Cyclic Automated Model Building (CAB) Applied to Nucleic Acids

: Obtaining high-quality models for nucleic acid structures by automated model building programs (AMB) is still a challenge. The main reasons are the rather low resolution of the di ﬀ raction data and the large number of rotatable bonds in the main chains. The application of the most popular and documented AMB programs (e.g., PHENIX.AUTOBUILD, NAUTILUS and ARP / wARP) may provide a good assessment of the state of the art. Quite recently, a cyclic automated model building (CAB) package was described; it is a new AMB approach that makes the use of BUCCANEER for protein model building cyclic without modifying its basic algorithms. The applications showed that CAB improves the e ﬃ ciency of BUCCANEER. The success suggested an extension of CAB to nucleic acids—in particular, to check if cyclically including NAUTILUS in CAB may improve its e ﬀ ectiveness. To accomplish this task, CAB algorithms designed for protein model building were modiﬁed to adapt them to the nucleic acid crystallochemistry. CAB was tested using 29 nucleic acids (DNA and RNA fragments). The phase estimates obtained via molecular replacement (MR) techniques were automatically submitted to phase reﬁnement and then used as input for CAB. The experimental results from CAB were compared with those obtained by NAUTILUS, ARP / wARP and PHENIX.AUTOBUILD.


Introduction
Automated model building (AMB) programs try to replace the visual interpretation of the three-dimensional electron density map, which is usually time consuming and subjective, with automatic procedures to speed up the structure determination process and to minimize the modelling errors.
Several successful and well-documented AMB programs are available for proteins (among others, we cite BUCCANEER [1], ARP/wARP [2], PHENIX.AUTOBUILD [3]). Equivalent tools for nucleic acids exist, but most of them are still in progress. Indeed, quite often, such AMB programs aid in detecting errors in crystallographic models [4], or extend and rebuild existing nucleotides chains [5] or perform semi-automatic building [6].
Because the number of solved nucleic acid structures is rapidly increasing, more efforts were spent recently on the specific difficulties in the electron density interpretation due to lower resolution data [7] and the large number of rotatable bonds in the main chain (two in the protein main chain, six in nucleic acids). As a consequence, the conformation at low resolution is often ambiguous, particularly for large nucleic acid structures, and is typically determined at resolutions worse than 2.5Å. It is not uncommon that phosphate and base planes are reliably located, but sugars and part of the backbone are not seen

CAB Algorithm for Locating Ligand Heavy Atoms
We suppose that a set of observed structure factor amplitudes with refined φ r phases and w r weights are available as the starting point for any AMB application. They were first obtained by applying REMO09 [14] to the test structures, and then refined via the SYNERGY approach [15]. The name of the latter procedure arises from the fact that it combines mainstream phase refinement procedures (DM by Cowtan [16]) and out-of-mainstream phase refinement techniques. SYNERGY includes free lunch [17,18], low density Fourier transform [19], vive la difference [20,21], phantom derivative [22,23] and phase-driven model refinement [24]. SYNERGY, as well as REMO09, was included in a modified version of SIR2014 [25].
In nucleic acid structures, sometimes the ligands' scattering power is a not negligible part of the total scattering power, and often ligands contain (or are constituted by) heavy atoms. If no ligand is taken into account, the final R value may be large even if the nucleic acid model is good. This is mainly due to the fact that any AMB program is more focused on building nucleic acid models than defining the ligand substructure. Because the latter contribution does not enter into the model structure factor calculation, an additional systematic discrepancy occurs between the observed structure factor amplitudes and the calculated nucleic acid amplitudes, thereby causing larger values of R (and of R f ) and, therefore, a larger distrust of the user.
We then decided to modify the standard CAB approach by searching for ligands, including heavy atoms, in the first step. In this step, another task may also be accomplished: the identification of the P atoms belonging to the nucleic acid backbone. This decision is supported by the observation that often the average phase error corresponding to φ r phases (say <|∆φ r |>) is low, even if the average phase error at the molecular replacement level is large. This is mainly due to the effectiveness of the SYNERGY step. Indeed, after SYNERGY it may be easier to locate heavy atoms and also to recognize a good percentage of the P atomic positions.
A useful premise for the location of heavy atoms is the following: the number and positions of the heavy atoms are unknown, while their atomic species are assumed to be known due to the known chemical composition of the ligands. Only atoms with an atomic number equal or larger than 20 are considered "heavy". The above condition is suggested by the following criterion: heavy atoms are not subjected to constraints or restraints during the least-squares refinement, so the optimization of their positions is feasible only if the atomic species is sufficiently heavy. If ligands contain two or more heavy atom species, we associate them with the heaviest atomic species. The following automatic algorithm is used: (i) An observed electron density map is calculated by using φ r phases and w r weights. The first candidates for being P atoms in the target structure are the P atoms of the model structure, as refined by SYNERGY. If no heavy atoms are present in ligands, steps (ii) and (iii) are skipped; (ii) The highest N peaks (where N = 30 × number of nucleotides in the target sequence) are selected and sorted with respect to the intensity (I). All peaks closer than DIST from model atoms are rejected, where DIST corresponds to the covalent radius of the heaviest species in the target. Non-crystallographic occupancy I(i)/I(1) and the heaviest atomic species in the target are associated to the ith peak; (iii) The structure parameters of 10 peaks with the largest I(i)/I(1) values are refined, one atom at a time, together with the nucleic acid model previously determined, by five REFMAC cycles. Then, a new R value is obtained. If it is smaller than the previous one, the heavy atom is accepted as a reliable candidate of the heavy atom substructure and the next peak is processed; otherwise, the procedure for locating heavy atoms stops.
At the end of the steps (ii) and (iii), a list of heavy atom candidates are available. The final R (and R f ) values and the new phase estimates (denoted by φ rh ) are expected to be better than the corresponding values obtained at the end of SYNERGY. Correspondently, the new average phase error (say <|∆ φ rh |>) is expected to be smaller.
Heavy atom candidates added to the SYNERGY model are used to calculate new structure factors by which a new electron density map is calculated. Steps ii) and iii) are repeated and a new model is obtained, which now includes the final estimates of P and heavy atoms' positions. A new phase set (φ rh ) and a new average phase error (<|∆ φ rh |>) correspond to such models.
There is a special reason why P atoms' positions are examined. In an experimental version of NAUTILUS (Cowtan, personal communication 2020), it is possible to use two new tools to improve the NAUTILUS default model building: a more extended library of nucleic acid model structures and the previous knowledge of the positions of the triples (O 3 ', P, O 5 '). A good percentage of such triples may be correctly identified when SYNERGY and the above described algorithm for heavy atom location end with a small R value. Then, a list of triples (O 3 ', P, O 5 ') is automatically passed to NAUTILUS, which may build the final model of the target nucleic acid more efficiently.

The Recursive Algorithm
We briefly summarize, in this section, the main CAB algorithms that make the application of NAUTILUS cyclic. The different characteristics of nucleic acids with respect to proteins suggest that CAB algorithms cannot be the same for the two types of structures.
We suppose that REMO09 molecular replacement techniques were applied to the test structures, and then the corresponding phases were refined by SYNERGY. Let φ r and w r be phases and weights at the end of the above procedure: they constitute the input for NAUTILUS, CAB, ARP/wARP and PHENIX.AUTOBUILD. Furthermore, let φ b and w b be phases and weights obtained after the first application of NAUTILUS. We divided the CAB procedure into the following steps: STEP 1: When a ligand contains heavy atoms, the procedure for locating them starts (see Section 2). φ rh and w rh are phases and weights corresponding to the combination of the nucleic acid model and the heavy atoms; R rh is the corresponding crystallographic residual and R frh the R free value. φ rh and w rh coincide with φ r and w r when ligand heavy atoms are not found; STEP 2: φ rh and w rh are used automatically to start the first NAUTILUS application, which provides a new molecular model of the nucleic acid. The Fourier inversion of the new model leads to a set of calculated structure factors to which the contribution of the ligand heavy atoms is added. Let R b and R fb be the corresponding crystallographic residuals, φ b and w b the corresponding model phases and weights. If R b is smaller than 0.30, then CAB stops and the model is considered not worthy of further improvement; STEP 3: The w rh and w b distributions are fitted through histogram matching, to put them on the same statistical basis. Then, the tangent is calculated, to derive a set of combined φ c phases. S C is the parameter that defines how φ rh and φ b should be combined. If the φ b phases are supposed to be reliable, then S C is expected to be large; if the user is not confident of their quality, then S C has to be small. At this stage, the quality of the φ b phases may be estimated through the R b value. We heuristically decided to linearly relate S C to R b via Equation (2) (owing to the different quality of the problem, this equation does not coincide with that used for proteins): with the conditions that if R b < 0.30, then S C = 1, and if R b > 0.5, then S C = 0.35. The reason is the following: when R b is sufficiently small, then the φ b phases are expected to be reliable and their weights deserve to stay on the same scale of the φ rh phases. If R b is large, then the contribution of the φ b phases to the tangent expression (1) has to be depleted. The weight may be applied to the φ c phases. However, if R b is large, then it is very likely that the weakly weighted φ c phases are badly estimated. Accordingly, we decided to eliminate in Equation (1) a percentage (PERC) of the φ b phases (those with lower weights) defined by the following equation: with the conditions that if R fb > 0.6, then PERC = 0.60, and if R fb < 0.35, no reflection is eliminated. Equation (3) is equivalent to assigning w b = 0 to 60% of the weakest estimates from NAUTILUS when R fb is equal or larger than 0.6, and to assigning w b = 0 to the 12% of the weakest estimates from NAUTILUS when R fb = 0.40. φ c phases and w c weights thus obtained are used as input values for the next NAUTILUS run to produce new φ b phases and w b weights, which are again combined according to Equation (1) with φ rh phases and w rh weights in up to six cyclic NAUTILUS runs. The procedure stops if R b < 0.30; STEP 4: The cyclic procedure described in STEP 3 is a useful tool that offers a variety of electron density maps to NAUTILUS algorithms, to increase the chance of a good interpretation. The six maps, however, are close to each other: a large variety of maps could make success easier. A total of 12 supplementary cycles are thus introduced in the procedure. The φ b phases and w b weights obtained at the cycle n are combined via a tangent expression with the φ c phases and w c weights obtained at the end of the (n − 1)-th tangent cycle. The procedure stops if R b < 0.30.
At the end of the above procedure, the minimum R b value is selected (and denoted as R C ); the corresponding model is considered the most accurate.
A special case, not very rare in nucleic acid crystallography, occurs when the model and target sequences are identical or differ by one nucleotide. Among the 29 test structures, seven cases (4xqz, 5ihd, 5jua, 5nt5, 5t4w, 2a0p, 5tpg) have identical nucleotides and four cases (3n4o, 1iha, 2fd0, 4enc) differ by one nucleotide. In the latter case, the lengths of the two sequences may be the same or may differ by one nucleotide. If R rh < 0.35 and R rh < R C , then SYNERGY and STEP 1 models are preferred to the CAB model (see Section 4).

Applications
In a previous paper [26], we selected from the Protein Data Bank (PDB) 38 nucleic acid structures for which phase solution attempts were made via molecular replacement (MR) techniques: we downloaded the observed diffraction data, unit cell dimensions, the space group symmetry, nucleotide sequence, and the structural models. We submitted the test structures to default runs of REMO09; for nine of them, REMO09 did not provide a sufficiently good model (i.e., the average phase error for such structures was larger than 80 • ). The remaining 29 structures, quoted in Table 1, are used as test cases for our applications: the first 16 of them are DNA, the other 13 are RNA fragments. For each structure, we show their PDB code (PDB), the space group (SG), and the data resolution (RES) in Table 1. The number of nucleotides in the asymmetric unit is reported in the form n·N, where n is the number of chains in the asymmetric unit and N is the number of nucleotides per chain. N is replaced by a sum of two numbers if two chains with a different type or number of nucleotides are present. The model used in the MR step (column model) is reported as p·CODE, where CODE is the PDB code of the molecular fragment and p is the number of fragments originally used in the MR process. The information on the ligands is given in the corresponding column, in the form of m·CODE, where CODE is the PDB code of a ligand and m is the number of ligands in the asymmetric unit of the target structure. The chemical formula for each ligand CODE is also specified at the bottom of Table 1, from which the possible presence of heavy atoms may be deduced.
Model phases obtained by REMO09 and refined by SYNERGY (φ r and w r , respectively) constitute the input for CAB, ARP/wARP and PHENIX.AUTOBUILD. Because we are interested in procedures for the automatic crystal structure solution, we used default directives for all three AMB programs. The available documentation for these programs suggested the following instructions (adapted to the 3eil test structure, as an example): We are conscious that the above instructions do not correspond to the optimized ways of applying the mentioned AMB programs. Indeed, any of them may be more effective if suitable instructions are introduced to treat special types of DNA or RNA and/or to explore different building approaches. The directives we used are only simple tools for automatic runs, which, if successful, constitute important achievements by themselves. Table 1. For the 29 nucleic acid test structures, the following abbreviations are used: Protein Data Bank (PDB) for the structure code, space group (SG) and the diffraction data resolution (RES) (Å). The number of nucleotides in the asymmetric unit (nN) is reported by the symbol n·N, where n is the number of chains per asymmetric unit, N is the number of nucleotides per chain. N is replaced by a sum of two numbers if chains with different type or different number of nucleotides are present. The model used in the MR step (column model) is reported as p·CODE, where CODE is the PDB code of the molecular fragment and p is the number of fragments originally used in the MR process. The information on the ligands is given in the corresponding column, in the form of m·CODE, where CODE is the PDB code of a ligand, and m is the number of ligands in the asymmetric unit of the target structure. The chemical formula for each ligand CODE is also specified at the bottom of the In Table 2, we show the experimental results obtained by NAUTILUS and CAB, both obtained by using the new NAUTILUS library and the knowledge of the positions of the triples (O 3 ', P, O 5 '), when detected after the SYNERGY step. The results obtained without such tools were poorer and are not shown for brevity. <|∆φ r |> • is the average phase error at the end of SYNERGY, R r and R f are the corresponding crystallographic residual (for all of the data) and Rfree value. R N , R fN , R C and R fC are the R and R f values at the end of NAUTILUS and CAB, respectively. During any AMB process, only the residuals R and R f are known. They are efficient figures of merit for establishing the overall accuracy of the proposed models, but they are not sufficient for assessing their true quality. We, therefore, used two a posteriori additional figures of merit, MA and MA M , to check the quality of the models provided by NAUTILUS and CAB (denoted as MA N and MA MN for the NAUTILUS case, and MA C and MA MC for CAB).  3ce5  50  41  43  54  59  36  16  52  53  41  18  3eil  46  31  36  47  50  59  43  36  38  82  76  3n4o  33  23  26  44  45  55  36  23  26  91  69  3tok  49  35  35  57  58  44  15  52  56  72  24  4gsg  53  34  38  45  45  17  9  42  46  44  17  4ms5  59  46  64  56  57  0  4  37  41  78  57  4xqz  48  32  35  58  58  30  22  27  30  80  94  5dwx  58  41  44  57  58  18  5  48  59  32  25  5i4s  35  25  29  36  37  59  49  35  34 82 51  5ihd  39  34  36  51  52  50  39  25  29  100  92  5ju4  26  26  28  37  37  95  83  26  28  100  100  5lj4  29  25  29  44  48  86  58  41  45  82  58  5mvt  28  29  28  38  37  82  79  31  31  95  92  5nt5  24  27  28  46  47  86  64  27  28  100  99  5t4w  25  25  29  43  42  86  64  25  29  100  96  1iha  41  34  35  36  37  94  77  23  25  88  81  1z7f  34  32  34  42  43  69  71  30 Table 2 suggests the following conclusions: (1) MA N and MA C only deal with the quality of the P chains; their usefulness as figures of merit has to be confirmed by MA MN and by MA MC , respectively, which define the overall quality of the structural model. Even if there is a good correlation between MA and MA M for all the tested AMB programs, their indications do not always agree; (2) The inequality MA N > MA C is rare (only in four cases, 5lj4, 1iha, 2pn4, 3d2v), and in all cases MA MN ≤ MA MC ; (3) For a high percentage of test structures, the quality of the NAUTILUS model is largely improved by CAB (examples are not given for brevity). Frequently, quite poor initial models are transformed by CAB into almost complete models. These cases correspond to poor values of MA N and MA MN , and to large values of MA C and MA MC ; (4) In all test cases, R N ≥ R C . That increases the confidence of CAB users in the quality of the built model. In some cases, the final residuals are large because of the unmodeled contribution to the diffraction from ligands that are missing in the model; (5) Eleven test cases (in bold) represent situations where the model and target sequences are identical or differ by only one residue and where the conditions R rh < 0.35 and R rh < R C are satisfied. The program automatically checks the sequence relationships and verifies if the numerical conditions are satisfied. As stated before, in all eleven cases, the program automatically chooses the models at the end of STEP 1 rather than the final CAB models. As an example, in Figure 1  In Table 3, we quote MAC and MAMC values obtained with and without the application of the algorithm for the sequence control, to allow the reader to understand how these values differ from each other. It is easily seen that MAC and MAMC values without the control are much worse;
In Table 4   In Table 3, we quote MA C and MA MC values obtained with and without the application of the algorithm for the sequence control, to allow the reader to understand how these values differ from each other. It is easily seen that MA C and MA MC values without the control are much worse; Table 3. MA C and MA MC for the eleven structures for which model and target sequences are equal or differ in one position, and for which the conditions R rh < 0.35 and R rh < R C are satisfied. WITH and WITHOUT indicate if the control on the sequences has been applied or not. MA values are percentages.

With
Without PDB MA C MA MC MA C MA MC   3n4o  91  69  77  38  4xqz  80  94  43  30  5ihd  100  92  70  47  5ju4  100  100  95  83  5nt5  100  99  100  87  5t4w  100  96  91  64  1iha  88  81  81  77  2a0p  100  99  100  99  2fd0  95  85  95  81  4enc  98  95  83  78  5tgp 100 100 100 75 (6) CAB (and NAUTILUS, of course) usually fails when ∆φ r • is close or larger than 50 • (this is the case for 3ce5, 3tok, 4gsg, 4ms5, 5dwx, 3d2v), even if two cases can be found in which it has success (3eil and 3fs0). This error limit is usually exceeded when CAB is applied to proteins. In Table 4 we show, for all test structures, the values of R, R f , MA and MA M obtained after the application of ARP/wARP (say R A , R fA , MA A and MA MA , respectively), and the analogous values obtained by the application of PHENIX.AUTOBUILD (say R P , R fP , MA P and MA MP , respectively). Comparing the quartet R A , R fA , MA A and MA MA with the quartet R P , R fP , MA P and MA MP clearly suggests the larger effectiveness of PHENIX.AUTOBUILD: usually R P < R A , R fP < R fA , MA P > MA A and MA MP > MA MA . The cpu time, however, is much larger for PHENIX.AUTOBUILD.  35  34  44  50  20  36  39  50  49  5ihd  39  51  51  10  5  52  56  25  19  5ju4  26  49  58  59  14  35  33  100  84  5lj4  29  41  52  55  25  40  41  68  65  5mvt  28  45  51  50  22  46  44  91  60  5nt5  24  35  48  91  43  35  38  100  84  5t4w  25  31  45  91  49  31  33  95  83  1iha  41  41  41  75  51  36  33  81  64  1z7f  34  40  46  69  32  35  36  91  82  2a0p  31  39  53  86  40  31  37  93  93  2fd0  33  45  52  73  30  37  36  95  80  2pn4  40  47  55  32  13  42  48  57  52  3d2v  57  56  57  6  3  47  48  26  23  3fs0  63  0  0  0  0  29  34  74  69  4enc  28  33  46  79  34  40  41  71  67  5kvj  49  39  55  59  20  35  40  84  63  5l4o  40  44  53  46  16  45  50  54  49  5nz6  45  34  38  53  29  35  37  78  56  5tgp  26  45  51  86  45  34  33  100  89  5uz6  34  34  40  91  53  33  33  91  82  6az4  51  42  46  38  15  39  40  67  53 PHENIX.AUTOBUILD and CAB results may be easily compared via their corresponding quartet (R, R f , MA, MA M ). Usually R P > R C , R fP > R fC , MA P < MA C , MA MP < MA MC , but there are also few cases in which PHENIX.AUTOBUILD alone performes better. Figures 2 and 3 synthetically represent the results quoted in Tables 2 and 4. Figure 2 shows the MA values corresponding to the default application of NAUTILUS, CAB, ARP/wARP and PHENIX.AUTOBUILD. In this condition, ARP/wARP seems the least efficient program: MA A > MA C only in one case (5dwx) and MA A = MA C also in one case (4gsg). The NAUTILUS and PHENIX.AUTOBUILD lines are closer to the CAB line. For NAUTILUS, MA N > MA C in four cases (5kvj, 5l4o, 5uz6, 6az4) and MA N = MA C in three cases (1iha, 2pn4, 3d2v). For PHENIX.AUTOBUILD, MA P > MA C in only two cases (3ce5, 3tok) and MA P = MA C in four cases (5ju4, 5nt5, 2fd0, 5tgp).  Tables 2 and 4. As previously stated, MA values are not in themselves indisputable estimates of the quality of the built models, because they register only the correctness of the P atoms. MAM may be considered a more general figure of merit involving all the non-H atoms. The MAM values obtained by the four tested programs are plotted in Figure 3. A common feature, no matter the algorithm used for AMB, is that usually MA > MAM; the P positions are more easily located than the other atoms. Even in this case, ARP/wARP seems the least efficient program, while the NAUTILUS and PHENIX.AUTOBUILD lines are closer to the CAB line, but the quality of the CAB models is markedly   Tables 2 and 4. As previously stated, MA values are not in themselves indisputable estimates of the quality of the built models, because they register only the correctness of the P atoms. MAM may be considered a more general figure of merit involving all the non-H atoms. The MAM values obtained by the four tested programs are plotted in Figure 3. A common feature, no matter the algorithm used for AMB, is that usually MA > MAM; the P positions are more easily located than the other atoms. Even in this case, ARP/wARP seems the least efficient program, while the NAUTILUS and PHENIX.AUTOBUILD lines are closer to the CAB line, but the quality of the CAB models is markedly  Tables 2 and 4. As previously stated, MA values are not in themselves indisputable estimates of the quality of the built models, because they register only the correctness of the P atoms. MA M may be considered a more general figure of merit involving all the non-H atoms. The MA M values obtained by the four tested programs are plotted in Figure 3. A common feature, no matter the algorithm used for AMB, is that usually MA > MA M ; the P positions are more easily located than the other atoms.
Even in this case, ARP/wARP seems the least efficient program, while the NAUTILUS and PHENIX.AUTOBUILD lines are closer to the CAB line, but the quality of the CAB models is markedly higher. In most cases, the CAB percentage of non-H atoms at a distance less than 0.6Å from the published positions is greater than 50.
A final observation is mandatory. This paper is mainly concerned with the full automation of the model building tools. However, the role of CAB for nucleic acids in the present scientific panorama may be better appreciated by including it in Table 5, where the most popular automated or semi-automated tools for model building are cited. Table 5. Most popular automated or semi-automated tools for model building of nucleic acids. SUB1: automated model building into electron density map from sequence; SUB2: guided semi-automated model building into electron density maps; SUB3: completing and rebuilding existing models into electron density maps; SUB4: building models from sequences without electron density.

Discussion
The CAB approach, originally designed for making the BUCCANEER application to proteins cyclic, was modified for use as an AMB tool for nucleic acids. In the new CAB version, we included NAUTILUS; the purpose was to improve the AMB effectiveness without changing NAUTILUS algorithms.
We applied CAB to a set of 29 nucleic acids (DNA and RNA) and compared the models thus obtained with those available after the mere application of NAUTILUS. We also applied ARP/wARP and PHENIX.AUTOBUILD to the same set of test structures. The procedures were fully automatic: a set of default instructions were given as inputs to any AMB program. Obviously, more appropriate input directives may improve the experimental results described in this paper. The results thus obtained show that the CAB cyclic approach remarkably increases NAUTILUS effectiveness and it is quite competitive with ARP/wARP and PHENIX.AUTOBUILD.
The AMB programs tested in this paper clearly show that their efficiency for nucleic acids is much smaller than for proteins. This partly depends on the particular difficulties to overcome for nucleic acids (see Section 1), but also on the smaller efforts spent in this field. This conclusion is supported by the following observation: quite often, SYNERGY ends with <|∆φ r |> • ≤ 40 • (16 times out of 29). This situation is usually very favourable for AMB programs when applied to proteins; on the contrary, Tables 2 and 3 show that MA and MA M values are often far from the expected values. Further efforts are needed for a complete and satisfactory AMB automation. Some of these efforts may be spent on improving the φ r phases, but most of them should concern the improvement of the AMB algorithms.
CAB for nucleic acids is part of an experimental version of SIR2014. Its full use requires that an experimental version of NAUTILUS, on which it is based, is also available. Hopefully, CAB will be released in late 2020.