research papers
Realspace PHENIX for cryoEM and crystallography
in^{a}Molecular Biophysics and Integrated Bioimaging Division, Lawrence Berkeley National Laboratory, Berkeley, CA 94720, USA, ^{b}Department of Physics and International Centre for Quantum and Molecular Structures, Shanghai University, Shanghai 200444, People's Republic of China, ^{c}Cambridge Institute for Medical Research, University of Cambridge, Wellcome Trust/MRC Building, Hills Road, Cambridge CB2 0XY, England, ^{d}Bioscience Division, Los Alamos National Laboratory, Los Alamos, NM 87545, USA, ^{e}New Mexico Consortium, Los Alamos, NM 87545, USA, ^{f}Faculté des Sciences et Technologies, Université de Lorraine, BP 239, 54506 VandoeuvrelesNancy, France, ^{g}Centre for Integrative Biology, IGBMC, CNRS–INSERM–UdS, 1 Rue Laurent Fries, BP 10142, 67404 Illkirch, France, and ^{h}Department of Bioengineering, University of California Berkeley, Berkeley, California, USA
^{*}Correspondence email: pafonine@lbl.gov
This article describes the implementation of realspace phenix.real_space_refine program from the PHENIX suite. The use of a simplified target function enables very fast calculation, which in turn makes it possible to identify optimal datarestraint weights as part of routine refinements with little runtime cost. of atomic models against lowresolution data benefits from the inclusion of as much additional information as is available. In addition to standard restraints on covalent geometry, phenix.real_space_refine makes use of extra information such as secondarystructure and rotamerspecific restraints, as well as restraints or constraints on internal molecular symmetry. The rerefinement of 385 cryoEMderived models available in the Protein Data Bank at resolutions of 6 Å or better shows significant improvement of the models and of the fit of these models to the target maps.
in theKeywords: realspace ; cryoEM; crystallography; map interpolation; atomiccentered targets; PHENIX.
1. Introduction
Improvements in the cryoelectron microscopy (cryoEM) technique have led to a rapid increase in the number of highresolution threedimensional reconstructions that can be interpreted with atomic models (Fig. 1). This has prompted a number of new developments in PHENIX (Adams et al., 2010) to support the method, from model building (Terwilliger, Adams et al., 2018), map improvement (Terwilliger, Sobolev et al., 2018) and (Afonine et al., 2013) to model validation (Afonine et al., 2018). In this manuscript, we focus on atomic model using a map (primarily cryoEM, but the same algorithms and software are also applicable to crystallographic maps).
Model ; Watkin, 2008; Afonine et al., 2012, 2015). Firstly, the model, i.e. a mathematical construct that explains the experimental data, with an associated set of refinable parameters: in this case an atomic model with coordinates whose positions can be varied to improve the fit to the data. Seondly, the target function that links the model parameters to the experimental data: this function scores modeltodata fit and therefore guides Finally, an optimization method that changes the values of refinable model parameters such that the model agreement with the experimental data is improved. In PHENIX, gradient methods are used through LBFGS (Liu & Nocedal, 1989) for this goal. If the target function is expressed through diffraction intensities or structure factors, is usually referred to as reciprocalspace, or Fourierspace, (FSR). Alternatively, a target function may be formulated in terms of a map: a Fourier synthesis in the case of crystallography or a threedimensional reconstruction from projections in the case of cryoEM. Such is referred to as realspace (RSR). In both cases the targets are the sums over a large number of similar terms corresponding to either reflections (FSR) or map grid points (RSR). A key methodological difference is that for RSR each term depends on only a few atoms, while for FSR each term depends on all model parameters. Most modern macromolecular programs were developed for crystallographic data and therefore perform in at least as their main mode of operation (see Table 1 in Afonine et al., 2015). This work focuses on the realspace of coordinates of atomic models.
is an optimization problem and as such it requires the definition of three entities (for reviews, see Tronrud, 2004In cryoEM studies realspace ) and does not change in a fundamental way as the atomic model is improved. This is not the case for crystallography, where the experimental data are diffraction intensities, and the associated and vital phase information has to be obtained indirectly. In crystallography, obtaining the best phases typically involves their calculation from atomic models, in turn making the resulting maps modelbiased (see, for example, Hodel et al., 1992). Although FSR methods are predominant in crystallographic RSR is attractive in some contexts as it makes it possible to refine parts of the model locally and fast, and model incompleteness does not influence as it does for FSR (Lunin et al., 2002). For this reason RSR has been particularly popular in the context of interactive modelbuilding software such as FRODO, O (Jones, 1978; Jones et al., 1991), MAIN (Turk, 2013) and Coot (Emsley & Cowtan, 2004; Emsley et al., 2010).
is a natural choice because a threedimensional map is the output of the singleparticle imagereconstruction method (see, for example, Frank, 2006In the case of cryoEM an atomic model can also be refined using a reciprocalspace target. This can be achieved by converting the map into Fourier coefficients. These Fourier coefficients can then be used in reciprocalspace et al., 2011; Baker et al., 2013; Brown et al., 2015). We note, however, that unless the map is converted to the full corresponding set of Fourier coefficients (and not a subset containing only a sphere limited to the stated resolution) this conversion may not be lossless.
using standard protocols that are well established for crystallographic structure (see, for example, ChengTo address the emerging structurerefinement needs of the rapidly growing field of cryoEM, the phenix.real_space_refine program (Afonine et al., 2013), which is capable of the of atomic models against maps, has been introduced into the PHENIX suite. It is not limited to cryoEM and can also be used in crystallographic (Xray, electron or neutron). In this paper, we describe the implementation of the phenix.real_space_refine program and demonstrate its performance by applications to simulated data and to cryoEM models in the PDB (Bernstein et al., 1977; Berman et al., 2000) and corresponding maps in the EMDB (Henrick et al., 2003). This is a work in progress, and further details and advances will be reported as the program evolves. To date, phenix.real_space_refine has been used in a number of documented structural studies (see, for example, Fischer et al., 2015; ShalevBenami et al., 2016; Chua et al., 2016; Ahmed et al., 2016; Yang et al., 2016; Gao et al., 2016; Chen et al., 2016; Bhardwaj et al., 2016; Lokareddy et al., 2017; Hryc et al., 2017; Ahmed et al., 2017; Demo et al., 2017; Paulino et al., 2017; Liu et al., 2017).
2. Methods
2.1. flowchart
Fig. 2 shows the modelrefinement flowchart as it is implemented in phenix.real_space_refine. This is very similar to the reciprocalspace workflow implemented in phenix.refine (see Fig. 1 in Afonine et al., 2012).
The program begins by reading a model file, in PDB or mmCIF format, map data (as an actual map in MRC/CCP4 format or as Fourier map coefficients in MTZ format) and other parameters, such as resolution (if a map is provided) or additional restraint definitions for novel ligands, internal molecular symmetry (e.g. NCS in crystallography) or secondary structure. Once inputs have been read, the program proceeds to calculations that constitute a set of tasks repeated multiple times (macrocycles). Tasks to be performed during the are defined by the program automatically and/or by the user. In its default mode the program will only perform gradientdriven minimization of the entire model. Other nondefault tasks allow optimization using simulated annealing (SA; Brünger et al., 1987), morphing (Terwilliger et al., 2013), rigidbody (see Afonine et al., 2009 and references therein) and systematic residue sidechain optimizations using grid searches in torsion χangle space (Oldfield, 2001). Parts of the model related by internal symmetry are determined automatically, if available, or can be defined by the user. In the presence of such internal symmetry, restraints or constraints can be applied between the coordinates of related molecules. The operators relating molecules can also be refined. The result of i.e. the refined model, is output as a file in PDB or mmCIF format.
Central to almost all tasks performed within a i.e. efficient convergence to an improved model. Also of the same importance is the assessment of progress by quantifying model quality and the goodness of modeltomap fit throughout the entire process. Some relevant points are discussed below.
macrocycle is the target function. Its choice is the key for the success of2.2. target function
Macromolecular cryoEM or crystallographic experimental data are almost always of insufficient quality to refine parameters of atomic models individually. To make
practical, restraints or constraints are almost always used in order to incorporate extra information into and the corresponding procedures are called restrained or In the target function is a sum of databased and restraintsbased components:The first term scores the modeltodata fit and the second term incorporates a priori information about the model. The weight w_{restraints} balances the contribution of restraints to maximize the modeltodata fit while also obeying the a priori information, and an optimal choice of its value is crucial. does not change the target function but rather changes (reduces) the set of independent parameters that can vary. Examples include rigidbody the use of a riding model (Sheldrick & Schneider, 1997) to parameterize the positions of H atoms in or the implementation of RSR by Diamond (1971) using torsion angles as variables.
2.2.1. Modeltomap target (T_{data})
In RSR, the T_{data} term scores the fit of the model being refined to a target map. In cryoEM the map is a threedimensional reconstruction, while in crystallography it may be, for example, a 2mF_{obs} − DF_{model} map (Read, 1986).
It is possible to express the difference between the two maps in the integral form (see, for example, Diamond, 1971)^{1}
For (2) we suppose that the original target map is optimally scaled to the model map (Diamond, 1971; Chapman, 1995). In the following, we will consider the target to be essentially unchanged by manipulations that shift its value by a constant or a scale factor, as such manipulations do not change the position of the minimum of the target. If the Euclidean norms of ρ_{tar}(r) and ρ_{calc}(r) are conserved during [i.e. if = constant, as will be the case when the target map itself does not change, and if = constant, which will be true if the overlap of atomic densities does not change] then minimization of (2) is equivalent to minimization of the anticorrelation target, which does not need the maps to be optimally scaled,
Assuming the target ρ_{tar} and modelcalculated ρ_{calc} maps are provided on the same grid, a continuous integration in (2) and (3) can be replaced with a numeric integration over the regular grid on which the maps are available (see, for example, Diamond, 1971),
or
respectively. The set G of grid nodes used to calculate the targets (i.e. the integration volume) is either the whole map or an envelope (mask) surrounding the whole atomic model or its part that is subject to refinement.
To match the finite resolution of the target map in (5) accurately, several steps are required to compute the model map. Firstly, the model map distribution is calculated using one of the available approximations (Sears, 1992; Maslen et al., 1992; Waasmaier & Kirfel, 1995; GrosseKunstleve et al., 2004; Peng et al., 1996; Peng, 1998). A set of Fourier coefficients is then calculated from the distribution up to the resolution limit specified by the target map.^{2} Finally, a subset of these coefficients is used to calculate the model Fourier synthesis ρ_{calc} that can then be used in (5). This synthesis is a representation of a model image at a given resolution. A typical may require hundreds or even thousands of such modelimage calculations, which are computationally expensive, involving two Fourier transforms.
Alternatively, a model map may be calculated from the atomic model directly as a sum of individual contributions of M atoms, with each contribution being a Fourier image (or its approximation) of the corresponding atom at a given resolution (see, for example, Diamond, 1971; Lunin & Urzhumtsev, 1984; Chapman, 1995; Mooij et al., 2006; Sorzano et al., 2015). While this is much faster than the previous method, it may be less accurate and still be computationally expensive, especially for large models.
A numeric integration over the whole map (5) can be simplified by the integration exploring the volume directly around the atomic centers r_{m}, m = 1, … M:
Here, are the values interpolated from the nearby grid node values ρ_{tar}(n) to the atomic centers r_{m} (Appendices A and B). Neglecting the local variation of the model map at the atomic centers (e.g. at low resolution) and thus supposing ρ_{calc}(r_{m}) ≃ constant for all m, the target simplifies further as (Rossmann, 2000; Rossmann et al., 2001)
The hypothesis ρ_{calc}(r_{m}) ≃ constant seems to be reasonable at low resolution, when a calculated map can be considered to be rather flat. On the other hand, minimization of (7) is essentially a fitting of atoms to the nearest peaks of the target map, which seems to be appropriate at high resolution as well. We show below (§3) that indeed this target function is efficient over a large resolution range; Appendix B supports this observation through the equivalence of targets (7) and (5) when taking map blurring/sharpening into account. If the difference in atomic size cannot be neglected, this target function can be modified to
where w_{m} is an atomspecific weight. For example, w_{m} can be the electron number of the corresponding atom or it can be set negative for O atoms of Asp and Glu residues in the case of cryoEM or for atoms that have a negative scattering length (such as hydrogen) in the case of neutron diffraction data. Clearly, for most of the macromolecular structures under consideration here these atomcentered targets are nearly the same, and for simplicity in the following we refer only to (7) unless otherwise stated. The computational cost of (7) is proportional, with a very small coefficient, to the number of atoms and therefore these targets are much faster to calculate compared with (5), making it advantageous for the of large models. Unlike (4) or (5), the computational cost of (7) or (8) does not depend on the resolution or mapsampling rate. Essentially, target (5) optimizes the fit of the shape between modelcalculated and experimental maps, while target (7) simply guides atoms to the nearest peaks in the experimental map. Therefore, using (5) can produce a more accurate modeltomap fit. An optimal protocol may consist of using target (7) for routine refinements and using (5) for the final refinement.
2.2.2. Restraints (T_{restraints})
In T_{restraints} with some weight (1). This extra term restrains model parameters to be similar, but not necessarily identical, to some reference values. At high to medium resolutions of approximately 3 Å or better, a standard set of restraints as implemented in PHENIX includes (GrosseKunstleve & Adams, 2004) restraints on lengths and angles, dihedral angles, planarity and restraints, and a nonbonded repulsion term. However, at lower resolutions the amount of experimental data is insufficient to preserve the geometry characteristics of a higher level of structural organization (such as secondary structure), and therefore including extra information (restraints or constraints) to help to produce a chemically meaningful model is desirable. These extra restraints or constraints may include similarity of related copies (NCS in the case of crystallography), restraints on secondary structure and restraints to one or more external reference models (for implementation details in PHENIX, see Headd et al., 2012, 2014; Sobolev et al., 2015). phenix.real_space_refine can use the following extra restraints and constraints.
extra information is introduced through the term

2.2.3. Relative weight
The relative weight w_{restraints} is chosen such that the model fits the map as well as possible while maintaining reasonable deviations from ideal lengths and angles. In PHENIX, w_{restraints} for RSR is determined by systematically trying a range of plausible values and performing a short for each trial value. A similar procedure in FSR would be very computationally expensive because for each trial value of w_{restraints} the whole structure would need to be used. In RSR this is computationally feasible using (7) but not (5). The weightcalculation procedure implemented in phenix.real_space_refine splits the model into a set of randomly chosen segments, each one a few residues long. After trial refinements of each segment with different weights, the best weight is defined as the one that results in a model possessing reasonable bond and angle rootmeansquare deviations (r.m.s.d.s) and that has the best modeltomap fit among all trial weights. The obtained array of best weights for all fragments is filtered for outliers and the average weight is calculated and defined as the best weight for the final This calculation typically takes less than a minute on an ordinary computer and is independent of the size of the structure or map. Instead of computing an average single weight for the entire model, this protocol can be extended (work in progress) to calculate and use different weights for different parts of the map, accounting for variations in local map quality.
2.3. Evaluation of progress and results
It is recognized that model validation (see, for example, Brändén & Jones, 1990; Read et al., 2011; Wlodawer & Dauter, 2017) is a critical step in and a number of corresponding tools have been developed in crystallography (see, for example, Chen et al., 2010; Read et al., 2011; Gore et al., 2017; Williams et al., 2018 and references therein) and some in cryoEM studies (see, for example, Henderson et al., 2012; Tickle, 2012; Lagerstedt et al., 2013; Barad et al., 2015; Pintilie et al., 2016; Joseph et al., 2017, Afonine et al., 2018). Generally, the process consists of assessing data, model quality and modeltodata fit quality, and is performed locally and globally. At the stage of refining a model we assume that the intrinsic data quality has already been evaluated, and only model quality and modeltodata fit need to be monitored.
The methods and tools to evaluate the geometric quality of a model are the same in crystallography and in cryoEM. For example, the PHENIX comprehensive validation program provides an extensive report on model quality, making extensive use of the MolProbity validation algorithms (Chen et al., 2010; Richardson et al., 2018). In crystallography, the modeltodata fit is quantified by crystallographic R and R_{free} (Brünger, 1992) factors, which are global reciprocalspace metrics. In cryoEM, model and data validation is currently performed by the comparison of complex Fourier coefficients in resolution shells; these coefficients are calculated from the model and from the full map or halfmaps; different masks can be applied prior to calculation of these coefficients. Also in real space the modeltodata fit can be evaluated locally or globally by various correlation coefficients between a modelcalculated map and the experimentally derived map (Urzhumtsev et al., 2014; Afonine et al., 2018). Some of these tools are used in §3.2, where models extracted from the PDB are refined against experimental cryoEM maps.
3. Results
3.1. Test refinements with simulated data
Below, we illustrate the performance of with geometry restraints included with optimal weights and data term (7). We begin with several numerical tests using simulated data. The advantage of such tests is that one can study individual effects in a setting where the answer is known.
at different resolutions and map sharpnesses and using atomic models with various amounts of error in the coordinates. All refinements were performed using target (1)3.1.1. Preparing simulated data
A model from the PDB (PDB entry 3vb1) was chosen as a test model. The following manipulations were made to this model prior to test calculations: (i) the model was placed in a sufficiently large P1 (ii) alternative conformations were replaced with a single conformation and (iii) model geometry was regularized using the phenix.geometry_minimization tool until convergence. In the following, we refer to this model as a reference model. Several Fourier maps at different resolutions d_{high} (1, 2, 3, 4, 5 and 6 Å) were calculated from the reference model considering three different overall B factors of 0, 100 and 200 Å^{2}; these maps mimic ρ_{tar} (18 maps in total). The maps were calculated on a grid with the step equal to d_{high}/4. Additionally, we calculated the same maps on a much finer grid with a step of 0.2 Å; the same step was used for all maps independent of their resolution.
3.1.2. of the exact reference model
Firstly, we refined the reference model against finiteresolution maps calculated from this model, as described in §3.1.1. While the reference model corresponds to the minimum of (5), this is not the case for (7) because map peaks in finite resolution Fourier images do not necessarily correspond to atomic centers. Therefore, it is expected that using (7) may shift the model from its original, correct, position. The goal of this test is to provide an estimate of the magnitude of these shifts after For each refined model we calculated the rootmeansquare deviation (r.m.s.d.) from the reference model. Fig. 3 summarizes the result of this test. We observe the following.
3.1.3. of perturbed reference models
Here, we describe tests that are similar to those in §3.1.2 except that instead of refining the reference model we refined perturbed reference models. These perturbed models were obtained by running moleculardynamics (MD) simulations using the phenix.dynamics tool until a prescribed r.m.s.d. compared with the reference model was achieved. Given the stochastic nature of MD, it is possible to obtain many different models with the same r.m.s.d. from the reference model. Owing to the limited convergence radius of and the finite resolution of the data, of these models will not produce exactly the same refined models. Therefore, to ensure more robust statistics, for each chosen r.m.s.d. we generated an ensemble of 100 models. The r.m.s.d. values between the perturbed and reference models were chosen to be 0.5, 1.0, 1.5 and 2.0 Å. We then refined each of these 100 × 4 = 400 models against each of 18 maps (§3.1.1) calculated on a grid with a spacing of d_{high}/4. For each refined model (from 100 × 4 × 6 × 3 = 7200 refined models) we calculated the r.m.s.d. from the reference model and then the average r.m.s.d. over the corresponding ensemble of 100 models. Fig. 4 summarizes the results of this test. We observe the following.

3.2. using data from the PDB and EMDB
3.2.1. CryoEM maps
Threedimensional reconstructions (cryoEM maps) represent the et al., 2017). Furthermore, such moieties may be susceptible to radiation damage and therefore may have a weaker footprint in the reconstructions. This may have an implication for realspace that uses target (7) [or (5) if the form factors do not reproduce the negative features] because this target favors atomic shifts towards positive map peaks. To investigate this effect, we surveyed map values at atomic positions considering reconstructions at 3 Å or better and map–model correlation better than 0.8. This selected nine (map, model) pairs. Prior to calculations, we normalized all selected maps to have zero mean value and a standard deviation of 1. Fig. 5(a) shows the distribution of map values for four groups of atoms: mainchain atoms, sidechain O atoms of Asp and Glu residues that may be negatively charged (OD1, OD2, OE1 and OE2), sidechain atoms of Arg and Lys residues that may be positively charged (NH1, NH2 and NZ) and all other sidechain atoms. We observe that sidechain O atoms of Asp and Glu residues indeed have systematically weaker map values, with about 8% of atoms having values below a threshold of −1 times the r.m.s. of the map. Negative map values for all other kinds of atoms are greater than −0.5 r.m.s. and may be considered as noise. We note that the size and flexibility of Asp, Glu, Arg and Lys side chains are likely to contribute to systematically weaker densities for these side chains. We repeated the same analysis for maps of lower resolution (3–4 Å; Fig. 5b). Here, the number of reliably observed atoms with negative features in the map is less than 1%.
of the sample. Therefore, these maps are expected to have negative features around negatively charged moieties such as aspartate and glutamate (see, for example, HrycThis analysis shows that for the majority of cryoEM models (resolution of 3 Å or worse) the concern about negative features in the map is rather small and is unlikely to affect the results of significantly. On the other hand, the rapidly increasing number of higher resolution cryoEM maps (better than 3 Å) is likely to highlight the limitation of (7) and to demand further improvements of the target [such as using (8) with properly chosen weights].
using (7)3.2.2. Default refinement
In order to test the suggested methods and demonstrate their utility, we rerefined 385 cryoEM models from the PDB that are reported at a resolution of 6 Å or better, that have model–map correlation greater than 0.3 and that contain only residues and ligands that are known to the PHENIX restraint library. A number of metrics were analyzed: the modeltomap CC_{mask} calculated in the map region around the model (for an exact definition, see Afonine et al., 2018), the number of Ramachandran plot and rotamer outliers, excessive C^{β} deviations, the MolProbity clashscore (Chen et al., 2010) and the EMRinger score (Barad et al., 2015; calculated for 277 entries with maps at 4.5 Å resolution or better), all calculated for the initial models from the PDB and for the models after Default parameters were used in all refinements that, in addition to standard restraints, also include rotamer, C^{β} deviations and Ramachandran plot restraints, as well as NCS constraints where applicable (see §2.2.2). The program ran successfully, generating a refined model for all cases and highlighting the robustness of the algorithms and their implementation. In all cases we observe a substantial overall improvement of geometry metrics, such as reduced or fully eliminated Ramachandran plot and rotamer outliers, C^{β} deviations and MolProbity clashscore, as well as improvement of the modeltodata (map) fit (Fig. 6). Clearly, the removal of some outliers can be attributed to the use of rotamer, C^{β} deviations and Ramachandran plot restraints. Therefore, we also used an orthogonal validation metric to assess model improvement: EMRinger (Barad et al., 2015). We observe that the overall average EMRinger score for the initial models is 1.73 and that for the refined models is 2.26. The improvement of the EMRinger score for the refined models indicates that the aminoacid side chains are more chemically realistic and better fit the map. Detailed validation or analysis of individual results is outside the scope of this work, but will be important in the future to assess the impact of stereochemical restraints on models, particularly when the starting models are of very poor quality.
3.2.3. against sharpened maps
Our tests using simulated data (§3.1) have indicated that map sharpening or blurring may be useful in To investigate this with the real experimental data we performed the following test. We selected models similarly to as described in §3.2.2, additionally requiring that independent halfmaps had also been deposited by the researcher. This resulted in 76 entries. We performed test refinements against the first of the two halfmaps and evaluated the refined modeltodata fit using the original second halfmap that had not been used in any calculations. In two independent refinements, the first halfmap was taken either as deposited or modified with phenix.auto_sharpen (Terwilliger, Sobolev et al., 2018) to automatically optimally sharpen or blur the map. Fig. 7 shows the model–map correlation CC_{mask} for models refined against the original and sharpened first halfmaps; the original second halfmaps were used to compute the correlations. Overall, the CCs across all 76 cases are similar for against the original first halfmap and the sharpened first halfmap. The refined models fit slightly but systematically better when using sharpened maps if the original model–map CC is low (<0.5) and systematically slightly worse if the original model–map correlation is higher (CC > 0.5). This agrees with the observation that target (7) allows the removal of large errors but may slightly distort exact models (§3.1.2). Also, we note that the MolProbity scores for models refined against sharpened maps are systematically better, but the difference is small.
3.2.4. Rerefinement of the TRPV1 structure
The structure of the TRPV1 ion channel (PDB entry 3j5p; EMDB code EMD5778) was determined by singleparticle cryoEM (Liao et al., 2013) at a resolution of 3.28 Å. The model was built manually and was not subjected to As the model was not refined it contains substantial geometry violations: the clashscore is high (∼100) and about one third of the side chains are identified as rotamer outliers (Table 1). More recently, the better resolved part of this structure has been reevaluated using the same data (Barad et al., 2015; PDB entry 3j9j; ankyrin domain not included). This involved some rebuilding and using algorithms implemented in the Rosetta suite (DiMaio et al., 2015). The resulting model has a much improved clashscore and EMRinger score (Barad et al., 2015) and no rotamer outliers, yet the number of Ramachandran plot outliers has increased compared with the original model (Table 1). We performed a of PDB entry 3j5p (the portion that matches PDB entry 3j9j) using phenix.real_space_refine with all default settings and automatically, with no manual intervention, using the original, deposited map. The took about 3 min on a Macintosh laptop.^{3} Overall, the refined model is similar to PDB entry 3j9j (virtually no rotamer or Ramachandran plot outliers), the EMRinger score is improved further and the modeltomap correlation (CC_{mask}) is increased compared with both PDB entries 3j5p and 3j9j. Notably, the MolProbity clashscore decreased from 100.8 to 5.6 as a result of the resolution of numerous steric clashes (Fig. 8).

Modeling experimental data at resolutions below atomic (around 1–1.5 Å and better) may not be unambiguous (Terwilliger et al., 2007). Therefore, it may be instructive to perform several trial refinements, each using the exact same settings but different (perturbed) input models. Here, we generated an ensemble of 100 perturbed models by running moleculardynamics simulation (using phenix.dynamics tool) until the r.m.s. deviation between the starting and simulated models reached 3 Å (Fig. 9a). We then refined all models using phenix.real_space_refine until convergence. This resulted in 100 refined models that are overall similar but vary locally (Fig. 9b). This highlights the fact that a singlemodel representation of experimental data is an approximation and should not be taken too literally (for example, when it comes to measuring and reporting distances between atoms). Also, this test demonstrates the rather large convergence radius of phenix.real_space_refine: the average map–model correlation (CC_{mask}) across all 100 refined models is 0.80, with the smallest and largest values being 0.79 and 0.81.
4. Conclusions
PHENIX tool, phenix.real_space_refine, that was specifically designed to perform such realspace refinements. RSR is a natural choice for cryoEM, unlike crystallography, where realspace methods are complementary to Fourierspace and are somewhat limited since crystallographic maps are almost always modelbiased. Nevertheless, while this work was inspired by rapid advances in the field of cryoEM and the increasing number of threedimensional reconstructions that allow atomic models to be refined (as opposed to rigidbody docked), the implementation is not limited to cryoEM and crystallographic maps can also be used.
of an atomic model against a map is increasingly important as the technique of cryoEM rapidly develops. We have described the algorithms implemented in a newThe proposed realspace for the final cycles and efficient modeling of atomic displacements.
procedure is fast owing to the use of an atomcentered target function that has been shown to be efficient at all tested resolutions from 1 to 6 Å. Several options for key calculation steps, such as map interpolation, gradient calculation and preliminary processing of the target (experimental) map, are available with the default choices selected on the basis of extensive test calculations. The realspace algorithm includes a fast and efficient search for the optimal relative weight of restraints, a procedure that is extremely challenging for reciprocalspace The algorithm is robust, with no failures for any of the cryoEM maps tested. For all test model refinements improvements are observed; in some cases these improvements are significant. Future developments of the algorithms will include methods to account for local variation in map resolution and a fast and accurate calculation of (5)APPENDIX A
Realspace targets and convolution
We show here that if the atoms all have the same shape, sampling a map at the positions of atomic centers, as in (7), can be made equivalent to the correlation function obtained by integrating or summing over the product of calculated and target densities, as in (3) or (5). Consider a simplified structure composed of a single atom. Looking for its best position according to (3) or (5) corresponds to seeking the position where the weighted average of the target map values (weighted by the atomic shape) inside a sphere centered at the trial atomic position is maximal. This calculation and check for the maximal value could be performed point by point. Alternatively, one can first calculate such averages for all grid points, replace the initial map values by these sums and then simply choose the maximum. From a mathematical point of view this averaging can be considered as a convolution and, if calculated simultaneously for the whole map, can be performed rapidly (Leslie, 1987; Urzhumtsev et al., 1989). Checking the values of the averaged, i.e. blurred, map for their maximum corresponds to using targets (7) or (8). Below, we give a formal interpretation of these realspace targets.
Let Z_{0}f_{0}(s; B_{0}) be a scattering factor of some isotropic atom characterized by a B_{0} value and the electron number Z_{0}. Let Z_{0}ρ_{0}(r; B_{0}) be an image of this atom in the corresponding model map if it is placed at the origin. Both Z_{0}f_{0}(s; B_{0}) and Z_{0}ρ_{0}(r; B_{0}) are spherically symmetric and related by Fourier transformation. If a hypothetical structure is composed of a single atom positioned at r_{0}, the corresponding model map is
which can be seen as a convolution of a point scatterer at position r_{0} with the atomic shape. Owing to the spherical symmetry of ρ_{0}(r; B_{0}), the target function (3)
can be interpreted as a convolution of the target map with ρ_{0}(r; B_{0}) taken at point r_{0}. Let {F_{tar}(s)} be the set of Fourier coefficients corresponding to the target map ρ_{tar}(r). By the convolution theorem, (10) is equal to the Fourier series of the corresponding Fourier coefficients,
Here, the map ρ_{tar_0}(r; B_{0}) is a Fourier series calculated with the coefficients F_{tar}(s)f_{0}(s; B_{0}). In other words, instead of blurring the model map with the atomic shape and calculating the pointbypoint product of the two maps, one may blur the experimental map and leave the model map unblurred, i.e. as a point map.
For a multiatom model
At resolutions typical for biocrystallography the shapes of macromolecular atoms are similar. If we additionally suppose that all of the atoms of the structure have the same (or similar) atomic displacement parameters B_{m} = B_{0}, then
using the function ρ_{tar_0}(r; B_{0}) calculated once in advance. This shows that in calculating (8) we in fact implicitly sharpen the target map using ρ_{tar}(r) instead of ρ_{tar_0}(r; B_{0}). Even when using (8) as the target, it is likely to be beneficial to choose an optimal sharpening factor, just as the signal in map correlations can be improved.
If the difference in atomic B values cannot be neglected, one can calculate in advance a few maps ρ_{tar_0}(r; B_{k}) for a range of Bfactor values B_{k}, k = 1, …, K, and use the appropriate ρ_{tar_0}(r_{m}; B_{k}) for a particular atom m,
If the atomic shapes are significantly different, as is the case for H atoms in neutron maps or negatively charged side chains in cryoEM maps at high resolution, the approximation (13) can be used with Z_{m} being a negative value, or the target map can be convoluted with the respective atomic shape (which can be negative) before the sum over the relevant atoms is calculated.
APPENDIX B
Threedimensional interpolation used
B1. General remarks
Using the atomcentered targets (7) and (8) requires an efficient and accurate interpolation of the maps calculated on threedimensional regular grids. Not only the interpolated function values are needed but also the gradient. In this work, two options have been considered: trilinear (https://en.wikipedia.org/wiki/Trilinear_interpolation) and tricubic (https://en.wikipedia.org/wiki/Tricubic_interpolation). Both interpolation procedures, including the gradient calculation, are available through the cctbx software library (GrosseKunstleve et al., 2002). Trilinear interpolation is the simplest and the easiest to understand. Its major disadvantage is that, by construction, the minimum of the interpolated function is always at one of the corners of the box of interpolation. Since the map grid step is usually larger that the accuracy of atomic positions required, this can impact the optimization procedure and results. For this reason, the tricubic interpolation has been chosen as the default method. Other interpolations have also been tried but are not discussed in this work. In the following, we first describe the interpolation procedures inside the unit cube and then adapt the results and the procedures to an arbitrary regular tridimensional grid.
B2. Tricubic interpolation inside a unit cube
Let us consider an interpolation inside a unit cube, 0 ≤ x < 1, 0 ≤ y < 1, 0 ≤ z < 1. We search for a function in the form
This function is cubic with respect to any of its three variables, giving expressions for the partial derivatives
One can calculate all 64 coefficients in advance and use them for further calculations (Lekien & Marsden, 2005). Alternatively, one can build an interpolation for the coordinate x, then for the coordinate y and finally for the coordinate z (in any order of variables). To build interpolation (16) eight values from the cube corners are insufficient and either values from the neighboring grid points (the corners of the neighboring cubes) or derivatives in the corners of the unit cube are required. In the following, f_{pqr} with integers p, q, r stand for the grid function values f(p, q, r).
Firstly, we define a cubic interpolation
of a function f(x) of one variable in the interval (0, 1) for which its values are known in the integer grid nodes, f_{−1} = f(−1), f_{0} = f(0), f_{1} = f(1), f_{2} = f(2). We notate this interpolation by int3(x; f_{−1}, f_{0}, f_{1}, f_{2}) and its derivative by gint3(x; f_{−1}, f_{0}, f_{1}, f_{2}), as they are called in cctbx:
The coefficients of this approximation are derived below. The procedure of the tricubic interpolation then becomes a suite of operations:
where p and q are integers −1, 0, 1 or 2, then
and finally
The last three expressions are redundant and only one of them can be calculated. However, the expressions previous to them are necessary to calculate partial derivatives as
The coefficients of the onedimensional cubic interpolation (17) can be chosen using various considerations. The possibility taken as the default choice in the current software version is to build a cubic function such that it and its first derivative coincide with f(x) and with f′(x), respectively, at points 0 and 1. Since the f′(0) and f′(1) values are unknown, they are estimated as
B3. Tricubic interpolation on a regular grid
Now let a function f(x, y, z) be defined in fractional coordinates on a grid with the step d_{x} = N_{x}^{−1}, d_{y} = N_{y}^{−1}, d_{z} = N_{z}^{−1}. Let us consider a point (x_{g}, y_{g}, z_{g}) and a box of this grid that this point belongs to,
with n_{x}, n_{y}, n_{z} being integer numbers. We introduce intermediate variables rescaling this `box' to a unit cube as
and apply the procedure (19)–(21) described above. According to (26), the respective derivatives are
Footnotes
^{1}It is a widely known consequence of Parseval's theorem [see, for example, Diamond (1971) or Arnold & Rossmann (1988)] that this is equivalent to a leastsquares target between a full set of the corresponding complex Fourier coefficients; CNS (Brünger et al., 1998) describes this as a `vector LS target'.
^{2}In crystallography, the set of the calculated Fourier coefficients usually coincides with that of the experimentally measured intensities.
^{3}For comparison of the CPU required by the two methods, we refer to Kim & Sanbonmatsu (2017).
Funding information
This work was supported by the NIH (grant GM063210 to PDA, RJR and TT) and the PHENIX Industrial Consortium. This work was supported in part by the US Department of Energy under Contract No. DEAC0205CH11231. AU acknowledges the support and the use of resources of the French Infrastructure for Integrated Structural Biology FRISBI ANR10INBS05 and of InstructERIC. RJR is supported by a Principal Research Fellowship funded by the Wellcome Trust (Grant 082961/ Z/07/Z).
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