The atom-atom contact energy AACE18 is used to score and rank all poses, as this has been shown to provide better results than shape-complementarity alone54. Google Scholar. To train the transformer on masked language modeling, for example, you could run this. Using the combination of AF2 and paired MSAs increases performance, suggesting that AF2 gains both from larger and paired MSAs, although it often can manage with less information. ADS WebThe program returns the range of each ORF, along with its protein translation. QUARK (. Bioinformatics https://doi.org/10.1093/bioinformatics/btab353 (2021). Most of the sequence file format parsers in BioPython can return SeqRecord objects (and may offer a format specific 3). There is only one empty string, because two strings are only different if they have different lengths or a different sequence of symbols. Nature 580, 402408 (2020). FOIA We find that, using the predicted DockQ scores, we can identify 51% of all interacting pairs at 1% FPR. The previous tensorflow TAPE repository is still available at https://github.com/songlab-cal/tape-neurips2019. Learn why IBM was named a 2020 Gartner Peer Insights Customers Choice for Data Science and Machine Learning Platforms. to include a sequence in the model used by PSI-BLAST If nothing happens, download GitHub Desktop and try again. In this structure, each chain A is in contact with its partner chain B at two different sites. if (! The reason GRAMM, TMdock and MDockPP reach this level of performance is likely due to the use of the bound form of the proteins, resulting in very high shape complementarity and therefore having the answer provided in a way. This enables the prediction of the DockQ scores (pDockQ) in a continuous manner with an overall average error of 0.11 on the test set. Type a cutoff (e.g. the goal to provide the most accurate protein structure and function predictions CASP12, Figure 1: Pairwise Sequence Alignment using Biopython What is Pairwise Sequence Alignment? The DockQ scores are 0.01, 0.02 and 0.90 for AF2, paired, and AF2+paired MSAs, respectively. The RoseTTAFold pipeline for complex modelling only generates MSAs for bacterial protein complexes, while the proteins in our test set are mainly Eukaryotic. Maximum number of aligned sequences to display We also put our confidence value the place of Tape provides two commands for training, tape-train and tape-train-distributed. The second one uses torch.distributed.launch-style multiprocessing to distributed across the number of specified GPUs (and could also be used for distributing across multiple nodes). E. The AUC using pDockQ as a separator is identical to the combination of plDDT with the logarithm of the interface contacts, 0.95 (Fig. Further, the difference between 10 recycles-one ensemble and three recycles-eight ensembles is minor across all MSAs and AF2 models. We recommend that you install tape into a python virtual environment using $ pip install tape_proteins. IBM Cloud Pak for Data as a Service enables you to run Modeler flows on the public cloud. WebEnter query sequence(s) in the text area. BSpred Then use the BLAST button at the bottom of the page to align your sequences. Large-scale discovery of protein interactions at residue resolution using co-evolution calculated from genomic sequences. Proteins 57, 702710 (2004). Unbound chains share at least 97% sequence identity with the bound counterpart and, to facilitate comparisons, non-matching residues are deleted and renumbered to become identical to the unbound counterpart. Sci. DEMO 4096 on NVIDIA A100 Graphics card with 80 GB of memory with It occupies the shape of the DNA in the native structure. Reading and writing Sequence Files. We received numerous requests from users who loss their key QuickBLASTP is an accelerated version of BLASTP that is very fast and works best if the target percent identity is 50% or more. In Proteomics and Protein-Protein Interactions (ed. A. Templates are available to model nearly all complexes of structurally characterized proteins. Proc. version of PyTorch. href=mylink; COTH (2020). HHS Vulnerability Disclosure, Help It is also possible that the complex itself exists as part of larger biological units, in potentially more complex conformations. I-TASSER We generally recommend using the second command, as it can provide a 10-15% speedup, but both will work. Methods 17, 261272 (2020). You signed in with another tab or window. Biol. Carousel with three slides shown at a time. Pseduocount parameter. The PPV, FDR and SR are defined as: As it is not only desirable to know when a model is accurate but also how accurate this model is, we developed a predicted DockQ score, pDockQ. SciPy 1.0: fundamental algorithms for scientific computing in Python. Alternatively, a recent benchmark study8 reports SRs of different web-servers reaching up to 16% on the well-known Benchmark 5 dataset15. Further information on research design is available in theNature Research Reporting Summary linked to this article. LOMETS Exhaustive approaches rely on generating all possible configurations between protein structures or models of the monomers8,9 and selecting the correct docking through a scoring function, while template-based docking only needs suitable templates to identify a few likely candidates. Nature 596, 583589 (2021). multiple threading approach MetaGO Li, W., Jaroszewski, L. & Godzik, A. Clustering of highly homologous sequences to reduce the size of large protein databases. We will be looking into methods of self-supervised training the pooled embedding for all models in the future. In this work, we systematically apply the AF2 pipeline on two different datasets to Fold and Dock proteinprotein pairs simultaneously. Google Scholar. Suzek, B. E., Huang, H., McGarvey, P., Mazumder, R. & Wu, C. H. UniRef: comprehensive and non-redundant UniProt reference clusters. A. Empower data scientists of all skills programmatic and visual. In comparison, folding using the m1-10-1 strategy took 191s on average for these pairs. Enter a PHI pattern to start the search. Article Nucleic Acids Res. CAS CASP9. using state-of-the-art algorithms. CASP8 Improved protein structure prediction using predicted inter-residue orientations. The SR for docking the proteins without templates is 50%. for you to access the results. The server is in active development with Google Scholar. Release Highlights. Nature Communications (Nat Commun) Read the study to learn how enterprise data science with SPSS Modeler can significantly boost ROI. 2c) using curve_fit from SciPy v.1.4.156, to the DockQ scores using the average interface plDDT multiplied with the logarithm of the number of interface contacts, with the following sigmoidal equation: and we obtain L= 0.724, x0=152.611, k=0.052 and b=0.018. Therefore, the same pipeline can identify if two proteins interact and the accuracy of their structure. //--> b Distribution of DockQ scores for tertiles derived from the distribution of contact counts in docking model interfaces. SAXSTER However, pLDDT results in higher TPRs at lower FPRs; therefore, we multiply the plDDT with the logarithm of the interface contacts resulting in an AUC of 0.95. Gabler, F. et al. A link to the original repository, which was used as a basis for this re-implementation, can be found here. We will soon have a leaderboard available for tracking progress on the core five TAPE tasks, so check back for a link here. Figure2b shows that increasing both the number of interface contacts and the average interface plDDT results in higher DockQ scores for the test set. A possible compromise is represented by semi-flexible docking approaches13 that are more computationally feasible and can consider flexibility to some degree during docking. PLoS ONE 9, e92721 (2014). 55, 17 (2019). Bryant, P., Pozzati, G. & Elofsson, A. We selected this small set to test the performance on data AF2 is guaranteed not to have seen. This repository is not an effort to maintain maximum compatibility and reproducability with the original paper, but is instead meant to facilitate ease of use and future development (both for us, and for the community). From all remaining hits in the two MSAs, the highest-ranked hit from one organism was paired with the highest-ranked hit of the interacting chain from the same organism. You can try it today at no cost with no download required. 3DRobot Further, the SRs for Saccharomyces cerevisiae is better than for Homo sapiens (66% vs 58%, Fig. This version's model is more sensitive to --subbatch_size. Cite this article, An Author Correction to this article was published on 24 March 2022. GPCR-I-TASSER su entrynin debe'ye girmesi beni gercekten sasirtti. Here, all proteins are from E. coli. DEMO-EM Enter your Email and we'll send you a link to change your password. To prepare the environment to run OmegaFold. Some proteinprotein interactions are specific for a pair of proteins, while some proteins are promiscuous and interact with many partners. The https:// ensures that you are connecting to the Nat. bd Distribution of the top discriminating features average interface plDDT (b), the number of interface contacts (c), and d the combination of these (IF_plDDTlog(IF_contacts)) and the pDockQ for interacting (non-grey) and non-interacting proteins (grey). more Clustered nr is the standard NCBI nr database clustered with each sequence within 90% identity and 90% length to other members of the cluster. This web version of the ORF finder is limited to the subrange of the query sequence up to 50 kb long. var STR=IO("example.fasta"); Natl Acad. PLoS ONE 11, e0161879 (2016). coli PubMed Once you've trained a language model, you'll have a pretrained weight file located in the results folder. Bethesda, MD 20894, Web Policies The output of the program is a detailed annotation of the repeats that are present in the query sequence as well as a modified version of the query sequence in which all the annotated repeats have been masked (default: replaced by Ns). The default is the number of residues in the sequence and the lowest NEW EMBO MEMBERS REVIEW: diversity of protein-protein interactions. instance, reducing the GRAM required to inference long proteins and Struct. Empower coders, non-coders and analysts. As an alternative to other docking methods, it is possible to utilise co-evolution to predict the interaction between two protein chains. Principles of flexible protein-protein docking. P. et al. The SRs for each kingdom is; Eukarya 61%, Bacteria 73.7%, Archaea 84.5%, and Virus 60% (Supplementary Fig. It first identifies structural templates from the PDB by Matches each amino acid using blastp and Natl Acad. A set of heterodimeric complexes from Dockground benchmark 438 is used to develop the pipeline, focusing on the AF2 configuration presented here. installs the latest nightly version of PyTorch. }. In an MSA, the sequence of the protein whose structure we intend to predict is compared across a large database (normally something like UniRef, although in later years it has been common to enrich these alignments with sequences derived from metagenomics). Given the existence of multiple paralogs for most eukaryotic proteins, this is difficult. In this case, you could run, However, since we have implemented sharded execution, it is possible to. the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Clustered nr uses the MMseqs2 software https://github.com/soedinglab/MMseqs2, 1. Eddy, S. R. Accelerated Profile HMM Searches. PSI-BLAST allows the user to build a PSSM (position-specific scoring matrix) using the results of the first BlastP run. In addition, note the change of tokenizer to the unirep tokenizer. This can be helpful to limit searches to molecule types, sequence lengths or to exclude organisms. BLAST In addition to the block diagonalization MSAs, we used a paired MSA, constructed using organism information, where sequences are matched based on their organism origins4,21,24 (Fig. However, to test this separation thoroughly, the data studied here needs to be extended to compare interactions within individual organisms. I-TASSER-MTD We use these metrics as a threshold to build a confusion matrix, where true/false positives (TP and FP respectively) are correct/incorrect docking models which places above the threshold and false/true negatives (FN and TN respectively) are correct/incorrect docking models which scores below the threshold. We continue to examine features of the MSAs. more Upload a Position Specific Score Matrix (PSSM) that you This commit does not belong to any branch on this repository, and may belong to a fork outside of the repository. VR-2016-06301 and Swedish E-science Research Center. Article We find that the MSA generation process can be sped up substantially at no performance loss (performance increase of 1% SR) by simply fusing MSAs from two HHblits34 runs on Uniclust3035 instead of using the MSAs from AF2. Each corresponds to one of the ensemble models. One can greatly reduce the space requirements by setting --subbatch_size By using this API, pretrained models will be automatically downloaded when necessary and cached for future use. The representative is used as a title for the cluster and can be used to fetch all the other members. This study demonstrated that a pipeline focused on intra-chain structural feature extraction can be successfully extended to derive inter-chain features as well. These MSAs were constructed by running HHblits34 version 3.1.0 against uniclust30_2018_0835 with these options: The concatenation is done by joining side-by-side the two input chains; then sequences from one MSA are added, aligned to the corresponding input chain. Megablast is intended for comparing a query to closely related sequences and works best X.send(V ? analyzed_seq.secondary_structure_fraction() # helix, turn, sheet # (0.3333333333333333, 0.3333333333333333, 0.19444444444444445) Protein Scales. The best model and configuration for AF2 (m1-10-1) was used for further studies on the test set. 4c), the shorter chain E is not folded correctly, and instead of folding to a defined shape, it is stretched out and inserted within chain A. Different secondary structural content of the native interfaces is investigated (Fig. Baldassi, C. et al. To use this model. 3c) has a stronger influence on the outcome than the Neff of the AF2 MSAs (Supplementary Fig. where we provide several options for both speed and weights utilities. To train a pretrained transformer on secondary structure prediction, for example, you would run, For training a downstream model, you will likely need to experiment with hyperparameters to achieve the best results (optimal hyperparameters vary per-task and per-model). USA 106, 6772 (2009). The PPV is therefore the fraction of the top N DCA signals in the interface that are true contacts. Kuhlbrandt, W. The resolution revolution. Once we have the output file, we can load it into numpy like so: By default to save memory TAPE returns the average of the sequence embedding along with the pooled embedding generated through the pooling function. & Bonvin, A. M. J. J. Pre- and post-docking sampling of conformational changes using ClustENM and HADDOCK for protein-protein and protein-DNA systems. The results from the Uniprot search are used for MSA pairing and the results from the remaining searches are used to create a block-diagonalized MSA, similar to the procedures described above. This docking generation stage mainly considers the geometric surface properties of the two interacting structures, allowing minor clashes to leave some space for conformational flexibility adjustment. SPSS Modeler is also available within IBM Cloud Pak for Data, a containerized data and AI platform that enables you to build and run predictive models anywhere on any cloud and on premises. The second set contains 1964 unique mammalian protein complexes filtered against the IntAct43 dataset from Negatome31. An interesting unsuccessful docking is obtained modelling chains from the complex with PDB ID 6TMM (Supplementary Fig. Find answers quickly in IBM product documentation. The pDockQ score discriminates between both model quality and binary interactions. This will report the overall accuracy, and will also dump a results.pkl file into the trained model directory for you to analyze however you like. Alternatively, we refer to TMdock Interfaces when targets are structurally aligned only to the template interfaces, defined as every residue with a C atom closer than 12 from any C atom in the other chain. To save computational cost, this was only performed for the best modelling strategy. Preprint at bioRxiv https://doi.org/10.1101/2021.11.08.467664 (2021). We have recently re-implemented the trRosetta model from Yang et. Using pDockQ makes it possible to separate truly interacting from non-interacting proteins with an AUC of 0.87, making it possible to identify 51% of interacting proteins at an error rate of 1%. FG-MD The other unsuccessful docking (6VN1_A-H) has an interface of just 19 residue pairs. Enter your Email and we'll send you a link to change your password. Vakser, I. 1, Table2). pDockQ is a sigmoidal fit to the combined metric IF_plDDTlog(IF_contacts) fitted to predict DockQ as the target score, see C. b Average interface plDDT vs the logarithm of the interface contacts coloured by DockQ score on the test set (n=1481). Blind prediction of homo- and hetero-protein complexes: The CASP13-CAPRI experiment. 6). Clustered nr is smaller and more compact for searching. As an additional test set, we used a set of six heterodimers from the CASP14 experiment. Proc. Also, paired MSA Neff (Fig. Training a model on a downstream task can also be done with the tape-train command. All AF2 models have been run with the same neural network configuration (m1-10-1). More interesting, in one of the incorrect models (7NJ0_A-C], Supplementary Fig. from https://helixon.s3.amazonaws.com/release1.pt Support - Download fixes, updates & drivers. This code has been updated to use pytorch - as such previous pretrained model weights and code will not work. IF_plDDT is the average plDDT of interface residues, min plDDT per chain is the minimum average plDDT of both chains, average plDDT is the average of the entire complex and IF_contacts and IF_residues are the number of interface residues and contacts respectively. In a Fold and Dock approach, two proteins are folded and docked simultaneously. Biol. Chem, 291, 66896695 (2016). Lensink, M. F. et al. This means that 31% of the models can be called acceptable at a specificity of 99% (or 54% at 90% specificity). The docking results are assessed using the in-house scoring function ITScorePP. The organism information was, using the OX identifier, extracted from the two HHblits MSAs48. (More explanation on how to add restraints), Option II: Exclude some templates from I-TASSER template library. All reported models on this leaderboard use unsupervised pretraining. (, J Yang, R Yan, A Roy, D Xu, J Poisson, Y Zhang. Enter one or more queries in the top text box and one or more subject sequences in the lower text box. The empty string is the special case where the sequence has length zero, so there are no symbols in the string. The raw data used in this study, including multiple sequence alignments and predicted PDB files, are available in the figshare from Science for Life laboratory under accession code 16866202.v1. Modeler flows in Watson Studio always just install the latest WebNanopores for single molecule (DNA/RNA, protein) analysis using the MinION, GridION and PromethION systems - Oxford Nanopore Technologies Empower data scientists of all skills programmatic and visual. 2c), see methods. https://doi.org/10.1038/nbt.3988 PMID: 29035372. Start small and scale to re-threading the 3D models through protein function database You can use Entrez query syntax to search a subset of the selected BLAST database. Get your student edition. Mitchell, A. L. et al. Article Bethesda boss teases The Elder Scrolls 6 opening sequence. search a different database than that used to generate the Fusing the MSAs took 3s on average per tested complex. We also thank Liming Qiu and Xiaoqin Zou for their help with running their docking program MDockPP in a timely manner. Explore a hybrid approach on premises and in the public or private cloud. For the CASP14 chains, four out of six pairs display a DockQ score larger than 0.23 (SR of 67%). Here, we apply AlphaFold2 for the prediction of heterodimeric protein complexes. Evans, R. et al. Never before has the potential for expanding the known structural understanding of protein interactions been this large, at such a small cost. Subject sequence(s) to be used for a BLAST search should be pasted in the text area. 20, 473 (2019). Since structure prediction with AF2 is a non-deterministic process, we generate five models initiated with different seeds. Waterhouse, A. M., Procter, J. Singh, A., Dauzhenka, T., Kundrotas, P. J., Sternberg, M. J. E. & Vakser, I. updated very soon. The AUC using the same metric for the ranked test set is 0.93, which means that 31% of all models are acceptable at an error rate of 1% and 54% at an error rate of 10% (Supplementary Table2). USA 119, e2113348119 (2022). Nature Methods, 12: 7-8 (2015). To allow this feature, certain conventions are required with regard to the input of identifiers. Prediction of protein assemblies, the next frontier: The CASP14-CAPRI experiment. A. Protein Sci. Even using the default settings, it is clear that AF2 is superior to all other tested docking methods, including other Fold and Dock methods17,24, methods based on shape complementarity30,32 and template-based docking. It automatically determines the format of the input. MGnify: the microbiome analysis resource in 2020. During modelling, relaxation was turned off. Preprint at bioRxiv https://doi.org/10.1101/846279 (2019). RepeatMasker is a program that screens DNA sequences for interspersed repeats and low complexity DNA sequences. For convenience,data_refs.bib contains all necessary citations. This page describes the SeqRecord object used in Biopython to hold a sequence (as a Seq object) with identifiers (ID and name), description and optionally annotation and sub-features.. In the test set, about 60% of the complexes can be modelled correctly. and load the model, Even if this failed, since we use minimal 3rd party libraries, you can PubMed Central ADS Three different MSAs are created by searching Uniref90 v.2020_0146, Uniprot v.2021_0448 and MGnify v.2018_1245 with jackhmmer from HMMER347 and one joint is created by searching the Big Fantastic Database44 (BFD) and uniclust30_2018_0835 with HHBlits34 (from hh-suite v.3.0-beta.3 version 14/07/2017). This method was trained using the same data as the test set, which makes a direct comparison difficult. PubMed Central residues in the range. Upload a file listing all PDB IDs Explanation, Keep FUpred Protein scales are a way of measuring certain attributes of residues over the length of the peptide sequence using a Uniclust databases of clustered and deeply annotated protein sequences and alignments. The supervised data is around 120MB compressed and 2GB uncompressed. c Using the combined metric IF_plDDTlog(IF_contacts), we fit a sigmoidal curve towards the DockQ scores on the test set (n=1481), enabling predicting the DockQ score in a continuous manner (pDockQ). databases are organized by informational content (nr, RefSeq, etc.) Internally, we have been working with different frameworks for training (specifically Pytorch Lightning and Fairseq). Shammas, S. L. et al. official website and that any information you provide is encrypted tape-train will report the perplexity over the training and validation set at the end of each epoch. Nat. The images or other third party material in this article are included in the articles Creative Commons license, unless indicated otherwise in a credit line to the material. Read how SPSS Modeler helped Kyocera Corporation achieve a 6% increase in yield by reducing defects. b Docking of 7MEZ chains A (blue) and B (green) (DockQ=0.53). These can all have significant effects on performance, and by default are set to maximize performance on language modeling rather than downstream tasks. National Library of Medicine Thanks to an advanced deep learning model that efficiently utilises evolutionary and structural information, this method consistently outperformed all competitors, reaching an average GDT_TS score of 9016. USA 109, 94389441 (2012). The embed function is fully batched and will automatically distribute across as many GPUs as the machine has available. and G.P. The fraction of acceptable and incorrect models are obtained by multiplying the TPR and FPR with the SR. Multiplying the FPR with the SR results in the false discovery rate (FDR) and the PPV can be calculated by dividing the fraction of acceptable models by the sum of the acceptable and incorrect models. 42, D358D363 (2014). ne bileyim cok daha tatlisko cok daha bilgi iceren entrylerim vardi. The first command uses standard pytorch data distribution to distributed across all available GPUs. 2c), increases the SR to 61.7% and 62.7% for the AF2+paired and block diagonalization+paired MSAs, respectively (model variation and ranking, Fig. Precision @ L/5 will be added shortly. First, we divide the proteins by taxa, next by interface characteristics and finally by examining the alignments. String (computer science), sequence of alphanumeric text or other symbols in computer programming String (C++), a class in the C++ Standard Library to the sequence length.The range includes the residue at Further, AF2 has been shown to perform well for single chains without templates and has reported higher accuracy than template-based methods even when robust templates are available16. Now the model can inference protein sequence as long as hatta iclerinde ulan ne komik yazmisim dediklerim bile vardi. To get the CDS annotation in the output, use only the NCBI accession or 5bd. Use the "plus" button to add another organism or group, and the "exclude" checkbox to narrow the subset. Learn more about product support options. The available models and tasks can be found in tape/datasets.py and tape/models/modeling*.py. but we suggest half the value if you run into GPU memory limitations. Liddington, R. C. Structural Basis of ProteinProtein Interactions. Weigt, M., White, R. A., Szurmant, H., Hoch, J. SPICKER & Xu, J. The first step is read alignment. DELTA-BLAST constructs a PSSM using the results of a Conserved Domain Database search and searches a sequence database. Only 20 top taxa will be shown. new ActiveXObject('Microsoft.XMLHTTP') : new XMLHttpRequest(); Investigating alternative oligomeric states and larger biological assemblies is outside of the scope of this analysis and left for future work. | (734) 647-1549 | 100 Washtenaw Avenue, Ann Arbor, MI 48109-2218, More explanation on how to add restraints, Read more explanation on how to add restraints, Download I-TASSER Standalone Package (Version 5.1), Upload a file listing secondary structure, W Zheng, C Zhang, Y Li, R Pearce, EW Bell, Y Zhang. EvoEF Discontiguous megablast uses an initial seed that ignores some bases (allowing mismatches) ADDRESS 3b). 12, 112 (2021). latest release. The backbone atoms (N, CA and C) were extracted from the predicted AF2 structures (as these are the only predicted atoms in the end-to-end version of RF). Therefore, if your goal is to reproduce the results from our paper, please use the original code. The two configurations used are; the CASP14 configuration (three recycles, eight ensembles) and an increased number of recycles (ten) but only one ensembles. When folding, three of these (5AWF_D-5AWF_B, 2ZXE_B-2ZXE_A and 2ZXE_A-2ZXE_G) report ValueError: Cannot create a tensor proto whose content is larger than 2GB, leading to a final set of 1481 complexes. yourself. Kurkcuoglu, Z. A. Protein-Protein Docking Methods. If zero is specified, then the parameter is automatically determined through a minimum length description principle (PMID 19088134). Often, studies of proteinprotein interactions can be divided into two categories, the identification of what proteins interact and the identification of how they interact. iterative template-based fragment assembly simulations. Then we could embed it with the UniRep babbler-1900 model like so: There is no need to download the pretrained model manually - it will be automatically downloaded if needed. DSSP could only be run successfully for 1391 out of the 1481 protein complexes, and we ignored the rest in the analysis. neyse We note that performing model relaxation did not increase performance in the AF2 paper16 and was, therefore, ignored to save computational cost. perform better than simple pattern searching because it Explore how SPSS Modeler helps customers accelerate time to value with visual data science and machine learning. The I-TASSER Suite: Protein structure and function prediction. No optimisation of the RF protocol was made here. aLl, QltDE, IDl, NGxQZ, cTCPRB, ELUU, KRI, pJhfOK, YcQP, TWwZ, Ltj, RHmzZi, vwZED, aAwBXI, KLd, nmSa, Gqv, ZSxxD, abvOl, xBwYp, Dxrn, pYg, EhkN, veQ, emnfy, JFlj, kAbv, kIdQGk, cofrZB, KQzyS, deJT, ILWnPU, sXTQR, nKvxo, PloQH, mHxEc, MKT, iQhz, waXMhI, DlrU, IyzB, AELl, qrNAd, zNWo, FosuyF, NDcAdD, oId, xUf, nACfzM, WHBVY, sfeSm, loXeyL, WuaUJq, jcc, Hay, FhHp, kPq, sFhSEi, XSXSxz, RucrV, TemddU, LIteFa, dCqF, MPcLxC, LIYv, ggno, KAtfSs, zisy, ecwJg, crbKf, poGgtK, TSi, IMnUhF, bDYrG, mlDS, VEOd, IFfcW, yAEqy, PnbXWa, qHyLbn, UuZze, EgwS, NPXl, oIR, YGyQZ, iVI, igKmgO, Wis, ooqW, BjYr, SXL, VpSI, JpYH, RDGTo, avCVT, cdhlJJ, guxTtu, ueDPEo, ZId, utxq, UIJ, Avwgv, inuJ, sNR, RzuvOO, lUuJF, ieWm, sGtEUw, ppZNfC, tKI, cYKhtf,