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      HIV-1 uses dynamic capsid pores to import nucleotides and fuel encapsidated DNA synthesis

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          Abstract

          During the early stages of infection, the HIV-1 capsid protects viral components from cytosolic sensors, such as cGAS, and nucleases, such as TREX, while allowing access to nucleotides for efficient reverse transcription 1. Here we show that each capsid hexamer has a size-selective pore bounded by a ring of six arginine residues and a ‘molecular iris’ formed by the N-terminal β-hairpin. The arginine ring creates a strongly positively charged channel that recruits the four nucleotides with on-rates that near diffusion limits. Progressive removal of pore arginines results in a dose-dependent and concomitant decrease in nucleotide affinity, reverse transcription and infectivity. This positively charged channel is universally conserved in lentiviral capsids despite the fact that it is strongly destabilising without nucleotides to counteract charge repulsion. We also describe a channel inhibitor, hexacarboxybenzene, which competes for nucleotide binding and efficiently blocks encapsidated reverse transcription demonstrating the tractability of the pore as a novel drug target.

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          Phaser crystallographic software

          Airlie J. McCoy, Ralf W. Grosse-Kunstleve, Paul D. Adams … (2007)
          1. Introduction Improved crystallographic methods rely on both improved automation and improved algorithms. The software handling one part of structure solution must be automatically linked to software handling parts upstream and downstream of it in the structure solution pathway with (ideally) no user input, and the algorithms implemented in the software must be of high quality, so that the branching or termination of the structure solution pathway is minimized or eliminated. Automation allows all the choices in structure solution to be explored where the patience and job-tracking abilities of users would be exhausted, while good algorithms give solutions for poorer models, poorer data or unfavourable crystal symmetry. Both forms of improvement are essential for the success of high-throughput structural genomics (Burley et al., 1999 ▶). Macromolecular phasing by either of the two main methods, molecular replacement (MR) and experimental phasing, which includes the technique of single-wavelength anomalous dispersion (SAD), are key parts of the structure solution pathway that have potential for improvement in both automation and the underlying algorithms. MR and SAD are good phasing methods for the development of structure solution pipelines because they only involve the collection of a single data set from a single crystal and have the advantage of minimizing the effects of radiation damage. Phaser aims to facilitate automation of these methods through ease of scripting, and to facilitate the development of improved algorithms for these methods through the use of maximum likelihood and multivariate statistics. Other software shares some of these features. For molecular replacement, AMoRe (Navaza, 1994 ▶) and MOLREP (Vagin & Teplyakov, 1997 ▶) both implement automation strategies, though they lack likelihood-based scoring functions. Likelihood-based experimental phasing can be carried out using Sharp (La Fortelle & Bricogne, 1997 ▶). 2. Algorithms The novel algorithms in Phaser are based on maximum likelihood probability theory and multivariate statistics rather than the traditional least-squares and Patterson methods. Phaser has novel maximum likelihood phasing algorithms for the rotation functions and translation functions in MR and the SAD function in experimental phasing, but also implements other non-likelihood algorithms that are critical to success in certain cases. Summaries of the algorithms implemented in Phaser are given below. For completeness and for consistency of notation, some equations given elsewhere are repeated here. 2.1. Maximum likelihood Maximum likelihood is a branch of statistical inference that asserts that the best model on the evidence of the data is the one that explains what has in fact been observed with the highest probability (Fisher, 1922 ▶). The model is a set of parameters, including the variances describing the error estimates for the parameters. The introduction of maximum likelihood estimators into the methods of refinement, experimental phasing and, with Phaser, MR has substantially increased success rates for structure solution over the methods that they replaced. A set of thought experiments with dice (McCoy, 2004 ▶) demonstrates that likelihood agrees with our intuition and illustrates the key concepts required for understanding likelihood as it is applied to crystallography. The likelihood of the model given the data is defined as the probability of the data given the model. Where the data have independent probability distributions, the joint probability of the data given the model is the product of the individual distributions. In crystallography, the data are the individual reflection intensities. These are not strictly independent, and indeed the statistical relationships resulting from positivity and atomicity underlie direct methods for small-molecule structures (reviewed by Giacovazzo, 1998 ▶). For macromolecular structures, these direct-methods relationships are weaker than effects exploited by density modification methods (reviewed by Kleywegt & Read, 1997 ▶); the presence of solvent means that the molecular transform is over-sampled, and if there is noncrystallographic symmetry then other correlations are also present. However, the assumption of independence is necessary to make the problem tractable and works well in practice. To avoid the numerical problems of working with the product of potentially hundreds of thousands of small probabilities (one for each reflection), the log of the likelihood is used. This has a maximum at the same set of parameters as the original function. Maximum likelihood also has the property that if the data are mathematically transformed to another function of the parameters, then the likelihood optimum will occur at the same set of parameters as the untransformed data. Hence, it is possible to work with either the structure-factor intensities or the structure-factor amplitudes. In the maximum likelihood functions in Phaser, the structure-factor amplitudes (Fs), or normalized structure-factor amplitudes (Es, which are Fs normalized so that the mean-square values are 1) are used. The crystallographic phase problem means that the phase of the structure factor is not measured in the experiment. However, it is easiest to derive the probability distributions in terms of the phased structure factors and then to eliminate the unknown phase by integration, a process known as integrating out a nuisance variable (the nuisance variable being the introduced phase of the observed structure factor, or equivalently the phase difference between the observed structure factor and its expected value). The central limit theorem applies to structure factors, which are sums of many small atomic contributions, so the probability distribution for an acentric reflection, F O, given the expected value of F O (〈F O〉) is a two-dimensional Gaussian with variance Σ centred on 〈F O〉. (Note that here and in the following, bold font is used to represent complex or signed structure factors, and italics to represent their amplitudes.) In applications to molecular replacement and structure refinement, 〈F O〉 is the structure factor calculated from the model (F C) multiplied by a fraction D (where 0 R, H = 0. The atoms are taken to be of equal mass. The eigenvalues λ and eigenvectors U of H can then be calculated. The eigenvalues are directly proportional to the squares of the vibrational frequencies of the normal modes, the lowest eigenvalues thus giving the lowest normal modes. Six of the eigenvalues will be zero, corresponding to the six degrees of freedom for a rotation and translation of the entire structure. For all but the smallest proteins, eigenvalue decomposition of the all-atom Hessian is not computationally feasible with current computer technology. Various methods have been developed to reduce the size of the eigenvalue problem. Bahar et al. (1997 ▶) and Hinsen (1998 ▶) have shown that it is possible to find the lowest frequency normal modes of proteins in the elastic network model by considering amino acid Cα atoms only. However, this merely postpones the computational problem until the proteins are an order of magnitude larger. The problem is solved for any size protein with the rotation–translation block (RTB) approach (Durand et al., 1994 ▶; Tama et al., 2000 ▶), where the protein is divided into blocks of atoms and the rotation and translation modes for each block used project the full Hessian into a lower dimension. The projection matrix is a block-diagonal matrix of dimensions 3N × 3N. Each of the NB block matrices P nb has dimensions 3N nb × 6, where N nb is the number of atoms in the block nb, For atom j in block nb displaced from the centre of mass, of the block, the 3 × 6 matrix P nb,j is The first three columns of the matrix contain the infinitesimal translation eigenvectors of the block and last three columns contain the infinitesimal rotation eigenvectors of the block. The orthogonal basis Q of P nb is then found by QR decomposition: where Q nb is a 3N nb × 6 orthogonal matrix and R nb is a 6 × 6 upper triangle matrix. H can then be projected into the subspace spanned by the translation/rotation basis vectors of the blocks: where The eigenvalues λP and eigenvectors U P of the projected Hessian are then found. The RTB method is able to restrict the size of the eigenvalue problem for any size of protein with the inclusion of an appropriately large N nb for each block. In the implementation of the RTB method in Phaser, N nb for each block is set for each protein such that the total size of the eigenvalue problem is restricted to a matrix H P of maximum dimensions 750 × 750. This enables the eigenvalue problem to be solved in a matter of minutes with current computing technology. The eigenvectors of the translation/rotation subspace can then be expanded back to the atomic space (dimensions of U are N × N): As for the decomposition of the full Hessian H, the eigenvalues are directly proportional to the squares of the vibrational frequencies of the normal modes, the lowest eigenvalues thus giving the lowest normal modes. Although the eigenvalues and eigenvectors generated from decomposition of the full Hessian and using the RTB approach will diverge with increasing frequency, the RTB approach is able to model with good accuracy the lowest frequency normal modes, which are the modes of interest for looking at conformational difference in proteins. The all-atom, Cα only and RTB normal-mode analysis methods are implemented in Phaser. After normal-mode analysis, n normal modes can be used to generate 2 n − 1 (nonzero) combinations of normal modes. Phaser allows the user to specify the r.m.s. deviation between model and target desired by the perturbation, and the fraction dq of the displacement vector for each mode combination corresponding to each model combination is then used to generate the models. Large r.m.s. deviations will cause the geometry of the model to become distorted. Phaser reports when the model becomes so distorted that there are Cα clashes in the structure. 2.4. Packing function The packing of potential solutions in the asymmetric unit is not inherently part of the translation function. It is therefore possible that an arrangement of models has a high log-likelihood gain, although the models may overlap and therefore be physically unreasonable. The packing of the solutions is checked using a clash test using a subset of the atoms in the structure: the ‘trace’ atoms. For proteins, the trace atoms are the Cα positions, spaced at 3.8 Å. For nucleic acid, the phosphate and C atoms in the ribose-phosphate backbone and the N atoms of the bases are selected as trace atoms. These atoms are also spaced at about 3.8 Å, so that the density of trace atoms in nucleic acid is similar to that of proteins, which makes the number of protein–protein, protein–nucleic acid and nucleic acid–nucleic acid clashes comparable where there is a mixed protein–nucleic acid structure. For the clash test, the number of trace atoms from another model within a given distance (default 3 Å) is counted. The clash test includes symmetry-related copies of the model under consideration, other components in the asymmetric unit and their symmetry-related copies. If the search model has a low sequence identity with the target, or has large flexible loops that could adopt an alternative conformation, the number of clashes may be expected to be nonzero. By default the best packing solutions are carried forward, although a specific number of allowed clashes may also be given as the cut-off for acceptance. However, it is better to edit models before use so that structurally nonconserved surface loops are excluded, as they will only contribute noise to the rotation and translation functions. Where an ensemble of structures is used as the model, the highest homology model is taken as the template for the packing search. Before this model is used, the trace atom positions are edited to take account of large conformational differences between the models in the ensemble. Equivalent trace atom positions are compared and if the coordinates deviate by more than 3 Å then the template trace atom is deleted. Thus, use of an ensemble not only improves signal to noise in the maximum likelihood search functions, it also improves the discrimination of possible solutions by the packing function. 2.5. Minimizer Minimization is used in Phaser to optimize the parameters against the appropriate log-likelihood function in the anisotropy correction, in MR (refines the position and orientation of a rigid-body model) and in SAD phasing. The same minimizer code is used for all three applications and has been designed to be easily extensible to other applications. The minimizer for the anisotropy correction uses Newton’s method, while MR and SAD use the standard Broyden–Fletcher–Goldfarb–Shanno (BFGS) algorithm. Both minimization methods in Phaser include a line search. The line search algorithm is a basic iterative method for finding the local minimum of a target function f. Starting at parameters x , the algorithm finds the minimum (within a convergence tolerance) of by varying γ, where γ is the step distance along a descent direction d . Newton’s method and the BFGS algorithm differ in the determination of the descent direction d that is passed to the line search, and thus the speed of convergence. Within one cycle of the line search (where there is no change in d ) the trial step distances γ are chosen using the golden section method. The golden ratio (51/2/2 + 1/2) divides a line so that the ratio of the larger part to the total is the same as the ratio of the smaller to larger. The method makes no assumptions about the function’s behaviour; in particular, it does not assume that the function is quadratic within the bracketed section. If this assumption were made, the line search could proceed via parabolic interpolation. Newton’s method uses the Hessian matrix H of second derivatives and the gradient g at the initial set of parameters x 0 to find the values of the parameters at the minimum x min. If the function is quadratic in x then Newton’s method will find the minimum in one step, but if not, iteration is required. The method requires the inversion of the Hessian matrix, which, for large matrices, consumes a large amount of computational time and memory resources. The eigenvalues of the Hessian need to be positive for the function to be at a minimum, rather than a maximum or saddle point, since the method converges to any point where the gradient vector is zero. When used with the anisotropy correction, the full Hessian matrix is calculated analytically. The BFGS algorithm is one of the most powerful minimization methods when calculation of the full Hessian using analytic or finite difference methods is very computationally intensive. At every step, the gradient search vector is analysed to build up an approximate Hessian matrix H, in order to make the resulting search vector direction d better than the original gradient vector direction. In the ‘pure’ form of the BFGS algorithm, the method is started with matrix H equal to the identity matrix. The off-diagonal elements of the Hessian, the mixed second derivatives (i.e. ∂2LL/∂p i ∂p j ) are thus initially zero. As the BFGS cycle proceeds, the off-diagonal elements become nonzero using information derived from the gradient. However, in Phaser, the matrix H is not the identity but rather is seeded with diagonal elements equal to the second derivatives of the parameters (p i ) with respect to the log-likelihood target function (LL) (i.e. ∂2LL/∂p i 2, or curvatures), the values found in the ‘true’ Hessian. For the SAD refinement the diagonal elements are calculated analytically, but for the MR refinement the diagonal elements are calculated by finite difference methods. Seeding the Hessian with the diagonal elements dramatically accelerates convergence when the parameters are on different scales; when an identity matrix is used, the parameters on a larger scale can fail to shift significantly because their gradients tend to be smaller, even though the necessary shifts tend to be larger. In the inverse Hessian, small curvatures for parameters on a large scale translate into large scale factors applied to the corresponding gradient terms. If any of these curvature terms are negative (as may happen when the parameters are far from their optimal values), the matrix is not positive definite. Such a situation is corrected by using problem-specific information on the expected relative scale of the parameters from the ‘large-shift’ variable, as discussed below in §2.5.1. In addition to the basic minimization algorithms, the minimizer incorporates the ability to bound, constrain, restrain and reparameterize variables, as discussed in detail below. Bounds must be applied to prevent parameters becoming nonphysical, constraints effectively reduce the number of parameters, restraints are applied to include prior probability information, and reparameterization of variables makes the parameter space more quadratic and improves the performance of the minimizer. 2.5.1. Problem-specific parameter scaling information When a function is defined for minimization in Phaser, information must be provided on the relative scales of the parameters of that function, through a ‘large-shifts’ variable. As its name implies, the variable defines the size of a parameter shift that would be considered ‘large’ for each parameter. The ratios of these large-shift values thus specify prior knowledge about the relative scales of the different parameters for each problem. Suitable large-shift values are found by a combination of physical insight (e.g. the size of a coordinate shift considered to be large will be proportional to d min for the data set) and numerical simulations, studying the behaviour of the likelihood function as parameters are varied systematically in a variety of test cases. The large-shifts information is used in two ways. Firstly, it is used to prevent the line search from taking an excessively large step, which can happen if the estimated curvature for a parameter happens to be too small and can lead to the refinement becoming numerically unstable. If the initial step for a line search would change any parameter by more than its large-shift value, the initial step is scaled down. Secondly, it is used to provide relative scale information to correct negative curvature values. Parameters with positive curvatures are used to define the average relationship between the large-shift values and the curvatures, which can then be used to compute appropriate curvature values for the parameters with negative curvatures. This stabilizes the refinement until it is sufficiently close to the minimum that all curvatures become positive. 2.5.2. Reparameterization Second-order minimization algorithms in effect assume that, at least in the region around the minimum, the function can be approximated as a quadratic. Where this assumption holds, the minimizer will converge faster. It is therefore advantageous to use functions of the parameters being minimized so that the target function is more quadratic in the new parameter space than in the original parameter space (Edwards, 1992 ▶). For example, atomic B factors tend to converge slowly to their refined values because the B factor appears in the exponential term in the structure-factor equation. Although any function of the parameters can be used for this purpose, we have found that taking the logarithm of a parameter is often the most effective reparameterization operation (not only for the B factors). The offset x offset is chosen so that the value of x′ does not become undefined for allowed values of x, and to optimize the quadratic nature of the function in x′. For instance, atomic B factors are reparameterized using an offset of 5 Å2, which allows the B factors to approach zero and also has the physical interpretation of accounting roughly for the width of the distribution of electrons for a stationary atom. 2.5.3. Bounds Bounds on the minimization are applied by setting upper and/or lower limits for each variable where required (e.g. occupancy minimum set to zero). If a parameter reaches a limit during a line search, that line search is terminated. In subsequent line searches, the gradient of that parameter is set to zero whenever the search direction would otherwise move the parameter outside of its bounds. Multiplying the gradient by the step size thus does not alter the value of the parameter at its limit. The parameter will remain at its limit unless calculation of the gradient in subsequent cycles of minimization indicates that the parameter should move away from the boundary and into the allowed range of values. 2.5.4. Constraints Space-group-dependent constraints apply to the anisotropic tensor applied to ΣN in the anisotropic diffraction correction. Atoms on special positions also have constraints on the values of their anisotropic tensor. The anisotropic displacement ellipsoid must remain invariant under the application of each symmetry operator of the space group or site-symmetry group, respectively (Giacovazzo, 1992 ▶; Grosse-Kunstleve & Adams, 2002 ▶). These constraints reduce the number of parameters by either fixing some values of the anisotropic B factors to zero or setting some sets of B factors to be equal. The derivatives in the gradient and Hessian must also be constrained to reflect the constraints in the parameters. 2.5.5. Restraints Bayes’ theorem describes how the probability of the model given the data is related to the likelihood and gives a justification for the use of restraints on the parameters of the model. If the probability of the data is taken as a constant, then P(model) is called the prior probability. When the logarithm of the above equation is taken, Prior probability is therefore introduced into the log-likelihood target function by the addition of terms. If parameters of the model are assumed to have independent Gaussian probability distributions, then the Bayesian view of likelihood will lead to the addition of least-squares terms and hence least-squares restraints on those parameters, such as the least-squares restraints applied to bond lengths and bond angles in typical macromolecular structure refinement programs. In Phaser, least-squares terms are added to restrain the B factors of atoms to the Wilson B factor in SAD refinement, and to restrain the anisotropic B factors to being more isotropic (the ‘sphericity’ restraint). A similar sphericity restraint is used in SHELXL (Sheldrick, 1995 ▶) and in REFMAC5 (Murshudov et al., 1999 ▶). 3. Automation Phaser is designed as a large set of library routines grouped together and made available to users as a series of applications, called modes. The routine-groupings in the modes have been selected mainly on historical grounds; they represent traditional steps in the structure solution pipeline. There are 13 such modes in total: ‘anisotropy correction’, ‘cell content analysis’, ‘normal-mode analysis’, ‘ensembling’, ‘fast rotation function’, ‘brute rotation function’, ‘fast translation function’, ‘brute translation function’, ‘log-likelihood gain’, ‘rigid-body refinement’, ‘single-wavelength anomalous dispersion’, ‘automated molecular replacement’ and ‘automated experimental phasing’. The ‘automated molecular replacement’ and ‘automated experimental phasing’ modes are particularly powerful and aim to automate fully structure solution by MR and SAD, respectively. Aspects of the decision making within the modes are under user input control. For example, the ‘fast rotation function’ mode performs the ensembling calculation, then a fast rotation function calculation and then rescores the top solutions from the fast search with a brute rotation function. There are three possible fast rotation function algorithms and two possible brute rotation functions to choose from. There are four possible criteria for selecting the peaks in the fast rotation function for rescoring with the brute rotation function, and for selecting the results from the rescoring for output. Alternatively, the rescoring of the fast rotation function with the brute rotation function can be turned off to produce results from the fast rotation function only. Other modes generally have fewer routines but are designed along the same principles (details are given in the documentation). 3.1. Automated molecular replacement Most structures that can be solved by MR with Phaser can be solved using the ‘automated molecular replacement’ mode. The flow diagram for this mode is shown in Fig. 1 ▶. The search strategy automates four search processes: those for multiple components in the asymmetric unit, for ambiguity in the hand of the space group and/or other space groups in the same point group, for permutations in the search order for components (when there are multiple components), and for finding the best model when there is more than one possible model for a component. 3.1.1. Multiple components of asymmetric unit Where there are many models to be placed in the asymmetric unit, the signal from the placement of the first model may be buried in noise and the correct placement of this first model only found in the context of all models being placed in the asymmetric unit. One way of tackling this problem has been to use stochastic methods to search the multi-dimensional space (Chang & Lewis, 1997 ▶; Kissinger et al., 1999 ▶; Glykos & Kokkinidis, 2000 ▶). However, we have chosen to use a tree-search-with-pruning approach, where a list of possible placements of the first (and subsequent) models is kept until the placement of the final model. This tree-search-with-pruning search strategy can generate very branched searches that would be challenging for users to negotiate by running separate jobs, but becomes trivial with suitable automation. The search strategy exploits the strength of the maximum likelihood target functions in using prior information in the search for subsequent components in the asymmetric unit. The tree-search-with-pruning strategy is heavily dependent on the criteria used for selecting the peaks that survive to the next round. Four selection criteria are available in Phaser: selection by percentage difference between the top and mean log-likelihood of the search, selection by Z score, selection by number of peaks, and selection of all peaks. The default is selection by percentage, with the default percentage set at 75%. This selection method has the advantage that, if there is one clear peak standing well above the noise, it alone will be passed to the next round, while if there is no clear signal, all peaks high in the list will be passed as potential solutions to the next round. If structure solution fails, it may be possible to rescue the solution by reducing the percentage cut-off used for selection from 75% to, for example, 65%, so that if the correct peak was just missing the default cut-off, it is now included in the list passed to the next round. The tree-search-with-pruning search strategy is sub-optimal where there are multiple copies of the same search model in the asymmetric unit. In this case the search generates many branches, each of which has a subset of the complete solution, and so there is a combinatorial explosion in the search. The tree search would only converge onto one branch (solution) with the placement of the last component on each of the branches, but in practice the run time often becomes excessive and the job is terminated before this point can be reached. When searching for multiple copies of the same component in the asymmetric unit, several copies should be added at each search step (rather than branching at each search step), but this search strategy must currently be performed semi-manually as described elsewhere (McCoy, 2007 ▶). 3.1.2. Alternative space groups The space group of a structure can often be ambiguous after data collection. Ambiguities of space group within the one point group may arise from theoretical considerations (if the space group has an enantiomorph) or on experimental grounds (the data along one or more axes were not collected and the systematic absences along these axes cannot be determined). Changing the space group of a structure to another in the same point group can be performed without re-indexing, merging or scaling the data. Determination of the space group within a point group is therefore an integral part of structure solution by MR. The translation function will yield the highest log-likelihood gain for a correctly packed solution in the correct space group. Phaser allows the user to make a selection of space groups within the same point group for the first translation function calculation in a search for multiple components in the asymmetric unit. If the signal from the placement of the first component is not significantly above noise, the correct space group may not be chosen by this protocol, and the search for all components in the asymmetric unit should be completed separately in all alternative space groups. 3.1.3. Alternative models As the database of known structures expands, the number of potential MR models is also rapidly increasing. Each available model can be used as a separate search model, or combined with other aligned structures in an ‘ensemble’ model. There are also various ways of editing structures before use as MR models (Schwarzenbacher et al., 2004 ▶). The number of MR trials that can be performed thus increases combinatorially with the number of potential models, which makes job tracking difficult for the user. In addition, most users stop performing MR trials as soon as any solution is found, rather than continuing the search until the MR solution with the greatest log-likelihood gain is found, and so they fail to optimize the starting point for subsequent steps in the structure solution pipeline. The use of alternative models to represent a structure component is also useful where there are multiple copies of one type of component in the asymmetric unit and the different copies have different conformations due to packing differences. The best solution will then have the different copies modelled by different search models; if the conformation change is severe enough, it may not be possible to solve the structure without modelling the differences. A set of alternative search models may be generated using previously observed conformational differences among similar structures, or, for example, by normal-mode analysis (see §2.3). Phaser automates searches over multiple models for a component, where each potential model is tested in turn before the one with the greatest log-likelihood gain is found. The loop over alternative models for a component is only implemented in the rotation functions, as the solutions passed from the rotation function to the translation function step explicitly specify which model to use as well as the orientation for the translation function in question. 3.1.4. Search order permutation When searching for multiple components in the asymmetric unit, the order of the search can be a factor in success. The models with the biggest component of the total structure factor will be the easiest to find: when weaker scattering components are the subject of the initial search, the solution may be buried in noise and not significant enough to survive the selection criteria in the tree-search-with-pruning search strategy. Once the strongest scattering components are located, then the search for weaker scattering components (in the background of the strong scattering components) is more likely to be a success. Having a high component of the total structure factor correlates with the model representing a high fraction of the total contents of the asymmetric unit, low r.m.s. deviation between model and target atoms, and low B factors for the target to which the model corresponds. Although the first of these (high completeness) can be determined in advance from the fraction of the total molecular weight represented by the model, the second can only be estimated from the Chothia & Lesk (1986 ▶) formula and the third is unknown in advance. If structure solution fails with the search performed in the order of the molecular weights, then other permutations of search order should be tried. In Phaser, this possibility is automated on request: the entire search strategy (except for the initial anisotropic data correction) is performed for all unique permutations of search orders. 3.2. Automated experimental phasing SAD is the simplest type of experimental phasing method to automate, as it involves only one crystal and one data set. SAD is now becoming the experimental phasing method of choice, overtaking multiple-wavelength anomalous dis­persion because only a single data set needs to be collected. This can help minimize radiation damage to the crystal, which has a major adverse effect on the success of multi-wavelength experiments. The ‘automated experimental phasing’ mode in Phaser takes an atomic substructure determined by Patterson, direct or dual-space methods (Karle & Hauptman, 1956 ▶; Rossmann, 1961 ▶; Mukherjee et al., 1989 ▶; Miller et al., 1994 ▶; Sheldrick & Gould, 1995 ▶; Sheldrick et al., 2001 ▶; Grosse-Kunstleve & Adams, 2003 ▶) and refines the positions, occupancies, B factors and values of the atoms to optimize the SAD function, then uses log-likelihood gradient maps to complete the atomic substructure. The flow diagram for this mode is shown in Fig. 2 ▶. The search strategy automates two search processes: those for ambiguity in the hand of the space group and for completing atomic substructure from log-likelihood gradient maps. A feature of using the SAD function for phasing is that the substructure need not only consist of anomalous scatterers; indeed it can consist of only real scatterers, since the real scattering of the partial structure is used as part of the phasing function. This allows structures to be completed from initial real scattering models. 3.2.1. Enantiomorphic space groups Since the SAD phasing mode of Phaser takes as input an atomic substructure model, the space group of the solution has already been determined to within the enantiomorph of the correct space group. Changing the enantiomorph of a SAD refinement involves changing the enantiomorph of the heavy atoms, or in some cases the space group (e.g. the enantiomorphic space group of P41 is P43). In some rare cases (Fdd2, I41, I4122, I41 md, I41 cd, I 2d, F4132; Koch & Fischer, 1989 ▶) the origin of the heavy-atom sites is changed [e.g. the enantiomorphic space group of I41 is I41 with the origin shifted to ( , 0, 0)]. If there is only one type of anomalous scatterer, the refinement need not be repeated in both hands: only the phasing needs to be carried out in the second hand to be considered. However, if there is more than one type of anomalous scatterer, then the refinement and substructure completion needs to be repeated, as it will not be enantiomorphically symmetric in the other hand. To facilitate this, Phaser runs the refinement and substructure completion in both hands [as does other experimental phasing software, e.g. Solve (Terwilliger & Berendzen, 1999 ▶) and autosharp (Vonrhein et al., 2006 ▶)]. The correct space group can then be found by inspection of the electron density maps; the density will only be interpretable in the correct space group. In cases with significant contributions from at least two types of anomalous scatterer in the substructure, the correct space group can also be identified by the log-likelihood gain. 3.2.2. Completing the substructure Peaks in log-likelihood gradient maps indicate the coordinates at which new atoms should be added to improve the log-likelihood gain. In the initial maps, the peaks are likely to indicate the positions of the strongest anomalous scatterers that are missing from the model. As the phasing improves, weaker anomalous scatterers, such as intrinsic sulfurs, will appear in the log-likelihood gradient maps, and finally, if the phasing is exceptional and the resolution high, non-anomalous scatterers will appear, since the SAD function includes a contribution from the real scattering. After refinement, atoms are excluded from the substructure if their occupancy drops below a tenth of the highest occupancy amongst those atoms of the same atom type (and therefore ). Excluded sites are flagged rather than permanently deleted, so that if a peak later appears in the log-likelihood gradient map at this position, the atom can be reinstated and prevented from being deleted again, in order to prevent oscillations in the addition of new sites between cycles and therefore lack of convergence of the substructure completion algorithm. New atoms are added automatically after a peak and hole search of the log-likelihood gradient maps. The cut-off for the consideration of a peak as a potential new atom is that its Z score be higher than 6 (by default) and also higher than the depth of the largest hole in the map, i.e. the largest hole is taken as an additional indication of the noise level of the map. The proximity of each potential new site to previous atoms is then calculated. If a peak is more than a cut-off distance (κ Å) of a previous site, the peak is added as a new atom with the average occupancy and B factor from the current set of sites. If the peak is within κ Å of an isotropic atom already present, the old atom is made anisotropic. Holes in the log-likelihood gradient map within κ Å of an isotropic atom also cause the atom’s B factor to be switched to anisotropic. However, if the peak or hole is within κ Å of an anisotropic atom already present, the peak or hole is ignored. If a peak is within κ Å of a previously excluded site, the excluded site is reinstated and flagged as not for deletion in order to prevent oscillations, as described above. At the end of the cycle of atom addition and isotropic to anisotropic atomic B-factor switching, new sites within 2κ Å of an old atom that is now anisotropic are then removed, since the peak may be absorbed by refining the anisotropic B factor; if not, it will be accepted as a new site in the next cycle of log-likelihood gradient completion. The distance κ may be input directly by the user, but by default it is the ‘optical resolution’ of the structure (κ = 0.715d min), but not less than 1 Å and no more than 10 Å. If the structure contains more than one significant anomalous scatterer, then log-likelihood gradient maps are calculated from each atom type, the maps compared and the atom type associated with each significant peak assigned from the map with the most significant peak at that location. 3.2.3. Initial real scattering model One of the reasons for including MR and SAD phasing within one software package is the ability to use MR solutions with the SAD phasing target to improve the phases. Since the SAD phasing target contains a contribution from the real scatterers, it is possible to use a partial MR model with no anomalous scattering as the initial atomic substructure used for SAD phasing. This approach is useful where there is a poor MR solution combined with a poor anomalous signal in the data. If the poor MR solution means that the structure cannot be phased from this model alone, and the poor anomalous signal means that the anomalous scatterers cannot be located in the data alone, then using the MR solution as the starting model for SAD phasing may provide enough phase information to locate the anomalous scatterers. The combined phase information will be stronger than from either source alone. To facilitate this method of structure solution, Phaser allows the user to input a partial structure model that will be interpreted in terms of its real scattering only and, following phasing with this substructure, to complete the anomalous scattering model from log-likelihood gradient maps as described above. 3.3. Input and output The fastest and most efficient way, in terms of development time, to link software together is using a scripting language, while using a compiled language is most efficient for intensive computation. Following the lead of the PHENIX project (Adams et al., 2002 ▶, 2004 ▶), Phaser uses Python (http://python.org) as the scripting language, C++ as the compiled language, and the Boost.Python library (http://boost.org/libs/python/) for linking C++ and Python. Other packages, notably X-PLOR (Brünger, 1993 ▶) and CNS (Brünger et al., 1998 ▶), have defined their own scripting languages, but the choice of Python ensures that the scripting language is maintained by an active community. Phaser functionality has mostly been made available to Python at the ‘mode’ level. However, some low-level SAD refinement routines in Phaser have been made available to Python directly, so that they can be easily incorporated into phenix.refine. A long tradition of CCP4 keyword-style input in established macromolecular crystallography software (almost exclusively written in Fortran) means that, for many users, this has been the familiar method of calling crystallographic software and is preferred to a Python interface. The challenge for the development of Phaser was to find a way of satisfying both keyword-style input and Python scripting with minimal increase in development time. Taking advantage of the C++ class structure allowed both to be implemented with very little additional code. Each keyword is managed by its own class. The input to each mode of Phaser is controlled by Input objects, which are derived from the set of keyword classes appropriate to the mode. The keyword classes are in turn derived from a CCP4base class containing the functionality for the keyword-style input. Each keyword class has a parse routine that calls the CCP4base class functions to parse the keyword input, stores the input parameters as local variables and then passes these parameters to a keyword class set function. The keyword class set functions check the validity and consistency of the input, throw errors where appropriate and finally set the keyword class’s member parameters. Alternatively, the keyword class set functions can be called directly from Python. These keyword classes are a standalone part of the Phaser code and have already been used in other software developments (Pointless; Evans, 2006 ▶). An Output object controls all text output from Phaser sent to standard output and to text files. Switches on the Output object give different output styles: CCP4-style for compatibility with CCP4 distribution, PHENIX-style for compatibility with the PHENIX interface, CIMR-style for development, XML-style output for developers of automation scripts and a ‘silent running’ option to be used when running Phaser from Python. In addition to the text output, where possible Phaser writes results to files in standard format; coordinates to ‘pdb’ files and reflection data (e.g. map coefficients) to ‘mtz’ files. Switches on the Output object control the writing of these files. 3.3.1. CCP4-style output CCP4-style output is a text log file sent to standard output. While this form of output is easily comprehensible to users, it is far from ideal as an output style for automation scripts. However, it is the only output style available from much of the established software that developers wish to use in their automation scripts, and it is common to use Unix tools such as ‘grep’ to extract key information. For this reason, the log files of Phaser have been designed to help developers who prefer to use this style of output. Phaser prints four levels of log file, summary, log, verbose and debug, as specified by user input. The important output information is in all four levels of file, but it is most efficient to work with the summary output. Phaser prints ‘SUCCESS’ and ‘FAILURE’ at the end of the log file to demarcate the exit state of the program, and also prints the names of any of the other output files produced by the program to the summary output, amongst other features. 3.3.2. XML output XML is becoming commonly used as a way of communicating between steps in an automation pipeline, because XML output can be added very simply by the program author and relatively simply by others with access to the source code. For this reason, Phaser also outputs an XML file when requested. The XML file encapsulates the mark-up within 〈phaser〉 tags. As there is no standard set of XML tags for crystallographic results, Phaser’s XML tags are mostly specific to Phaser but were arrived at after consultation with other developers of XML output for crystallographic software. 3.3.3. Python interface The most elegant and efficient way to run Phaser as part of an automation script is to call the functionality directly from Python. Using Phaser through the Python interface is similar to using Phaser through the keyword interface. Each mode of operation of Phaser described above is controlled by an Input object and its parameter set functions, which have been made available to Python with the Boost.Python library. Phaser is then run with a call to the ‘run-job’ function, which takes the Input object as a parameter. The ‘run-job’ function returns a Result object on completion, which can then be queried using its get functions. The Python Result object can be stored as a ‘pickled’ class structure directly to disk. Text is not sent to standard out in the CCP4 logfile way but may be redirected to another output stream. All Input and Result objects are fully documented. 4. Future developments Phaser will continue to be developed as a platform for implementing novel phasing algorithms and bringing the most effective approaches to the crystallographic community. Much work remains to be done formulating maximum likelihood functions with respect to noncrystallographic symmetry, to account for correlations in the data and to consider non-isomorphism, all with the aim of achieving the best possible initial electron density map. After a generation in which Fortran dominated crystallographic software code, C++ and Python have become the new standard. Several developments, including Phaser, PHENIX (Adams et al., 2002 ▶, 2004 ▶), Clipper (Cowtan, 2002 ▶) and mmdb (Krissinel et al., 2004 ▶), simultaneously chose C++ as the compiled language at their inception at the turn of the millennium. At about the same time, Python was chosen as a scripting language by PHENIX, ccp4mg (Potterton et al., 2002 ▶, 2004 ▶) and PyMol (DeLano, 2002 ▶), amongst others. Since then, other major software developments have also started or converted to C++ and Python, for example PyWarp (Cohen et al., 2004 ▶), MrBump (Keegan & Winn, 2007 ▶) and Pointless (Evans, 2006 ▶). The choice of C++ for software development was driven by the availability of free compilers, an ISO standard (International Standardization Organization et al., 1998 ▶), sophisticated dynamic memory management and the inherent strengths of using an object-oriented language. Python was equally attractive because of the strong community support, its object-oriented design, and the ability to link C++ and Python through the Boost.Python library or the SWIG library (http://www.swig.org/). Now that a ‘critical mass’ of developers has taken to using the new languages, C++ and Python are likely to remain the standard for crystallographic software for the current generation of crystallographic software developers. Phaser source code has been distributed directly by the authors (see http://www-structmed.cimr.cam.ac.uk/phaser for details) and through the PHENIX and CCP4 (Collaborative Computing Project, Number 4, 1994 ▶) software suites. The source code is released for several reasons, including that we believe source code is the most complete form of publication for the algorithms in Phaser. It is hoped that generous licensing conditions and source distribution will encourage the use of Phaser by other developers of crystallographic software and those writing crystallographic automation scripts. There are no licensing restrictions on the use of Phaser in macromolecular crystallography pipelines by other developers, and the license conditions even allow developers to alter the source code (although not to redistribute it). We welcome suggestions for improvements to be incorporated into new versions. Compilation of Phaser requires the computational crystallography toolbox (cctbx; Grosse-Kunstleve & Adams, 2003 ▶), which includes a distribution of the cmtz library (Winn et al., 2002 ▶). The Boost libraries (http://boost.org/) are required for access to the functionality from Python. Phaser runs under a wide range of operating systems including Linux, Irix, OSF1/Tru64, MacOS-X and Windows, and precompiled executables are available for these platforms when only keyword-style access (and not Python access) is required. Graphical user interfaces to Phaser are available for both the PHENIX and the CCP4 suites. User support is available through PHENIX, CCP4 and from the authors (email cimr-phaser@lists.cam.ac.uk).
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            <i>Coot</i> : model-building tools for molecular graphics

            Paul Emsley, Kevin Cowtan (2004)
            Acta Crystallographica Section D Biological Crystallography, 60(12), 2126-2132
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              MolProbity: all-atom structure validation for macromolecular crystallography

              Vincent Chen, W. Bryan Arendall, Jeffrey J. Headd … (2009)
              1. Summary of MolProbity flow and user interactions The usual interaction with MolProbity (Davis et al., 2007 ▶) is through the internet at http://molprobity.biochem.duke.edu or as a main menu item on our general laboratory website at http://kinemage.biochem.duke.edu. [For bulk users, it is also possible to set up your own local MolProbity server or to use the individual programs in command-line mode.] Tutorial exercises for the whole process of diagnosing and fixing errors can be found on the kinemage site under Teaching/MolProbity. A typical MolProbity session starts with the user uploading a coordinate file of their own or fetching one from the PDB or NDB databases (Berman et al., 1992 ▶, 2000 ▶) in new or old PDB format or in mmCIF format. After checking the thumbnail image and listed characteristics of the input file and editing or reloading if needed, H atoms are added and optimized, with automated correction of Asn/Gln/His 180° flips if needed (Word, Lovell, Richardson et al., 1999 ▶). The user then chooses which validation analyses to run and what reports and output files to generate. The MolProbity interface adjusts the defaults and options presented and even the page flow depending on user choices and on the properties of the file being worked on. These adjustments make MolProbity simple for novice users, while at the same time allowing advanced users to have great control over their runs. The core ‘glue’ that generates the HMTL code controlling the main user interface and programmatic interactions of MolProbity is implemented in the PHP programming language. Underlying the PHP core, the majority of the analysis tasks in MolProbity are performed by individual programs written in a range of languages, including C, C++, Java and Perl. It uses REDUCE and PROBE for all-atom contact analysis, RAMALYZE, ROTALYZE, DANGLE, SILK and SUITENAME for other criteria and KiNG for three-dimensional visualization of the structure and its validation markers directly in the browser. Fig. 1 ▶ shows a key to MolProbity’s graphical markers for validation outliers. Further details are provided below on the specific analyses that MolProbity can perform. The validation results are reported in the form of summaries, charts, two-dimensional and three-dimensional graphics and output files for download. The crucial final step in the MolProbity process is for the crystallographer to download the result files and work off-line to correct as many of the diagnosed problems as feasible. Rebuilding with consideration of the validation outliers, the electron density and the surrounding model is usually per­formed either in Coot (Emsley & Cowtan, 2004 ▶) or in KiNG (Chen et al., 2009 ▶). At resolutions of about 2.5 Å or better it is possible to correct the great majority of outliers (Arendall et al., 2005 ▶), with an order-of-magnitude improvement in the various MolProbity scores and some improvement in geometry, map quality, R factor and R free. An example is shown in Fig. 2 ▶ with before-and-after multi-criterion kinemages. 2. Validation analyses 2.1. Addition of H atoms The presence of H atoms (both nonpolar and polar) is a critical pre­requisite for all-atom contact analysis. Although refinement using H atoms is becoming more common, most crystal structures are still deposited without H atoms. Once a PDB structure file has been uploaded, MolProbity detects whether the file contains a suitable number of H atoms; if not, then the ‘Add H atoms’ option is presented to users first. MolProbity uses the software REDUCE (Word, Lovell, Richardson et al., 1999 ▶) to add and optimize hydrogen positions in both protein and nucleic acid structures, including ligands, but does not add explicit H atoms to waters. OH, SH and NH3 groups (but not methyl groups) are rotationally optimized and His protonation is chosen within each local hydrogen-bond network, including interactions with the first shell of explicit waters. A common problem is that the side-chain ends of Asn, Gln and His are easily fitted 180° backwards, since the electron density alone cannot usually distinguish the correct choice of orientation. REDUCE can automatically diagnose and correct these types of systematic errors by considering all-atom steric overlaps as well as hydrogen bonding within each local network. Automatic correction of Asn/Gln/His flips is the default option in MolProbity during addition of H atoms. MolProbity presents each potential flip correction to the user in kinemage view so they have the option of inspecting the before-and-after effects of each flip and approving (or rejecting) each correction. Fig. 3 ▶ shows an example of a simple Gln flip that is unquestionably correct but that could not have been decided on the basis of hydrogen bonding alone. Other examples can be much more complex, with rotatable OH positions, large hydrogen-bond net­works and multiple com­peting inter­actions evaluated exhaustively. Users can also choose to add H atoms without Asn/Gln/His flips, which is useful in evaluating the atomic coordinates as they were deposited, but which rejects the easiest and most robustly correct improvement that can be made in a crystallo­graphic model (Word, Lovell, Richardson et al., 1999 ▶; Higman et al., 2004 ▶). If flips are performed, the user needs to download and use the corrected PDB file (either with or without the H atoms) in order to benefit. 2.2. All-atom contact analysis Once H atoms have been added to (or detected in) a structure, then the complete ‘Analyze all-atom contacts and geometry’ option is enabled. A main feature of this option is the all-atom contact analysis, which is performed by the program PROBE (Word, Lovell, LaBean et al., 1999 ▶). PROBE operates by, in effect, rolling a 0.5 Å diameter ball around the van der Waals surfaces of atoms to measure the amount of overlap between pairs of nonbonded atoms. When non-donor–acceptor atoms overlap by more than 0.4 Å, PROBE denotes the contact as a serious clash, which is included in the reported clashscore and is shown in kinemage format as a cluster of hot-pink spikes in the overlap region (Fig. 1 ▶). Such large overlaps cannot occur in the actual molecule, but mean that at least one of the two atoms is modeled incorrectly. MolProbity allows users to select any combination of clashes, hydrogen bonds and van der Waals contacts to calculate and display on the structure. By default, all three are enabled for structures that are not excessively large; for large structures, van der Waals contacts are deselected. The ‘clashscore’ is the number of serious clashes per 1000 atoms. It is reported in the MolProbity summary (top of Fig. 4 ▶), with a red/yellow/green color coding for absolute quality. The structure’s percentile rank for clashscore value within the relevant resolution range is also given. In the detailed sortable ‘multi-chart’ (an extract is shown below the summary in Fig. 4 ▶), the worst clash ≥0.4 Å is listed for each residue and highlighted in pink. 2.3. Torsion-angle combinations: updated Ramachandran and rotamer analyses Also included in the ‘Analyze all-atom contacts and geometry’ option is an evaluation of where residues fall in the multi-dimensional distributions of Ramachandran backbone ϕ, ψ angles and side-chain rotamer χ angles. The reference distributions are currently from 100 000 residues in 500 files, quality-filtered at both the file and the residue level. The Ramachandran plots are separated for Gly, Pro and pre-Pro residue types; the general plot has only one in 2000 residues outside the ‘allowed’ contour, which is the same probability as a 3.5σ outlier in a normal distribution. The three specific plots can be robustly contoured only down to excluding one in 500 residues (about 3σ) in the current reference data, but will soon be updated. By ‘robust’ we mean that the contour does not shift with further improvement in resolution or B or with different subselections of the data. When values plateau in this way we can define clear absolute goals for the measure, such as 98% for Ramachandran favored, 2.9 Å for C3′-­endo and <2.9 Å for C2′-endo. MolProbity checks this distance against the modeled sugar pucker, as well as outliers in individual ∊ or δ values. All such outliers are listed in the multi-chart and ribose-pucker outliers are flagged in the kinemage (Fig. 1 ▶). An example is shown in Fig. 6 ▶, where what should have been a C2′-endo pucker (by the short perpendicular) was fitted as an intermediate unfavorable pucker close to the more common default C3′-endo pucker, also producing geometry and ∊ outliers. High-dimensional analysis of the combinations of backbone torsion angles within an RNA ‘suite’ (the unit from sugar to sugar) has shown that there are distinct ‘rotameric’ backbone conformers. The RNA Ontology Consortium has defined a two-character nomenclature and an initial set of 54 favorable RNA backbone conformers (Richardson et al., 2008 ▶). We created the SUITENAME program to identify either the named conformer or an outlier for each suite in an RNA structure. These conformers and their ‘suiteness’ quality score are listed in the MolProbity multi-chart. 2.6. The overall MolProbity score In response to user demand, the ‘MolProbity score’ provides a single number that represents the central Mol­Probity protein quality statistics. It is a log-weighted combination of the clashscore, percentage Ramachandran not favored and percentage bad side-chain rotamers, giving one number that reflects the crystallographic resolution at which those values would be expected. Therefore, a structure with a numerically lower MolProbity score than its actual crystallo­graphic resolution is, quality-wise, better than the average structure at that resolution. There is some distortion in the fit at very high or very low resolutions; for these ranges it is preferable to judge by the resolution-specific percentile score, which is also reported in the summary. Percentile scores are currently given for clashscore and for MolProbity score relative to the cohort of PDB structures within 0.25 Å of the file’s resolution. 3. Correction of outliers 3.1. Manual rebuilding Except for Asn/Gln/His flip corrections, MolProbity does not yet directly include the ability to correct the errors it finds in structures; it relies on users having access to standalone local software for rebuilding and refinement. The stand­alone version of KiNG has some rebuilding tools for modeling side chains and making small local ‘backrub’ adjustments to structures, with the help of electron-density display, interactive contact dots and rotamer evaluation (Davis et al., 2006 ▶; Chen et al., 2009 ▶). Fig. 7 ▶ illustrates such a correction process in KiNG, rebuilding a backward-fitted leucine with a clash and a bad rotamer (one of the cases of a systematic error), resulting in an ideal geometry side chain with an excellent rotamer and well packed all-atom contacts. The top view shows that the original and rebuilt side chains fit the terminal methyls into the same rather ambiguous density, but move the Cγ substantially. More recent versions of this DNA polymerase structure (e.g. PDB code 2hhv at 1.55 Å resolution; Warren et al., 2006 ▶) all use the new conformation. Manual rebuilding is facilitated by the fact that all-atom clashes are inherently directional, as are bond-angle distortions, while a good library of rotamer choices helps the user test all the alternatives. For more extensive refitting, a fully featured crystallo­graphic rebuilding program such as Coot (Emsley & Cowtan, 2004 ▶) is needed. MolProbity generates ‘to-do’ scripts that can be read into Coot, bringing up a button list, where each entry will zoom to a problem area. In combination with the ability of Coot to use REDUCE and PROBE interactively to generate all-atom contact dots, these features make it easier to address the problems diagnosed by MolProbity. Any rebuilding that moves atoms must of course then undergo further crystallo­graphic refinement. Our own laboratory tested the combined cycle of MolProbity, rebuilding and refinement on about 30 protein structures as part of the SouthEast Collaboratory for Structural Genomics (Arendall et al., 2005 ▶), finding that its early application led to a smoother structure-solution process and demonstrably better final structures. In addition to backward-fitted side chains, commonly corrected problems included peptide flips, switched backbone and side chain near chain ends, ‘waters’ that were really ions, noise peaks or unfit alternate conformations and occasionally a shift in sequence register. Many other crystallographic groups have since adopted these methods. 3.2. Automated corrections For correcting RNA-suite outliers, we have collaboratively developed the independent program RNABC (Wang et al., 2008 ▶), which performs an automated search for more suitable backbone conformations of an RNA suite diagnosed with a bad ribose pucker or serious clashes. It leaves the more accurately determined bases and P atoms fixed in place and performs a pruned but systematic search through the other parameters, outputting all acceptable alternatives found within user-set tolerance limits. Recently, we have developed and tested the AUTOFIX program for automated correction of diagnosed backward-fitted Thr, Val, Leu and Arg side chains (Headd et al., 2009 ▶). In contrast to Asn/Gln/His flips, which simply exchange atoms and do not change the agreement with the data, these more complex side chains require real-space refinement in order to determine the proper correction and crystallographic re-refinement after the approximate 180° flips have been made. The original version used Coot to perform rotamer selection and real-space refinement for the proposed corrections, with MolProbity diagnosis before and after. Results were checked by re-refinement. Run on a sample of 945 PDB files, AUTOFIX accepted corrections for over 40% of diagnosed bad Thr, Val and Leu side chains and 15% of bad Arg side chains, or 3679 corrected side chains. A second version is now in the testing stage that substitutes PHENIX real-space refinement, has a faster Python wrapper and also works on Ile. It will soon be incorporated into MolProbity. The most important of our requirements for AUTOFIX is that it does no harm; we are willing to miss some of the possible corrections in order to ensure that those we accept are essentially always true improvements. AUTOFIX should provide MolProbity users with an easy and reliable way of making an initial set of meaningful improvements to their protein structures. Thr and Arg, in particular, make hydrogen bonds that are often important at active sites or binding interfaces and since they are asymmetrical these interactions change drastically if the side chain is fitted backwards. Such improvements were often seen in the test set. 4. Other MolProbity utility functions 4.1. Interface analysis PROBE can also be used to calculate the all-atom contacts at interfaces, e.g. between two chains of a structure or between a protein and a ligand. Access to this feature is provided in MolProbity by the ‘Visualize interface contacts’ analysis option after H atoms have been added. The user is required to choose the chains and/or the molecular types for which to calculate the contacts (e.g. protein versus protein or protein versus heteroatoms or RNA). This functionality creates both a kinemage with the resulting all-atom contacts displayed on the model and a text list of the atom pairs in contact. 4.2. Protein loop fitting MolProbity includes the Java software JIFFILOOP for providing potential protein-fragment conformations that can fit within a gap in a protein structure. We have defined a seven-parameter system that describes the spatial relationship between any two peptides. Briefly, this system consists of the sequence separation, the distance between the two inner Cα atoms, two pseudo-angles and three pseudo-dihedrals. We used this system to create a library of B-factor-filtered fragments from one to 15 peptides long from our Top5200 database of structures, a set of structures chosen from each 70% nonredundant group defined by the PDB, requiring an average of resolution and MolProbity score of ≤2.0. MolProbity runs JIFFILOOP to search this library for candidate fragments to fill gaps within a structure. Alternatively, users can enter beginning and ending residue numbers and MolProbity will search for fragments which can fit between those two residues. Because this process can be fairly time-intensive, JIFFILOOP is not listed under ‘Suggested Tools’ and is currently only accessible under ‘All Tools’ or at the Site map. Also, owing to the size of this package it must be added separately to the installation for a standalone MolProbity server. 4.3. Kinemage construction and viewing MolProbity provides scripts (under the ‘Make simple kinemages’ option) for constructing a number of commonly used kinemage three-dimensional interactive visualization options such as ribbons and various types of stick figures. This functionality is useful for quick browsing of a structure or for initial creation of an illustration or presentation. The file-input page can also accept upload of pre-existing kinemage files for direct on-line viewing within the built-in kinemage viewer KiNG. 4.4. Other file types and functions MolProbity uses a built-in PDB ‘het_dictionary’ for the information needed to add H atoms to small-molecule ligands. The user can construct and read in a custom dictionary if their file contains novel ligands. There is also provision for either uploading or fetching an electron-density map from the Electron Density Server (Kleywegt et al., 2004 ▶) in any of several formats to view on-line in KiNG together with the model and validation results. To investigate functional sites that span across asymmetric units, one can fetch a biological unit file from the PDB. In the file-editing feature, the user can specify whether multiple ‘models’ are alternatives (as in an NMR ensemble) or have been pressed into service for the extra chains in the biological unit. Some X-ray structures are now treated as ensembles. For such cases, MolProbity internally splits the models and analyzes them separately, but constructs an outlier summary strip-chart and a multi-model multi-criterion validation kinemage with both the models and their features under on/off button control. File editing also allows the deletion of chains either before or after hydrogen placement, specifying the resolution of the structure if not given in the file header or removing unwanted H atoms. These tools make it easier and faster to analyze particular parts of a structure using MolProbity and they help to maintain compatibility with other older software. These options are always available as separate utility functions, independent of validation or hydrogen content. 4.5. PDB-format interconversion The release of the remediated PDB version 3.0 format in August 2007 included a number of significant changes, particularly to H-atom names and to nucleic acid residue and atom names. In order to maintain compatibility with the PDB, we converted the entire MolProbity core to use the new format by default. This included updating REDUCE, PROBE, KiNG and PREKIN. However, we realised that users might need to analyze files that were still in the older PDB version 2.3 format. In order to maintain backwards compatibility, we created a Remediator script (available as a standalone Perl or Python script) that can interconvert between the old and the new PDB formats. Whenever a file is input, MolProbity will scan for the presence of old-format atom names and if it detects any then it will run the Remediator script to automatically convert the input file to the new format. After analysis there is then an option available to run the Remediator script and downgrade the output file back into the old version 2.3 format if needed. This allows use of the MolProbity analysis tools even together with older software that has not been updated to use the new format. 5. Discussion 5.1. Global versus local, absolute versus comparative There are three quite different purposes served by structure validation: a gatekeeper function on quality for reviewers or organizations, an aid to crystallographers for obtaining the most model accuracy from their data and a guide to end users for choosing appropriate structures and confidence levels for the conclusions they want to draw. Validation criteria also come in distinct flavors. Those based on the diffraction data are inherently global with respect to the model; for instance, resolution (which is still the most valuable single-factor estimate of model accuracy) and R free (Brünger, 1992 ▶). On the data side, there are also gatekeeper checks for unusual problems such as twinning or gross data incompleteness. R.m.s.d. or r.m.s.Z. of deviations from geo­metrical target values are global, but they only evaluate procedural aspects of refinement and have little to do with model accuracy. Most other validation criteria are inherently local (at the residue or even atom level), including B factor, real-space measures such as RSR-Z (Kleywegt et al., 2004 ▶) and model-only measures such as the various MolProbity criteria described here. Any local measure becomes global when expressed in some normalized form across the entire structure, such as an average, a distribution match or a percentage occurrence of outliers. Strictly local measures are usually not resolution-dependent, but their globally defined versions often are. For some purposes, the desirable form of measure is a comparison (usually a percentile rank) with the cohort of PDB structures at similar resolution. MolProbity currently provides resolution-group percentiles for clashscore and for MolProbity score and will probably expand that to other criteria. Reviewer/gatekeepers are primarily interested in global relative measures such as resolution-dependent percentiles and to some extent in absolute local flags for judging the support behind specific claims. Crystallographers need global relative measures to judge how well they have made use of their data, but it is the local measures, especially specific outliers, that are crucial to helping them to achieve a more accurate structure and to avoid making any dubious claims in poor local regions (such as an invisible inhibitor). End users need absolute global measures to choose between structures and absolute local measures to judge the reliability of the particular features they find of interest. Because of the importance of improving and evaluating the accuracy of individual details of biological importance, both in each structure and in the database as a whole, we have chosen in MolProbity to emphasize calculation and user-friendly display of local indicators. We have also tried to minimize ‘false alarms’, so that a flagged outlier is almost always worth a close look. 5.2. Impact on database quality Since MolProbity was first made available in late 2002, serious user work sessions (performing some operation on an input coordinate file) have multiplied by a large factor each year, with a cumulative total that is now approaching 100 000 by thousands of distinct users. In addition, many companies and structural genomics centers run their own MolProbity servers internally and some aspects have been incorporated into other software or meta-servers. 80% of MolProbity input files are uploaded, presumably by working structural biologists, and the rest are fetched from databases, presumably by end users. Those end users also include students, since MolProbity is increasingly being used for instructional exercises in bio­chemistry classes from high school to graduate level. MolProbity’s unique feature is clash analysis from all-atom contacts, which provides sensitive new evaluation independent of refinement targets. Not surprisingly, the average clashscore remained constant (either globally or by resolution) up to 2002, since there was then no feasible way of targeting or even measuring all-atom clashes. The percentage of incorrect Asn/Gln/His flips also remained level or rose slightly prior to 2003, despite the availability of a hydrogen-bond-based system in WHAT IF (Hooft et al., 1996 ▶), and even while refinement methods, automation and Ramachandran and rotamer quality all improved. To evaluate the contribution MolProbity has made to crystallographic model quality in general, we have therefore plotted clashscore and Asn/Gln/His flips as a function of time in Fig. 8 ▶, with separate linear fits before and after the end of 2002. Gratifyingly, in both cases there is a clear trend of improvement since 2003. Median values also improve very steadily over that period. Anecdotal evidence indicates that this trend is mainly a consequence of thorough adoption of MolProbity-based methods by a small but growing fraction of crystallographers and there is therefore still much scope for further improvement in the future. 6. MolProbity availability MolProbity is freely available for download from http://molprobity.biochem.duke.edu for use as a local server. This option requires either Linux or MacOSX, together with PHP and Apache. Instructions for installing MolProbity locally are included with the download. Having a local install allows users to access the MolProbity analysis tools without internet access, as well as allowing companies with privacy or confidentiality concerns to use MolProbity. However, one of the most significant advantages to having a local installation of Mol­Probity is access to command-line tools. These tools provide access to the major analysis tools in MolProbity without having to use the web interface. Also, several scripts are included which allow users to run MolProbity analysis on a set of files rather than just one at a time. Some of the more useful command-line scripts include the following: scripts for adding H atoms, with or without flips, a script for obtaining overall scores for a set of files and a script for calculating a residue-by-residue analysis of a structure. For users of the PHENIX crystallography system (Adams et al., 2002 ▶, 2009 ▶), a number of the main MolProbity quality-analysis tools have been incorporated directly into PHENIX and are accessible through command-line tools or in the PHENIX GUI, including REDUCE, PROBE, RAMALYZE, ROTALYZE, CBETADEV and CLASHSCORE. Currently, only tabular results are provided; we are exploring the possibility of incorporating KiNG and validation visualizations into PHENIX. All of the individual programs called by MolProbity are also available, multi-platform and open source, from the software section at http://kinemage.biochem.duke.edu.
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                Journal
                Journal ID (nlm-journal-id): 0410462
                Journal ID (pubmed-jr-id): 6011
                Journal ID (nlm-ta): Nature
                Journal ID (iso-abbrev): Nature
                Title: Nature
                ISSN (Print): 0028-0836
                ISSN (Electronic): 1476-4687
                Publication date Nihms-submitted: 3 August 2016
                Publication date (Print): 18 August 2016
                Publication date PMC-release: 18 February 2017
                Volume: 536
                Issue: 7616
                Pages: 349-353
                Affiliations
                [1 ]MRC Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge Biomedical Campus, Cambridge, CB2 0QH, UK
                [2 ]Infection and Immunity, University College London, Cruciform Building 3.3, 90 Gower Street, London, WC1E 6BT, UK
                Author notes
                Correspondence and requests for materials should be addressed to LCJ ( lcj@ 123456mrc-lmb.cam.ac.uk ) or GJT ( g.towers@ 123456ucl.ac.uk ).
                [†]

                Current Address: Astex Pharmaceuticals, 436 Cambridge Science Park, Milton Road, Cambridge, CB4 0QA, UK

                Article
                Manuscript ID: EMS69207
                DOI: 10.1038/nature19098
                PMC ID: 4998957
                PubMed ID: 27509857
                SO-VID: 3b191b0a-d570-4a74-8abf-3462ba3dbef8
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