The CCP4 suite: integrative software for macromolecular crystallography.

The Collaborative Computational Project No. 4 (CCP4) is a UK-led international collective with a mission to develop, test, distribute and promote software for macromolecular crystallography. The CCP4 suite is a multiplatform collection of programs brought together by familiar execution routines, a set of common libraries and graphical interfaces. The CCP4 suite has experienced several considerable changes since its last reference article, involving new infrastructure, original programs and graphical interfaces. This article, which is intended as a general literature citation for the use of the CCP4 software suite in structure determination, will guide the reader through such transformations, offering a general overview of the new features and outlining future developments. As such, it aims to highlight the individual programs that comprise the suite and to provide the latest references to them for perusal by crystallographers around the world.

Crystal structures of the molecular class A ß-lactamase TEM-171 and its complexes with tazobactam.

The resistance of bacteria to ß-lactam antibiotics is primarily caused by the production of ß-lactamases. Here, novel crystal structures of the native ß-lactamase TEM-171 and two complexes with the widely used inhibitor tazobactam are presented, alongside complementary data from UV spectroscopy and fluorescence quenching. The six chemically identical ß-lactamase molecules in the crystallographic asymmetric unit displayed different degrees of disorder. The tazobactam intermediate was covalently bound to the catalytic Ser70 in the trans-enamine configuration. While the conformation of tazobactam in the first complex resembled that in published ß-lactamase-tazobactam structures, in the second complex, which was obtained after longer soaking of the native crystals in the inhibitor solution, a new and previously unreported tazobactam conformation was observed. It is proposed that the two complexes correspond to different stages along the deacylation path of the acyl-enzyme intermediate. The results provide a novel structural basis for the rational design of new ß-lactamase inhibitors.

The XBI BioLab for life science experiments at the European XFEL.

The science of X-ray free-electron lasers (XFELs) critically depends on the performance of the X-ray laser and on the quality of the samples placed into the X-ray beam. The stability of biological samples is limited and key biomolecular transformations occur on short timescales. Experiments in biology require a support laboratory in the immediate vicinity of the beamlines. The XBI BioLab of the European XFEL (XBI denotes XFEL Biology Infrastructure) is an integrated user facility connected to the beamlines for supporting a wide range of biological experiments. The laboratory was financed and built by a collaboration between the European XFEL and the XBI User Consortium, whose members come from Finland, Germany, the Slovak Republic, Sweden and the USA, with observers from Denmark and the Russian Federation. Arranged around a central wet laboratory, the XBI BioLab provides facilities for sample preparation and scoring, laboratories for growing prokaryotic and eukaryotic cells, a Bio Safety Level 2 laboratory, sample purification and characterization facilities, a crystallization laboratory, an anaerobic laboratory, an aerosol laboratory, a vacuum laboratory for injector tests, and laboratories for optical microscopy, atomic force microscopy and electron microscopy. Here, an overview of the XBI facility is given and some of the results of the first user experiments are highlighted.

The accuracy of protein models automatically built into cryo-EM maps with ARP/wARP.

Recent developments in cryogenic electron microscopy (cryo-EM) have enabled structural studies of large macromolecular complexes at resolutions previously only attainable using macromolecular crystallography. Although a number of methods can already assist in de novo building of models into high-resolution cryo-EM maps, automated and reliable map interpretation remains a challenge. Presented here is a systematic study of the accuracy of models built into cryo-EM maps using ARP/wARP. It is demonstrated that the local resolution is a good indicator of map interpretability, and for the majority of the test cases ARP/wARP correctly builds 90% of main-chain fragments in regions where the local resolution is 4.0 Šor better. It is also demonstrated that the coordinate accuracy for models built into cryo-EM maps is comparable to that of X-ray crystallographic models at similar local cryo-EM and crystallographic resolutions. The model accuracy also correlates with the refined atomic displacement parameters.

The use of local structural similarity of distant homologues for crystallographic model building from a molecular-replacement solution.

The performance of automated protein model building usually decreases with resolution, mainly owing to the lower information content of the experimental data. This calls for a more elaborate use of the available structural information about macromolecules. Here, a new method is presented that uses structural homologues to improve the quality of protein models automatically constructed using ARP/wARP. The method uses local structural similarity between deposited models and the model being built, and results in longer main-chain fragments that in turn can be more reliably docked to the protein sequence. The application of the homology-based model extension method to the example of a CFA synthase at 2.7 Šresolution resulted in a more complete model with almost all of the residues correctly built and docked to the sequence. The method was also evaluated on 1493 molecular-replacement solutions at a resolution of 4.0 Šand better that were submitted to the ARP/wARP web service for model building. A significant improvement in the completeness and sequence coverage of the built models has been observed.

Sequence assignment for low-resolution modelling of protein crystal structures.

The performance of automated model building in crystal structure determination usually decreases with the resolution of the experimental data, and may result in fragmented models and incorrect side-chain assignment. Presented here are new methods for machine-learning-based docking of main-chain fragments to the sequence and for their sequence-independent connection using a dedicated library of protein fragments. The combined use of these new methods noticeably increases sequence coverage and reduces fragmentation of the protein models automatically built with ARP/wARP.

West-Life: A Virtual Research Environment for structural biology.

The West-Life project (https://about.west-life.eu/) is a Horizon 2020 project funded by the European Commission to provide data processing and data management services for the international community of structural biologists, and in particular to support integrative experimental approaches within the field of structural biology. It has developed enhancements to existing web services for structure solution and analysis, created new pipelines to link these services into more complex higher-level workflows, and added new data management facilities. Through this work it has striven to make the benefits of European e-Infrastructures more accessible to life-science researchers in general and structural biologists in particular.

Structural and functional insights into the unique CBS-CP12 fusion protein family in cyanobacteria.

Cyanobacteria are important photosynthetic organisms inhabiting a range of dynamic environments. This phylum is distinctive among photosynthetic organisms in containing genes encoding uncharacterized cystathionine ß-synthase (CBS)-chloroplast protein (CP12) fusion proteins. These consist of two domains, each recognized as stand-alone photosynthetic regulators with different functions described in cyanobacteria (CP12) and plants (CP12 and CBSX). Here we show that CBS-CP12 fusion proteins are encoded in distinct gene neighborhoods, several unrelated to photosynthesis. Most frequently, CBS-CP12 genes are in a gene cluster with thioredoxin A (TrxA), which is prevalent in bloom-forming, marine symbiotic, and benthic mat cyanobacteria. Focusing on a CBS-CP12 from Microcystis aeruginosa PCC 7806 encoded in a gene cluster with TrxA, we reveal that the domain fusion led to the formation of a hexameric protein. We show that the CP12 domain is essential for hexamerization and contains an ordered, previously structurally uncharacterized N-terminal region. We provide evidence that CBS-CP12, while combining properties of both regulatory domains, behaves different from CP12 and plant CBSX. It does not form a ternary complex with phosphoribulokinase (PRK) and glyceraldehyde-3-phosphate dehydrogenase. Instead, CBS-CP12 decreases the activity of PRK in an AMP-dependent manner. We propose that the novel domain architecture and oligomeric state of CBS-CP12 expand its regulatory function beyond those of CP12 in cyanobacteria.

Structure and function of the N-terminal domain of the yeast telomerase reverse transcriptase.

The elongation of single-stranded DNA repeats at the 3'-ends of chromosomes by telomerase is a key process in maintaining genome integrity in eukaryotes. Abnormal activation of telomerase leads to uncontrolled cell division, whereas its down-regulation is attributed to ageing and several pathologies related to early cell death. Telomerase function is based on the dynamic interactions of its catalytic subunit (TERT) with nucleic acids-telomerase RNA, telomeric DNA and the DNA/RNA heteroduplex. Here, we present the crystallographic and NMR structures of the N-terminal (TEN) domain of TERT from the thermotolerant yeast Hansenula polymorpha and demonstrate the structural conservation of the core motif in evolutionarily divergent organisms. We identify the TEN residues that are involved in interactions with the telomerase RNA and in the recognition of the 'fork' at the distal end of the DNA product/RNA template heteroduplex. We propose that the TEN domain assists telomerase biological function and is involved in restricting the size of the heteroduplex during telomere repeat synthesis.

A distance geometry-based description and validation of protein main-chain conformation.

Understanding the protein main-chain conformational space forms the basis for the modelling of protein structures and for the validation of models derived from structural biology techniques. Presented here is a novel idea for a three-dimensional distance geometry-based metric to account for the fine details of protein backbone conformations. The metrics are computed for dipeptide units, defined as blocks of Cαi-1-O i-1-Cαi -O i -Cαi+1 atoms, by obtaining the eigenvalues of their Euclidean distance matrices. These were computed for ∼1.3 million dipeptide units collected from nonredundant good-quality structures in the Protein Data Bank and subjected to principal component analysis. The resulting new Euclidean orthogonal three-dimensional space (DipSpace) allows a probabilistic description of protein backbone geometry. The three axes of the DipSpace describe the local extension of the dipeptide unit structure, its twist and its bend. By using a higher-dimensional metric, the method is efficient for the identification of Cα atoms in an unlikely or unusual geometrical environment, and its use for both local and overall validation of protein models is demonstrated. It is also shown, for the example of trypsin proteases, that the detection of unusual conformations that are conserved among the structures of this protein family may indicate geometrically strained residues of potentially functional importance.

Estimation of the protein-ligand interaction energy for model building and validation.

Macromolecular X-ray crystallography is one of the main experimental techniques to visualize protein-ligand interactions. The high complexity of the ligand universe, however, has delayed the development of efficient methods for the automated identification, fitting and validation of ligands in their electron-density clusters. The identification and fitting are primarily based on the density itself and do not take into account the protein environment, which is a step that is only taken during the validation of the proposed binding mode. Here, a new approach, based on the estimation of the major energetic terms of protein-ligand interaction, is introduced for the automated identification of crystallographic ligands in the indicated binding site with ARP/wARP. The applicability of the method to the validation of protein-ligand models from the Protein Data Bank is demonstrated by the detection of models that are `questionable' and the pinpointing of unfavourable interatomic contacts.

Synthesis, SAR and molecular docking study of novel non-ß-lactam inhibitors of TEM type ß-lactamase.

The novel classes of acylated phenoxyanilide and thiourea compounds were investigated for their ability to inhibit TEM type ß-lactamase enzyme. Two compounds 4g and 5c reveal the inhibition potency in micromolar range and show their action by non-covalent binding in the vicinity of the TEM-171 active site. The structure activity relationship around carbon chain length and different substituents in ortho- and para-positions of acylated phenoxyanilide as well as molecular modelling study has been performed.

Visual automated macromolecular model building.

Automated model-building software aims at the objective interpretation of crystallographic diffraction data by means of the construction or completion of macromolecular models. Automated methods have rapidly gained in popularity as they are easy to use and generate reproducible and consistent results. However, the process of model building has become increasingly hidden and the user is often left to decide on how to proceed further with little feedback on what has preceded the output of the built model. Here, ArpNavigator, a molecular viewer tightly integrated into the ARP/wARP automated model-building package, is presented that directly controls model building and displays the evolving output in real time in order to make the procedure transparent to the user.

Use of noncrystallographic symmetry for automated model building at medium to low resolution.

A novel method is presented for the automatic detection of noncrystallographic symmetry (NCS) in macromolecular crystal structure determination which does not require the derivation of molecular masks or the segmentation of density. It was found that throughout structure determination the NCS-related parts may be differently pronounced in the electron density. This often results in the modelling of molecular fragments of variable length and accuracy, especially during automated model-building procedures. These fragments were used to identify NCS relations in order to aid automated model building and refinement. In a number of test cases higher completeness and greater accuracy of the obtained structures were achieved, specifically at a crystallographic resolution of 2.3 Šor poorer. In the best case, the method allowed the building of up to 15% more residues automatically and a tripling of the average length of the built fragments.

Fragmentation-tree density representation for crystallographic modelling of bound ligands.

The identification and modelling of ligands into macromolecular models is important for understanding molecule's function and for designing inhibitors to modulate its activities. We describe new algorithms for the automated building of ligands into electron density maps in crystal structure determination. Location of the ligand-binding site is achieved by matching numerical shape features describing the ligand to those of density clusters using a "fragmentation-tree" density representation. The ligand molecule is built using two distinct algorithms exploiting free atoms with inter-atomic connectivity and Metropolis-based optimisation of the conformational state of the ligand, producing an ensemble of structures from which the final model is derived. The method was validated on several thousand entries from the Protein Data Bank. In the majority of cases, the ligand-binding site could be correctly located and the ligand model built with a coordinate accuracy of better than 1 Å. We anticipate that the method will be of routine use to anyone modelling ligands, lead compounds or even compound fragments as part of protein functional analyses or drug design efforts.

Aspartate aminotransferase: bridging carbohydrate and energy metabolism in Plasmodium falciparum.

In this mini-review we briefly examine and summarize evidence on the role of the plasmodial aspartate aminotransferase (AspAT) of the malarial parasite. Recent data have provided information on the products of the purine salvage pathway as well as the glycolytic and oxidative phosphorylation pathways, suggesting that the reaction catalyzed by AspAT is an essential step in all these biochemical processes. While the biological role of the oxidative phosphorylation cycle still remains to be demonstrated, the presence of a single protein that is functional in multiple pathways (i.e. amino acid/purine/pyrimidine biosynthesis and carbohydrate metabolism) provides a high potential for the development of novel strategies to combat the spread of multi-drug resistant malaria.

Crystal structure of D-serine dehydratase from Escherichia coli.

D-Serine dehydratase from Escherichia coli is a member of the ß-family (fold-type II) of the pyridoxal 5'-phosphate-dependent enzymes, catalyzing the conversion of D-serine to pyruvate and ammonia. The crystal structure of monomeric D-serine dehydratase has been solved to 1.97Å-resolution for an orthorhombic data set by molecular replacement. In addition, the structure was refined in a monoclinic data set to 1.55Å resolution. The structure of DSD reveals a larger pyridoxal 5'-phosphate-binding domain and a smaller domain. The active site of DSD is very similar to those of the other members of the ß-family. Lys118 forms the Schiff base to PLP, the cofactor phosphate group is liganded to a tetraglycine cluster Gly279-Gly283, and the 3-hydroxyl group of PLP is liganded to Asn170 and N1 to Thr424, respectively. In the closed conformation the movement of the small domain blocks the entrance to active site of DSD. The domain movement plays an important role in the formation of the substrate recognition site and the catalysis of the enzyme. Modeling of D-serine into the active site of DSD suggests that the hydroxyl group of D-serine is coordinated to the carboxyl group of Asp238. The carboxyl oxygen of D-serine is coordinated to the hydroxyl group of Ser167 and the amide group of Leu171 (O1), whereas the O2 of the carboxyl group of D-serine is hydrogen-bonded to the hydroxyl group of Ser167 and the amide group of Thr168. A catalytic mechanism very similar to that proposed for L-serine dehydratase is discussed.

On the routine use of soft X-rays in macromolecular crystallography. Part V. Molecular replacement and anomalous scattering.

Currently, about two thirds of all new macromolecular structures are determined by molecular replacement. In general the method works reliably, but it reaches its limits when the search model differs too much from the target structure in terms of coordinate deviations or completeness. Since anomalously scattering substructures are better conserved than the overall structure, these substructures and the corresponding anomalous intensity differences can be utilized to enhance the performance of molecular-replacement approaches. It is demonstrated that the combined and concomitant use of structure-factor amplitudes and anomalous differences constitutes a promising approach to push the limits of molecular replacement and to make more structures amenable to structure solution by this technique.

Crystal structure of small protein crambin at 0.48†Šresolution.

With the development of highly brilliant and extremely intense synchrotron X-ray sources, extreme high-resolution limits for biological samples are now becoming attainable. Here, a study is presented that sets the record in crystallographic resolution for a biological macromolecule. The structure of the small protein crambin was determined to 0.48 Šresolution on the PETRA II ring before its conversion to a dedicated synchrotron-radiation source. The results reveal a wealth of details in electron density and demonstrate the possibilities that are potentially offered by a high-energy source. The question now arises as to what the true limits are in terms of what can be seen at such high resolution. From what can be extrapolated from the results using crystals of crambin, this limit would be at approximately 0.40 Å, which approaches that for smaller compounds.

A moment invariant for evaluating the chirality of three-dimensional objects.

Chirality is an important feature of three-dimensional objects and a key concept in chemistry, biology and many other disciplines. However, it has been difficult to quantify, largely owing to computational complications. Here we present a general chirality measure, called the chiral invariant (CI), which is applicable to any three-dimensional object containing a large amount of data. The CI distinguishes the hand of the object and quantifies the degree of its handedness. It is invariant to the translation, rotation and scale of the object, and tolerant to a modest amount of noise in the experimental data. The invariant is expressed in terms of moments and can be computed in almost no time. Because of its universality and computational efficiency, the CI is suitable for a wide range of pattern-recognition problems. We demonstrate its applicability to molecular atomic models and their electron density maps. We show that the occurrence of the conformations of the macromolecular polypeptide backbone is related to the value of the CI of the constituting peptide fragments. We also illustrate how the CI can be used to assess the quality of a crystallographic electron density map.