highly cited articles

PHENIX: a comprehensive Python-based system for macromolecular structure solution

P. D. Adams, P. V. Afonine, G. Bunkóczi, V. B. Chen, I. W. Davis, N. Echols, J. J. Headd, L.-W. Hung, G. J. Kapral, R. W. Grosse-Kunstleve, A. J. McCoy, N. W. Moriarty, R. Oeffner, R. J. Read, D. C. Richardson, J. S. Richardson, T. C. Terwilliger and P. H. Zwart

Macromolecular X-ray crystallography is routinely applied to understand biological processes at a molecular level. However, significant time and effort are still required to solve and complete many of these structures because of the need for manual interpretation of complex numerical data using many software packages and the repeated use of interactive three-dimensional graphics. PHENIX has been developed to provide a comprehensive system for macromolecular crystallographic structure solution with an emphasis on the automation of all procedures. This has relied on the development of algorithms that minimize or eliminate subjective input, the development of algorithms that automate procedures that are traditionally performed by hand and, finally, the development of a framework that allows a tight integration between the algorithms.

Structure validation in chemical crystallography

A. L. Spek

Automated structure validation was introduced in chemical crystallography about 12 years ago as a tool to assist practitioners with the exponential growth in crystal structure analyses. Validation has since evolved into an easy-to-use checkCIF/PLATON web-based IUCr service. The result of a crystal structure determination has to be supplied as a CIF-formatted computer-readable file. The checking software tests the data in the CIF for completeness, quality and consistency. In addition, the reported structure is checked for incomplete analysis, errors in the analysis and relevant issues to be verified. A validation report is generated in the form of a list of ALERTS on the issues to be corrected, checked or commented on. Structure validation has largely eliminated obvious problems with structure reports published in IUCr journals, such as refinement in a space group of too low symmetry. This paper reports on the current status of structure validation and possible future extensions.

XDS

W. Kabsch

The usage and control of recent modifications of the program package XDS for the processing of rotation images are described in the context of previous versions. New features include automatic determination of spot size and reflecting range and recognition and assignment of crystal symmetry. Moreover, the limitations of earlier package versions on the number of correction/scaling factors and the representation of pixel contents have been removed. Large program parts have been restructured for parallel processing so that the quality and completeness of collected data can be assessed soon after measurement.

Features and development of Coot

P. Emsley, B. Lohkamp, W. G. Scott and K. Cowtan

Coot is a molecular-graphics application for model building and validation of biological macromolecules. The program displays electron-density maps and atomic models and allows model manipulations such as idealization, real-space refinement, manual rotation/translation, rigid-body fitting, ligand search, solvation, mutations, rotamers and Ramachandran idealization. Furthermore, tools are provided for model validation as well as interfaces to external programs for refinement, validation and graphics. The software is designed to be easy to learn for novice users, which is achieved by ensuring that tools for common tasks are `discoverable' through familiar user-interface elements (menus and toolbars) or by intuitive behaviour (mouse controls). Recent developments have focused on providing tools for expert users, with customisable key bindings, extensions and an extensive scripting interface. The software is under rapid development, but has already achieved very widespread use within the crystallographic community. The current state of the software is presented, with a description of the facilities available and of some of the underlying methods employed.

MolProbity: all-atom structure validation for macromolecular crystallography

V. B. Chen, W. B. Arendall III, J. J. Headd, D. A. Keedy, R. M. Immormino, G. J. Kapral, L. W. Murray, J. S. Richardson and D. C. Richardson

MolProbity is a structure-validation web service that provides broad-spectrum solidly based evaluation of model quality at both the global and local levels for both proteins and nucleic acids. It relies heavily on the power and sensitivity provided by optimized hydrogen placement and all-atom contact analysis, complemented by updated versions of covalent-geometry and torsion-angle criteria. Some of the local corrections can be performed automatically in MolProbity and all of the diagnostics are presented in chart and graphical forms that help guide manual rebuilding. X-ray crystallography provides a wealth of biologically important molecular data in the form of atomic three-dimensional structures of proteins, nucleic acids and increasingly large complexes in multiple forms and states. Advances in automation, in everything from crystallization to data collection to phasing to model building to refinement, have made solving a structure using crystallography easier than ever. However, despite these improvements, local errors that can affect biological interpretation are widespread at low resolution and even high-resolution structures nearly all contain at least a few local errors such as Ramachandran outliers, flipped branched protein side chains and incorrect sugar puckers. It is critical both for the crystallographer and for the end user that there are easy and reliable methods to diagnose and correct these sorts of errors in structures. MolProbity is the authors' contribution to helping solve this problem and this article reviews its general capabilities, reports on recent enhancements and usage, and presents evidence that the resulting improvements are now beneficially affecting the global database.

Generalized X-ray and neutron crystallographic analysis: more accurate and complete structures for biological macromolecules

P. D. Adams, M. Mustyakimov, P. V. Afonine and P. Langan

X-ray and neutron crystallographic techniques provide complementary information on the structure and function of biological macromolecules. X-ray and neutron (XN) crystallographic data have been combined in a joint structure-refinement procedure that has been developed using recent advances in modern computational methodologies, including cross-validated maximum-likelihood target functions with gradient-based optimization and simulated annealing. The XN approach for complete (including hydrogen) macromolecular structure analysis provides more accurate and complete structures, as demonstrated for diisopropyl fluorophosphatase, photoactive yellow protein and human aldose reductase. Furthermore, this method has several practical advantages, including the easier determination of the orientation of water molecules, hydroxyl groups and some amino-acid side chains.

Molecular replacement with MOLREP

A. Vagin and A. Teplyakov

MOLREP is an automated program for molecular replacement that utilizes a number of original approaches to rotational and translational search and data preparation. Since the first publication describing the program, MOLREP has acquired a variety of features that include weighting of the X-ray data and search models, multi-copy search, fitting the model into electron density, structural superposition of two models and rigid-body refinement. The program can run in a fully automatic mode using optimized parameters calculated from the input data.

Integration, scaling, space-group assignment and post-refinement

W. Kabsch

Important steps in the processing of rotation data are described that are common to most software packages. These programs differ in the details and in the methods implemented to carry out the tasks. Here, the working principles underlying the data-reduction package XDS are explained, including the new features of automatic determination of spot size and reflecting range, recognition and assignment of crystal symmetry and a highly efficient algorithm for the determination of correction/scaling factors.

ALINE: a WYSIWYG protein-sequence alignment editor for publication-quality alignments

C. S. Bond and A. W. Schüttelkopf

Marked-up sequence alignments typically provide the central figure in articles describing proteins, whether in the fields of biochemistry, bioinformatics or structural biology. The generation of these figures is often unwieldy: interactive programs are often aesthetically limited and the use of batch programs requires the repetitive iterative editing of scripts. ALINE is a portable interactive graphical sequence-alignment editor implemented in Perl/Tk which produces publication-quality sequence-alignment figures where `what you see is what you get'. ALINE is freely available for download from http://crystal.bcs.uwa.edu.au/px/charlie/software/aline/.

An emerging consensus for the structure of EmrE

V. M. Korkhov and C. G. Tate

The archetypical member of the small multidrug-resistance family is EmrE, a multidrug transporter that extrudes toxic polyaromatic cations from the cell coupled to the inward movement of protons down a concentration gradient. The architecture of EmrE was first defined from the analysis of two-dimensional crystals by cryoelectron microscopy (cryo-EM), which showed that EmrE was an unusual asymmetric dimer formed from a bundle of eight -helices. The most favoured interpretation of the structure was that the monomers were oriented in opposite orientations in the membrane in an antiparallel orientation. A model was subsequently built based upon the cryo-EM data and evolutionary constraints and this model was consistent with mutagenic data indicating which amino-acid residues were important for substrate binding and transport. Two X-ray structures that differed significantly from the cryo-EM structure were subsequently retracted owing to a data-analysis error. However, the revised X-ray structure with substrate bound is extremely similar to the model built from the cryo-EM structure (r.m.s.d. of 1.4 Å), suggesting that the proposed antiparallel orientation of the monomers is indeed correct; this represents a new structural paradigm in membrane-protein structures. The vast majority of mutagenic and biochemical data corroborate this structure, although cross-linking studies and recent EPR data apparently support a model of EmrE that contains parallel dimers.

Re-refinement from deposited X-ray data can deliver improved models for most PDB entries

R. P. Joosten, T. Womack, G. Vriend and G. Bricogne

The deposition of X-ray data along with the customary structural models defining PDB entries makes it possible to apply large-scale re-refinement protocols to these entries, thus giving users the benefit of improvements in X-ray methods that have occurred since the structure was deposited. Automated gradient refinement is an effective method to achieve this goal, but real-space intervention is most often required in order to adequately address problems detected by structure-validation software. In order to improve the existing protocol, automated re-refinement was combined with structure validation and difference-density peak analysis to produce a catalogue of problems in PDB entries that are amenable to automatic correction. It is shown that re-refinement can be effective in producing improvements, which are often associated with the systematic use of the TLS parameterization of B factors, even for relatively new and high-resolution PDB entries, while the accompanying manual or semi-manual map analysis and fitting steps show good prospects for eventual automation. It is proposed that the potential for simultaneous improvements in methods and in re-refinement results be further encouraged by broadening the scope of depositions to include refinement metadata and ultimately primary rather than reduced X-ray data.

Distal histidine conformational flexibility in dehaloperoxidase from Amphitrite ornata

Z. Chen, V. de Serrano, L. Betts and S. Franzen

The enzyme dehaloperoxidase (DHP) from the terebellid polychaete Amphitrite ornata is a heme protein which has a globin fold but can function as both a hemoglobin and a peroxidase. As a peroxidase, DHP is capable of converting 2,4,6-trihalophenols to the corresponding 2,6-dihaloquinones in the presence of hydrogen peroxide. As a hemoglobin, DHP cycles between the oxy and deoxy states as it reversibly binds oxygen for storage. Here, it is reported that the distal histidine, His55, exhibits conformational flexibility in the deoxy form and is consequently observed in two solvent-exposed conformations more than 9.5 Å away from the heme. These conformations are analogous to the open conformation of sperm whale myoglobin. The heme iron in deoxy ferrous DHP is five-coordinate and has an out-of-plane displacement of 0.25 Å from the heme plane. The observation of five-coordinate heme iron with His55 in a remote solvent-exposed conformation is consistent with the hypothesis that His55 interacts with heme iron ligands through hydrogen bonding in the closed conformation. Since His55 is also displaced by the binding of 4-iodophenol in an internal pocket, these results provide new insight into the correlation between heme iron ligation, molecular binding in the distal pocket and the conformation of the distal histidine in DHP. PDB reference: 3dr9

Decision-making in structure solution using Bayesian estimates of map quality: the PHENIX AutoSol wizard

T. C. Terwilliger, P. D. Adams, R. J. Read, A. J. McCoy, N. W. Moriarty, R. W. Grosse-Kunstleve, P. V. Afonine, P. H. Zwart and L.-W. Hung

Estimates of the quality of experimental maps are important in many stages of structure determination of macromolecules. Map quality is defined here as the correlation between a map and the corresponding map obtained using phases from the final refined model. Here, ten different measures of experimental map quality were examined using a set of 1359 maps calculated by re-analysis of 246 solved MAD, SAD and MIR data sets. A simple Bayesian approach to estimation of map quality from one or more measures is presented. It was found that a Bayesian estimator based on the skewness of the density values in an electron-density map is the most accurate of the ten individual Bayesian estimators of map quality examined, with a correlation between estimated and actual map quality of 0.90. A combination of the skewness of electron density with the local correlation of r.m.s. density gives a further improvement in estimating map quality, with an overall correlation coefficient of 0.92. The PHENIX AutoSol wizard carries out automated structure solution based on any combination of SAD, MAD, SIR or MIR data sets. The wizard is based on tools from the PHENIX package and uses the Bayesian estimates of map quality described here to choose the highest quality solutions after experimental phasing.

Analysis and validation of carbohydrate three-dimensional structures

T. Lütteke

Knowledge of the three-dimensional structures of the carbohydrate molecules is indispensable for a full understanding of the molecular processes in which carbohydrates are involved, such as protein glycosylation or protein-carbohydrate interactions. The Protein Data Bank (PDB) is a valuable resource for three-dimensional structural information on glycoproteins and protein-carbohydrate complexes. Unfortunately, many carbohydrate moieties in the PDB contain inconsistencies or errors. This article gives an overview of the information that can be obtained from individual PDB entries and from statistical analyses of sets of three-dimensional structures, of typical problems that arise during the analysis of carbohydrate three-dimensional structures and of the validation tools that are currently available to scientists to evaluate the quality of these structures.

On the combination of molecular replacement and single-wavelength anomalous diffraction phasing for automated structure determination

S. Panjikar, V. Parthasarathy, V. S. Lamzin, M. S. Weiss and P. A. Tucker

A combination of molecular replacement and single-wavelength anomalous diffraction phasing has been incorporated into the automated structure-determination platform Auto-Rickshaw. The complete MRSAD procedure includes molecular replacement, model refinement, experimental phasing, phase improvement and automated model building. The improvement over the standard SAD or MR approaches is illustrated by ten test cases taken from the JCSG diffraction data-set database. Poor MR or SAD phases with phase errors larger than 70° can be improved using the described procedure and a large fraction of the model can be determined in a purely automatic manner from X-ray data extending to better than 2.6 Å resolution.

X-ray structure determination at low resolution

A. T. Brunger, B. DeLaBarre, J. M. Davies and W. I. Weis

As an example of structure determination in the 3.5-4.5 Å resolution range, crystal structures of the ATPase p97/VCP, consisting of an N-terminal domain followed by a tandem pair of ATPase domains (D1 and D2), are discussed. The structures were originally solved by molecular replacement with the high-resolution structure of the N-D1 fragment of p97/VCP, whereas the D2 domain was manually built using its homology to the D1 domain as a guide. The structure of the D2 domain alone was subsequently solved at 3 Å resolution. The refined model of D2 and the high-resolution structure of the N-D1 fragment were then used as starting models for re-refinement against the low-resolution diffraction data for full-length p97. The re-refined full-length models showed significant improvement in both secondary structure and R values. The free R values dropped by as much as 5% compared with the original structure refinements, indicating that refinement is meaningful at low resolution and that there is information in the diffraction data even at 4 Å resolution that objectively assesses the quality of the model. It is concluded that de novo model building is problematic at low resolution and refinement should start from high-resolution crystal structures whenever possible.

electronic Ligand Builder and Optimization Workbench (eLBOW): a tool for ligand coordinate and restraint generation

N. W. Moriarty, R. W. Grosse-Kunstleve and P. D. Adams

The electronic Ligand Builder and Optimization Workbench (eLBOW) is a program module of the PHENIX suite of computational crystallographic software. It is designed to be a flexible procedure that uses simple and fast quantum-chemical techniques to provide chemically accurate information for novel and known ligands alike. A variety of input formats and options allow the attainment of a number of diverse goals including geometry optimization and generation of restraints.

On vital aid: the why, what and how of validation

G. J. Kleywegt

Limitations to the data and subjectivity in the structure-determination process may cause errors in macromolecular crystal structures. Appropriate validation techniques may be used to reveal problems in structures, ideally before they are analysed, published or deposited. Additionally, such techniques may be used a posteriori to assess the (relative) merits of a model by potential users. Weak validation methods and statistics assess how well a model reproduces the information that was used in its construction (i.e. experimental data and prior knowledge). Strong methods and statistics, on the other hand, test how well a model predicts data or information that were not used in the structure-determination process. These may be data that were excluded from the process on purpose, general knowledge about macromolecular structure, information about the biological role and biochemical activity of the molecule under study or its mutants or complexes and predictions that are based on the model and that can be tested experimentally.

Crystallographic model quality at a glance

L. Urzhumtseva, P. V. Afonine, P. D. Adams and A. Urzhumtsev

A crystallographic macromolecular model is typically characterized by a list of quality criteria, such as R factors, deviations from ideal stereochemistry and average B factors, which are usually provided as tables in publications or in structural databases. In order to facilitate a quick model-quality evaluation, a graphical representation is proposed. Each key parameter such as R factor or bond-length deviation from `ideal values' is shown graphically as a point on a `ruler'. These rulers are plotted as a set of lines with the same origin, forming a hub and spokes. Different parts of the rulers are coloured differently to reflect the frequency (red for a low frequency, blue for a high frequency) with which the corresponding values are observed in a reference set of structures determined previously. The points for a given model marked on these lines are connected to form a polygon. A polygon that is strongly compressed or dilated along some axes reveals unusually low or high values of the corresponding characteristics. Polygon vertices in `red zones' indicate parameters which lie outside typical values.

Structure of NS1A effector domain from the influenza A/Udorn/72 virus

S. Xia, A. F. Monzingo and J. D. Robertus

The nonstructural protein NS1A from influenza virus is a multifunctional virulence factor and a potent inhibitor of host immunity. It has two functional domains: an N-terminal 73-amino-acid RNA-binding domain and a C-terminal effector domain. Here, the crystallographic structure of the NS1A effector domain of influenza A/Udorn/72 virus is presented. Structure comparison with the NS1 effector domain from mouse-adapted influenza A/Puerto Rico/8/34 (PR8) virus strain reveals a similar monomer conformation but a different dimer interface. Further analysis and evaluation shows that the dimer interface observed in the structure of the PR8 NS1 effector domain is likely to be a crystallographic packing effect. A hypothetical model of the intact NS1 dimer is presented.PDB references: 3ee9 and 3ee8

REFMAC5 for the refinement of macromolecular crystal structures

G. N. Murshudov, P. Skubák, A. A. Lebedev, N. S. Pannu, R. A. Steiner, R. A. Nicholls, M. D. Winn, F. Long and A. A. Vagin

This paper describes various components of the macromolecular crystallographic refinement program REFMAC5, which is distributed as part of the CCP4 suite. REFMAC5 utilizes different likelihood functions depending on the diffraction data employed (amplitudes or intensities), the presence of twinning and the availability of SAD/SIRAS experimental diffraction data. To ensure chemical and structural integrity of the refined model, REFMAC5 offers several classes of restraints and choices of model parameterization. Reliable models at resolutions at least as low as 4 Å can be achieved thanks to low-resolution refinement tools such as secondary-structure restraints, restraints to known homologous structures, automatic global and local NCS restraints, `jelly-body' restraints and the use of novel long-range restraints on atomic displacement parameters (ADPs) based on the Kullback-Leibler divergence. REFMAC5 additionally offers TLS parameterization and, when high-resolution data are available, fast refinement of anisotropic ADPs. Refinement in the presence of twinning is performed in a fully automated fashion. REFMAC5 is a flexible and highly optimized refinement package that is ideally suited for refinement across the entire resolution spectrum encountered in macromolecular crystallography.

Recent developments in classical density modification

K. Cowtan

Classical density-modification techniques (as opposed to statistical approaches) offer a computationally cheap method for improving phase estimates in order to provide a good electron-density map for model building. The rise of statistical methods has lead to a shift in focus away from the classical approaches; as a result, some recent developments have not made their way into classical density-modification software. This paper describes the application of some recent techniques, including most importantly the use of prior phase information in the likelihood estimation of phase errors within a classical density-modification framework. The resulting software gives significantly better results than comparable classical methods, while remaining nearly two orders of magnitude faster than statistical methods.

Radiation damage in macromolecular crystallography: what is it and why should we care?

E. F. Garman

Radiation damage inflicted during diffraction data collection in macromolecular crystallography has re-emerged in the last decade as a major experimental and computational challenge, as even for crystals held at 100 K it can result in severe data-quality degradation and the appearance in solved structures of artefacts which affect biological interpretations. Here, the observable symptoms and basic physical processes involved in radiation damage are described and the concept of absorbed dose as the basic metric against which to monitor the experimentally observed changes is outlined. Investigations into radiation damage in macromolecular crystallography are ongoing and the number of studies is rapidly increasing. The current literature on the subject is compiled as a resource for the interested researcher.

Know your dose: RADDOSE

K. S. Paithankar and E. F. Garman

The program RADDOSE is widely used to compute the dose absorbed by a macromolecular crystal during an X-ray diffraction experiment. A number of factors affect the absorbed dose, including the incident X-ray flux density, the photon energy and the composition of the macromolecule and of the buffer in the crystal. An experimental dose limit for macromolecular crystallography (MX) of 30 MGy at 100 K has been reported, beyond which the biological information obtained may be compromised. Thus, for the planning of an optimized diffraction experiment the estimation of dose has become an additional tool. A number of approximations were made in the original version of RADDOSE. Recently, the code has been modified in order to take into account fluorescent X-ray escape from the crystal (version 2) and the inclusion of incoherent (Compton) scattering into the dose calculation is now reported (version 3). The Compton cross-section, although negligible at the energies currently commonly used in MX, should be considered in dose calculations for incident energies above 20 keV. Calculations using version 3 of RADDOSE reinforce previous studies that predict a reduction in the absorbed dose when data are collected at higher energies compared with data collected at 12.4 keV. Hence, a longer irradiation lifetime for the sample can be achieved at these higher energies but this is at the cost of lower diffraction intensities. The parameter `diffraction-dose efficiency', which is the diffracted intensity per absorbed dose, is revisited in an attempt to investigate the benefits and pitfalls of data collection using higher and lower energy radiation, particularly for thin crystals.

Experimental phasing with SHELXC/D/E: combining chain tracing with density modification

G. M. Sheldrick

The programs SHELXC, SHELXD and SHELXE are designed to provide simple, robust and efficient experimental phasing of macromolecules by the SAD, MAD, SIR, SIRAS and RIP methods and are particularly suitable for use in automated structure-solution pipelines. This paper gives a general account of experimental phasing using these programs and describes the extension of iterative density modification in SHELXE by the inclusion of automated protein main-chain tracing. This gives a good indication as to whether the structure has been solved and enables interpretable maps to be obtained from poorer starting phases. The autotracing algorithm starts with the location of possible seven-residue -helices and common tripeptides. After extension of these fragments in both directions, various criteria are used to decide whether to accept or reject the resulting poly-Ala traces. Noncrystallographic symmetry (NCS) is applied to the traced fragments, not to the density. Further features are the use of a `no-go' map to prevent the traces from passing through heavy atoms or symmetry elements and a splicing technique to combine the best parts of traces (including those generated by NCS) that partly overlap.

Overview of the CCP4 suite and current developments

M. D. Winn, C. C. Ballard, K. D. Cowtan, E. J. Dodson, P. Emsley, P. R. Evans, R. M. Keegan, E. B. Krissinel, A. G. W. Leslie, A. McCoy, S. J. McNicholas, G. N. Murshudov, N. S. Pannu, E. A. Potterton, H. R. Powell, R. J. Read, A. Vagin and K. S. Wilson

The CCP4 (Collaborative Computational Project, Number 4) software suite is a collection of programs and associated data and software libraries which can be used for macromolecular structure determination by X-ray crystallography. The suite is designed to be flexible, allowing users a number of methods of achieving their aims. The programs are from a wide variety of sources but are connected by a common infrastructure provided by standard file formats, data objects and graphical interfaces. Structure solution by macromolecular crystallography is becoming increasingly automated and the CCP4 suite includes several automation pipelines. After giving a brief description of the evolution of CCP4 over the last 30 years, an overview of the current suite is given. While detailed descriptions are given in the accompanying articles, here it is shown how the individual programs contribute to a complete software package.