and the observation of key contacts at the interface between dimers in the crystal.
PMCID: PMC4321490  PMID: 25664751
DNA–protein interactions; helix–turn–helix; restriction–modification; controller protein; gene regulation; EMSA
9.  Structure of the GH76 α-mannanase homolog, BT2949, from the gut symbiont Bacteroides thetaiotaomicron  
A high-resolution structure of a noncanonical α-mannanase relevant to human health and nutrition has been solved via heavy-atom phasing of a selenomethionine derivative.
The large bowel microbiota, a complex ecosystem resident within the gastrointestinal tract of all human beings and large mammals, functions as an essential, nonsomatic metabolic organ, hydrolysing complex dietary polysaccharides and modulating the host immune system to adequately tolerate ingested antigens. A significant member of this community, Bacteroides thetaiotaomicron, has evolved a complex system for sensing and processing a wide variety of natural glycoproducts in such a way as to provide maximum benefit to itself, the wider microbial community and the host. The immense ability of B. thetaiotaomicron as a ‘glycan specialist’ resides in its enormous array of carbohydrate-active enzymes, many of which are arranged into polysaccharide-utilization loci (PULs) that are able to degrade sugar polymers that are often inaccessible to other gut residents, notably α-mannan. The B. thetaiotaomicron genome encodes ten putative α-mannanases spread across various PULs; however, little is known about the activity of these enzymes or the wider implications of α-mannan metabolism for the health of both the microbiota and the host. In this study, SAD phasing of a selenomethionine derivative has been used to investigate the structure of one such B. thetaiotaomicron enzyme, BT2949, which belongs to the GH76 family of α-mannanases. BT2949 presents a classical (α/α)6-barrel structure comprising a large extended surface cleft common to other GH76 family members. Analysis of the structure in conjunction with sequence alignments reveals the likely location of the catalytic active site of this noncanonical GH76.
PMCID: PMC4321491  PMID: 25664752
GH76; BT2949; Bacteroides thetaiotaomicron
10.  The rotational order–disorder structure of the reversibly photoswitchable red fluorescent protein rsTagRFP 
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.
PMCID: PMC3919260  PMID: 24419376
OD structure; rotational order–disorder; fluorescent protein
11.  Lysine carboxylation: unveiling a spontaneous post-translational modification 
A computational method for the prediction of lysine carboxylation (KCX) in protein structures is described. The method accurately identifies misreported KCXs and predicts previously unknown KCX sites.
The carboxylation of lysine residues is a post-translational modification (PTM) that plays a critical role in the catalytic mechanisms of several important enzymes. It occurs spontaneously under certain physicochemical conditions, but is difficult to detect experimentally. Its full impact is unknown. In this work, the signature microenvironment of lysine-carboxylation sites has been characterized. In addition, a computational method called Predictor of Lysine Carboxyl­ation (PreLysCar) for the detection of lysine carboxylation in proteins with available three-dimensional structures has been developed. The likely prevalence of lysine carboxylation in the proteome was assessed through large-scale computations. The results suggest that about 1.3% of large proteins may contain a carboxylated lysine residue. This unexpected prevalence of lysine carboxylation implies an enrichment of reactions in which it may play functional roles. The results also suggest that by switching enzymes on and off under appropriate physicochemical conditions spontaneous PTMs may serve as an important and widely used efficient biological machinery for regulation.
PMCID: PMC3919261  PMID: 24419378
lysine carboxylation; spontaneous post-translational modifications; structural motif; metal-ion centers; PreLysCar; Predictor of Lysine Carboxylation
12.  Structural characterization of new deoxycytidine kinase inhibitors rationalizes the affinity-determining moieties of the molecules 
This article describes the structural and biochemical characterization of a new class of high-affinity and selective human deoxycytidine kinase inhibitors.
Deoxycytidine kinase (dCK) is a key enzyme in the nucleoside salvage pathway that is also required for the activation of several anticancer and antiviral nucleoside analog prodrugs. Additionally, dCK has been implicated in immune disorders and has been found to be overexpressed in several cancers. To allow the probing and modulation of dCK activity, a new class of small-molecule inhibitors of the enzyme were developed. Here, the structural characterization of four of these inhibitors in complex with human dCK is presented. The structures reveal that the compounds occupy the nucleoside-binding site and bind to the open form of dCK. Surprisingly, a slight variation in the nature of the substituent at the 5-position of the thiazole ring governs whether the active site of the enzyme is occupied by one or two inhibitor molecules. Moreover, this substituent plays a critical role in determining the affinity, improving it from >700 to 1.5 nM in the best binder. These structures lay the groundwork for future modifications that would result in even tighter binding and the correct placement of moieties that confer favorable pharmacodynamics and pharmacokinetic properties.
PMCID: PMC3919262  PMID: 24419380
deoxycytidine kinase; inhibitors; nucleotide salvage pathway
13.  A challenging interpretation of a hexagonally layered protein structure 
The authors describe the structure determination of a hexagonally layered protein structure that suffered from a complicated combination of translational non-crystallographic symmetry and hemihedral twinning. This case serves as a reminder that broken crystallographic symmetry resulting from doubling of a unit-cell axis often requires a new choice of origin.
The carboxysome is a giant protein complex that acts as a metabolic organelle in cyanobacteria and some chemoautotrophs. Its outer structure is formed by the assembly of thousands of copies of hexameric shell protein subunits into a molecular layer. The structure determination of a CcmK1 shell protein mutant (L11K) from the β-carboxysome of the cyanobacterium Synechocystis PCC6803 led to challenges in structure determination. Twinning, noncrystallographic symmetry and packing of hexameric units in a special arrangement led to initial difficulties in space-group assignment. The correct space group was clarified after initial model refinement revealed additional symmetry. This study provides an instructive example in which broken symmetry requires a new choice of unit-cell origin in order to identify the highest symmetry space group. An additional observation related to the packing arrangement of molecules in this crystal suggests that these hexameric shell proteins might have lower internal symmetry than previously believed.
PMCID: PMC4030663  PMID: 24419393
CcmK1 shell protein; carboxysome; hexameric shell proteins
14.  Two way street – complementary methods  
An introduction to the 2014 CCP4 Study Weekend.
PMCID: PMC4304680  PMID: 25615854
complementary methods; CCP4 Study Weekend
15.  Combining biophysical methods for the analysis of protein complex stoichiometry and affinity in SEDPHAT  
Global multi-method analysis for protein interactions (GMMA) can increase the precision and complexity of binding studies for the determination of the stoichiometry, affinity and cooperativity of multi-site interactions. The principles and recent developments of biophysical solution methods implemented for GMMA in the software SEDPHAT are reviewed, their complementarity in GMMA is described and a new GMMA simulation tool set in SEDPHAT is presented.
Reversible macromolecular interactions are ubiquitous in signal transduction pathways, often forming dynamic multi-protein complexes with three or more components. Multivalent binding and cooperativity in these complexes are often key motifs of their biological mechanisms. Traditional solution biophysical techniques for characterizing the binding and cooperativity are very limited in the number of states that can be resolved. A global multi-method analysis (GMMA) approach has recently been introduced that can leverage the strengths and the different observables of different techniques to improve the accuracy of the resulting binding parameters and to facilitate the study of multi-component systems and multi-site interactions. Here, GMMA is described in the software SEDPHAT for the analysis of data from isothermal titration calorimetry, surface plasmon resonance or other biosensing, analytical ultracentrifugation, fluorescence anisotropy and various other spectroscopic and thermodynamic techniques. The basic principles of these techniques are reviewed and recent advances in view of their particular strengths in the context of GMMA are described. Furthermore, a new feature in SEDPHAT is introduced for the simulation of multi-method data. In combination with specific statistical tools for GMMA in SEDPHAT, simulations can be a valuable step in the experimental design.
PMCID: PMC4304681  PMID: 25615855
16.  In crystallo optical spectroscopy (icOS) as a complementary tool on the macromolecular crystallography beamlines of the ESRF 
The current version of the Cryobench in crystallo optical spectroscopy facility of the ESRF is presented. The diverse experiments that can be performed at the Cryobench are also reviewed.
The analysis of structural data obtained by X-ray crystallo­graphy benefits from information obtained from complementary techniques, especially as applied to the crystals themselves. As a consequence, optical spectroscopies in structural biology have become instrumental in assessing the relevance and context of many crystallographic results. Since the year 2000, it has been possible to record such data adjacent to, or directly on, the Structural Biology Group beamlines of the ESRF. A core laboratory featuring various spectrometers, named the Cryobench, is now in its third version and houses portable devices that can be directly mounted on beamlines. This paper reports the current status of the Cryobench, which is now located on the MAD beamline ID29 and is thus called the ID29S-Cryobench (where S stands for ‘spectroscopy’). It also reviews the diverse experiments that can be performed at the Cryobench, highlighting the various scientific questions that can be addressed.
PMCID: PMC4304682  PMID: 25615856
in crystallo optical spectroscopy; Cryobench; UV-visible light absorption spectroscopy; fluorescence spectroscopy; Raman spectroscopy
17.  Challenges and solutions for the analysis of in situ, in crystallo micro-spectrophotometric data 
The particular challenge of the analysis of optical absorption and Raman spectroscopic data measured from protein crystals and how the SLS-APE software toolbox supports scientists in dealing with such data is described.
Combining macromolecular crystallography with in crystallo micro-spectrophotometry yields valuable complementary information on the sample, including the redox states of metal cofactors, the identification of bound ligands and the onset and strength of undesired photochemistry, also known as radiation damage. However, the analysis and processing of the resulting data differs significantly from the approaches used for solution spectrophotometric data. The varying size and shape of the sample, together with the suboptimal sample environment, the lack of proper reference signals and the general influence of the X-ray beam on the sample have to be considered and carefully corrected for. In the present article, how to characterize and treat these sample-dependent artefacts in a reproducible manner is discussed and the SLS-APE in situ, in crystallo optical spectroscopy data-analysis toolbox is demonstrated.
PMCID: PMC4304683  PMID: 25615857
micro-spectrophotometer; UV–visible absorption; Raman spectroscopy; single-crystal spectroscopy; software toolbox; data analysis; background correction
18.  Protein–ligand interactions investigated by thermal shift assays (TSA) and dual polarization interferometry (DPI) 
The biophysical characterization of protein–ligand interactions in solution using techniques such as thermal shift assay, or on surfaces using, for example, dual polarization interferometry, plays an increasingly important role in complementing crystal structure determinations.
Over the last decades, a wide range of biophysical techniques investigating protein–ligand interactions have become indispensable tools to complement high-resolution crystal structure determinations. Current approaches in solution range from high-throughput-capable methods such as thermal shift assays (TSA) to highly accurate techniques including microscale thermophoresis (MST) and isothermal titration calorimetry (ITC) that can provide a full thermodynamic description of binding events. Surface-based methods such as surface plasmon resonance (SPR) and dual polarization interferometry (DPI) allow real-time measurements and can provide kinetic parameters as well as binding constants. DPI provides additional spatial information about the binding event. Here, an account is presented of new developments and recent applications of TSA and DPI connected to crystallography.
PMCID: PMC4304684  PMID: 25615858
thermal shift assays; dual polarization interferometry; protein–ligand interactions
19.  The accurate assessment of small-angle X-ray scattering data 
A set of quantitative techniques is suggested for assessing SAXS data quality. These are applied in the form of a script, SAXStats, to a test set of 27 proteins, showing that these techniques are more sensitive than manual assessment of data quality.
Small-angle X-ray scattering (SAXS) has grown in popularity in recent times with the advent of bright synchrotron X-ray sources, powerful computational resources and algorithms enabling the calculation of increasingly complex models. However, the lack of standardized data-quality metrics presents difficulties for the growing user community in accurately assessing the quality of experimental SAXS data. Here, a series of metrics to quantitatively describe SAXS data in an objective manner using statistical evaluations are defined. These metrics are applied to identify the effects of radiation damage, concentration dependence and interparticle interactions on SAXS data from a set of 27 previously described targets for which high-resolution structures have been determined via X-ray crystallography or nuclear magnetic resonance (NMR) spectroscopy. The studies show that these metrics are sufficient to characterize SAXS data quality on a small sample set with statistical rigor and sensitivity similar to or better than manual analysis. The development of data-quality analysis strategies such as these initial efforts is needed to enable the accurate and unbiased assessment of SAXS data quality.
PMCID: PMC4304685  PMID: 25615859
SAXS data quality; SAXStats
20.  Uniqueness of models from small-angle scattering data: the impact of a hydration shell and complementary NMR restraints 
The potentialities and limitations of biomacromolecular modelling of small-angle scattering data are reviewed and discussed with a focus on the impact of complementary NMR restraints and a hydration shell.
Small-angle scattering (SAS) has witnessed a breathtaking renaissance and expansion over the past 15 years regarding the determination of biomacromolecular structures in solution. While important issues such as sample quality, good experimental practice and guidelines for data analysis, interpretation, presentation, publication and deposition are increasingly being recognized, crucial topics such as the uniqueness, precision and accuracy of the structural models obtained by SAS are still only poorly understood and addressed. The present article provides an overview of recent developments in these fields with a focus on the influence of complementary NMR restraints and of a hydration shell on the uniqueness of biomacromolecular models. As a first topic, the impact of incorporating NMR orientational restraints in addition to SAS distance restraints is discussed using a quantitative visual representation that illustrates how the possible conformational space of a two-body system is reduced as a function of the available data. As a second topic, the impact of a hydration shell on modelling parameters of a two-body system is illustrated, in particular on its inter-body distance. Finally, practical recommendations are provided to take both effects into account and promising future perspectives of SAS approaches are discussed.
PMCID: PMC4304686  PMID: 25615860
small-angle scattering; NMR restraints; hydration shell
21.  BioSAXS Sample Changer: a robotic sample changer for rapid and reliable high-throughput X-ray solution scattering experiments 
A robotic sample changer for solution X-ray scattering experiments optimized for speed and to use the minimum amount of material has been developed. This system is now in routine use at three high-brilliance European synchrotron sites, each capable of several hundred measurements per day.
Small-angle X-ray scattering (SAXS) of macromolecules in solution is in increasing demand by an ever more diverse research community, both academic and industrial. To better serve user needs, and to allow automated and high-throughput operation, a sample changer (BioSAXS Sample Changer) that is able to perform unattended measurements of up to several hundred samples per day has been developed. The Sample Changer is able to handle and expose sample volumes of down to 5 µl with a measurement/cleaning cycle of under 1 min. The samples are stored in standard 96-well plates and the data are collected in a vacuum-mounted capillary with automated positioning of the solution in the X-ray beam. Fast and efficient capillary cleaning avoids cross-contamination and ensures reproducibility of the measurements. Independent temperature control for the well storage and for the measurement capillary allows the samples to be kept cool while still collecting data at physiological temperatures. The Sample Changer has been installed at three major third-generation synchrotrons: on the BM29 beamline at the European Synchrotron Radiation Facility (ESRF), the P12 beamline at the PETRA-III synchrotron (EMBL@PETRA-III) and the I22/B21 beamlines at Diamond Light Source, with the latter being the first commercial unit supplied by Bruker ASC.
PMCID: PMC4304687  PMID: 25615861
small-angle X-ray scattering; BioSAXS Sample Changer; high-throughput; automation
22.  ISPyB for BioSAXS, the gateway to user autonomy in solution scattering experiments 
The ISPyB information-management system for crystallography has been adapted to include data from small-angle X-ray scattering of macromolecules in solution experiments.
Logging experiments with the laboratory-information management system ISPyB (Information System for Protein crystallography Beamlines) enhances the automation of small-angle X-ray scattering of biological macromolecules in solution (BioSAXS) experiments. The ISPyB interface provides immediate user-oriented online feedback and enables data cross-checking and downstream analysis. To optimize data quality and completeness, ISPyBB (ISPyB for BioSAXS) makes it simple for users to compare the results from new measurements with previous acquisitions from the same day or earlier experiments in order to maximize the ability to collect all data required in a single synchrotron visit. The graphical user interface (GUI) of ISPyBB has been designed to guide users in the preparation of an experiment. The input of sample information and the ability to outline the experimental aims in advance provides feedback on the number of measurements required, calculation of expected sample volumes and time needed to collect the data: all of this information aids the users to better prepare for their trip to the synchrotron. A prototype version of the ISPyBB database is now available at the European Synchrotron Radiation Facility (ESRF) beamline BM29 and is already greatly appreciated by academic users and industrial clients. It will soon be available at the PETRA III beamline P12 and the Diamond Light Source beamlines I22 and B21.
PMCID: PMC4304688  PMID: 25615862
small-angle X-ray scattering; proteins in solution; automation; laboratory information-management system
23.  Memprot: a program to model the detergent corona around a membrane protein based on SEC–SAXS data 
Systematic SAXS simulations have been analysed over a wide range of parameters in order to better understand the detergent corona around a membrane protein.
The application of small-angle X-ray scattering (SAXS) to structural investigations of transmembrane proteins in detergent solution has been hampered by two main inherent hurdles. On the one hand, the formation of a detergent corona around the hydrophobic region of the protein strongly modifies the scattering curve of the protein. On the other hand, free micelles of detergent without a precisely known concentration coexist with the protein–detergent complex in solution, therefore adding an uncontrolled signal. To gain robust structural information on such systems from SAXS data, in previous work, advantage was taken of the online combination of size-exclusion chromatography (SEC) and SAXS, and the detergent corona around aquaporin-0, a membrane protein of known structure, could be modelled. A precise geometrical model of the corona, shaped as an elliptical torus, was determined. Here, in order to better understand the correlations between the corona model parameters and to discuss the uniqueness of the model, this work was revisited by analyzing systematic SAXS simulations over a wide range of parameters of the torus.
PMCID: PMC4304689  PMID: 25615863
small-angle X-ray scattering; membrane proteins; SEC–SAXS; Memprot
24.  Acoustic transfer of protein crystals from agarose pedestals to micromeshes for high-throughput screening 
An acoustic high-throughput screening method is described for harvesting protein crystals and combining the protein crystals with chemicals such as a fragment library.
Acoustic droplet ejection (ADE) is an emerging technology with broad applications in serial crystallography such as growing, improving and manipulating protein crystals. One application of this technology is to gently transfer crystals onto MiTeGen micromeshes with minimal solvent. Once mounted on a micromesh, each crystal can be combined with different chemicals such as crystal-improving additives or a fragment library. Acoustic crystal mounting is fast (2.33 transfers s−1) and all transfers occur in a sealed environment that is in vapor equilibrium with the mother liquor. Here, a system is presented to retain crystals near the ejection point and away from the inaccessible dead volume at the bottom of the well by placing the crystals on a concave agarose pedestal (CAP) with the same chemical composition as the crystal mother liquor. The bowl-shaped CAP is impenetrable to crystals. Consequently, gravity will gently move the crystals into the optimal location for acoustic ejection. It is demonstrated that an agarose pedestal of this type is compatible with most commercially available crystallization conditions and that protein crystals are readily transferred from the agarose pedestal onto micromeshes with no loss in diffraction quality. It is also shown that crystals can be grown directly on CAPs, which avoids the need to transfer the crystals from the hanging drop to a CAP. This technology has been used to combine thermolysin and lysozyme crystals with an assortment of anomalously scattering heavy atoms. The results point towards a fast nanolitre method for crystal mounting and high-throughput screening.
PMCID: PMC4304690  PMID: 25615864
macromolecular crystallography; acoustic droplet ejection; crystal mounting; drug discovery; chemical biology; high-throughput screening
25.  Experimental phasing for structure determination using membrane-protein crystals grown by the lipid cubic phase method 
Very little information is available in the literature concerning the experimental heavy-atom phasing of membrane-protein structures where the crystals have been grown using the lipid cubic phase (in meso) method. In this paper, pre-labelling, co-crystallization, soaking, site-specific mercury binding to genetically engineered single-cysteine mutants and selenomethionine labelling as applied to an integral membrane kinase crystallized in meso are described. An assay to assess cysteine accessibility for mercury labelling of membrane proteins is introduced.
Despite the marked increase in the number of membrane-protein structures solved using crystals grown by the lipid cubic phase or in meso method, only ten have been determined by SAD/MAD. This is likely to be a consequence of the technical difficulties associated with handling proteins and crystals in the sticky and viscous hosting mesophase that is usually incubated in glass sandwich plates for the purposes of crystallization. Here, a four-year campaign aimed at phasing the in meso structure of the integral membrane diacylglycerol kinase (DgkA) from Escherichia coli is reported. Heavy-atom labelling of this small hydrophobic enzyme was attempted by pre-labelling, co-crystallization, soaking, site-specific mercury binding to genetically engineered single-cysteine mutants and selenomethionine incorporation. Strategies and techniques for special handling are reported, as well as the typical results and the lessons learned for each of these approaches. In addition, an assay to assess the accessibility of cysteine residues in membrane proteins for mercury labelling is introduced. The various techniques and strategies described will provide a valuable reference for future experimental phasing of membrane proteins where crystals are grown by the lipid cubic phase method.
PMCID: PMC4304691  PMID: 25615865
co-crystallization; cysteine mutagenesis; heavy atoms; in meso; LCP; lipid mesophase; phase determination; pre-labelling; selenomethionine; soaking

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