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Sequence co-evolution gives 3D contacts and structures of protein complexes
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Protein–protein interactions are fundamental to many biological processes. Experimental screens have identified tens of thousands of interactions, and structural biology has provided detailed functional insight for select 3D protein complexes. An alternative rich source of information about protein interactions is the evolutionary sequence record. Building on earlier work, we show that analysis of correlated evolutionary sequence changes across proteins identifies residues that are close in space with sufficient accuracy to determine the three-dimensional structure of the protein complexes. We evaluate prediction performance in blinded tests on 76 complexes of known 3D structure, predict protein–protein contacts in 32 complexes of unknown structure, and demonstrate how evolutionary couplings can be used to distinguish between interacting and non-interacting protein pairs in a large complex. With the current growth of sequences, we expect that the method can be generalized to genome-wide elucidation of protein–protein interaction networks and used for interaction predictions at residue resolution.https://doi.org/10.7554/eLife.03430.001
eLife digest
DNA is often referred to as the 'blueprint of life', as this molecule contains the instructions that are required to build a living organism from a single cell. But these instructions largely play out through the proteins that DNA encodes; and most proteins do not work alone. Instead they come together in different combinations, or complexes, and a single protein may participate in many complexes with different activities.
Proteins are so small that it is difficult to get clear information about what they look like. Visualizing protein complexes is even harder. Most protein–protein interactions remain poorly understood, even in the best-studied organisms such as humans, yeast, and bacteria.
Proteins are made from smaller molecules, called amino acids, strung together one after the other. The order in which different amino acids are arranged in a protein determines the protein's shape and ultimately its function. Like DNA, protein sequences can change over time. Sometimes, the sequence of one protein changes in a way that prevents it binding to another protein. If these two proteins must work together for an organism to survive, the second protein will often develop a compensating change that allows the protein–protein complex to reform.
Identifying pairs of changes in the sequences of pairs of proteins suggests that the two proteins interact and gives some information about how the proteins fit together. Different species can have copies of the same proteins that have slightly different sequences. Since the DNA sequences from many different organisms are already known, there are now many opportunities to find sites in pairs of proteins that have evolved together, or co-evolved, over time.
To find sites that seem to have co-evolved, Hopf et al. used a computer program based on an approach from statistical physics to look at pairs of proteins that were already known to form complexes. Co-evolving sites were found in over 300 pairs of proteins; including 76 where the structure of the complex was already known. When sites that were predicted to be co-evolving were then mapped to these known complex structures, the co-evolving sites were remarkably close to the true protein–protein contacts. This indicates that the information from the co-evolved sequences is sufficient to show how two proteins fit together.
Hopf et al. then turned their attention to 82 pairs of proteins that were thought to interact, but where a structure was unavailable. For 32 of these pairs, structures of the entire complex could be predicted, showing how the two proteins might interact. Furthermore, when other researchers subsequently worked out the structure of one of these complexes, the prediction was a good match to the solved complex structure.
The machinery of life is largely made up of proteins, which must interact in ever-changing but precise ways. The new methods developed by Hopf et al. provide a new way to discover and investigate the details of these interactions.https://doi.org/10.7554/eLife.03430.002
A large part of biological research is concerned with the identity, dynamics, and specificity of protein interactions. There have been impressive advances in the three-dimensional (3D) structure determination of protein complexes which has been significantly extended by homology-inferred 3D models (Mosca et al., 2012; Webb et al., 2014) (Hart et al., 2006; Zhang et al., 2012). However, there is still little, or no, 3D information for ∼80% of the currently known protein interactions in bacteria, yeast, or human, amounting to at least ∼30,000/∼6000 incompletely characterized interactions in human and Escherichia coli, respectively (Mosca et al., 2012; Rajagopala et al., 2014). With the rapid rise in our knowledge of genetic variation at the sequence level, there is an increased interest in linking sequence changes to changes in molecular interactions, but current experimental methods cannot match the increase in the demand for residue-level information of these interactions. One way to address the knowledge gap of protein interactions has been the use of hybrid, computational–experimental approaches that typically combine 3D structural information at varying resolutions, homology models, and other methods (de Juan et al., 2013), with force field-based approaches such as RosettaDock, residue cross-linking, and data-driven approaches that incorporate various sources of biological information (Kortemme and Baker, 2002; Dominguez et al., 2003; Kortemme and Baker, 2004; Kortemme et al., 2004; Svensson et al., 2004; Chaudhury et al., 2011; Schneidman-Duhovny et al., 2012; Velazquez-Muriel et al., 2012; Karaca and Bonvin, 2013; Rodrigues et al., 2013; Webb et al., 2014). However, most of these approaches depend on the availability of prior knowledge and many biologically relevant systems remain out of reach, as additional experimental information is sparse (e.g., membrane proteins, transient interactions, and large complexes). One promising computational approach is to use evolutionary analysis of amino acid co-variation to identify close residue contacts across protein interactions, which was first used 20 years ago (Gobel et al., 1994; Pazos and Valencia, 2001), and subsequently used also to identify protein interactions (Pazos et al., 1997; Pazos and Valencia, 2002). Others have used some evolutionary information to improve a machine learning approach to developing docking potentials (Faure et al., 2012; Andreani et al., 2013; Andreani and Guerois, 2014). These previous approaches relied on a local model of co-evolution that is less likely to disentangle indirect and therefore incorrect correlations from the direct co-evolution, as has been described in work on residue–residue interactions in single proteins (Marks et al., 2012). More recently, reports using a global model have been successful in identifying residue interactions from evolutionary co-variation, for instance between histidine kinases and response regulators (Burger & van Nimwegen, 2008; Skerker et al., 2008; Weigt et al., 2009), and this approach has only recently been generalized and used to predict contacts between proteins in complexes of unknown structure, in an independent effort parallel to this work (Ovchinnikov et al., 2014). In principle, just a small number of key residue–residue contacts across a protein interface would allow computation of 3D models and provide a powerful, orthogonal approach to experiments.
Since the recent demonstration of the use of evolutionary couplings (ECs) between residues to determine the 3D structure of individual proteins (Marks et al., 2011; Morcos et al., 2011; Aurell and Ekeberg, 2012; Jones et al., 2012; Kamisetty et al., 2013), including integral membrane proteins (Hopf et al., 2012; Nugent and Jones, 2012), we reason that an evolutionary statistical approach such as EVcouplings (Marks et al., 2011) could be used to determine co-evolved residues between proteins. To assess this hypothesis, we built an evaluation set based on all known binary protein interactions in E. coli that have 3D structures of the complex as recently summarized (Rajagopala et al., 2014). We develop a score for every predicted inter-protein residue pair based on the overall inter-protein EC score distributions resulting in accurate predictions for the majority of top ranked inter-protein EC pairs (inter-ECs) and sufficient to calculate accurate 3D models of the complexes in the docked subset (Figure 1A). This approach was then used to predict evolutionary couplings for 32 complexes of unknown 3D structures that have a sufficient number of sequences. Predictions include the structurally unsolved interactions between the a-, b-, and c-subunits of ATP synthase, which are supported by previously published experimental results.
We first investigated whether co-evolving residues between proteins are close in three dimensions by assessing blinded predictions of residue co-evolution against experimentally determined 3D complex structures. We follow this evaluation by then predicting co-evolved residue pairs of interacting proteins that have no known complex structure.
Extension of the evolutionary couplings method to protein complexes
To compute co-evolution across proteins, individual protein sequences must be paired up with each other that are presumed to interact, or being tested to see if they interact. Without this condition, proteins could be paired together that do not in fact interact with each other and therefore detection of co-evolution would be compromised. Given that the evolutionary couplings method depends on large numbers of diverse sequences (Hopf et al., 2012), some assumption must be made about which proteins interact with each other in homologous sequences in other species. Since it is challenging to know a priori whether particular interactions are conserved across many millions of years in thousands of different organisms, we use proximity of the two interacting partners on the genome as a proxy for this, with the goal of reducing incorrect pairings.
To assemble the broadest possible data sets to test the approach and make predictions, we take all known interacting proteins assembled in a published data set that contains ∼3500 high-confidence protein interactions in E. coli (Rajagopala et al., 2014). After removing redundancy and requiring close genome distance between the pairs of proteins this results in 326 interactions, see 'Materials and methods' (Figure 1B, Figure 1—figure supplement 1, Supplementary file 1 and 2),
The paired sequences are concatenated and statistical co-evolution analysis is performed using EVcouplings (Marks et al., 2011; Morcos et al., 2011; Aurell and Ekeberg, 2012), that applies a pseudolikelihood maximization (PLM) approximation to determine the interaction parameters in the underlying maximum entropy probability model (Balakrishnan et al., 2011; Ekeberg et al., 2013; Kamisetty et al., 2013), simultaneously generating both intra- and inter-EC scores for all pairs of residues within and across the protein pairs (Figure 1A). Evolutionary coupling calculations in previous work have indicated that this global probability model approach requires a minimum number of sequences in the alignment with at least 1 non-redundant sequence per residue (Marks et al., 2011; Morcos et al., 2011; Hopf et al., 2012; Jones et al., 2012; Kamisetty et al., 2013). Our current approach allows complexes with fewer available sequences to be assessed (minimum at 0.3 non-redundant sequences per residue) by using a new quality assessment score to assess the likelihood of the predicted contacts to be correct. The EVcomplex score is based on the knowledge that most pairs of residues are not coupled and true pair couplings are outliers in the high-scoring tail of the distribution (See 'Materials and methods', Figure 2A,B, Figure 2—figure supplement 1 and 2). The score can intuitively be understood as the distance from the noisy background of non-significant pair scores, normalized by the number of non-redundant sequences and the length of the protein ('Materials and methods', equations 1 and 2). If the number of sequences per residue is not controlled for, there is a large bias in the results, overestimating performance with low numbers of sequences (Figure 2B,C). The precise functional form of the correction for low numbers of sequences was chosen non-blindly after observing the dependencies in the test set.
Blinded prediction of known complexes
Evolutionary covariation reveals inter-protein contacts
Of the 329 interactions identified that are close on the E. coli genome, 76 have a sufficient number of alignable homologous sequences and known 3D structures either in E. coli or in other species. This set was used to test the inter-protein evolutionary coupling predictions (Supplementary file 1). The relationship between the EVcomplex score and the precision of the corresponding inter-protein ECs suggests that on average 74% (69%) of the predicted pairs with EVcomplex score greater than 0.8 will be accurate to within 10 Å (8 Å) of an experimental structure of the complex (Figure 2C). Most complexes have at least one inter-protein predicted contact above the selected score threshold of 0.8 (53/76 complexes). Three complexes have more than 20 predicted inter-protein residue contacts which are over 80% accurate, namely the histidine kinase and response regulator system (78 residue pairs), t-RNA synthetase (32 residue pairs), and the vitamin B importer complex (21 residue pairs), with precision over 80% (complex numbers 330, 019, 130 respectively, Figure 2D, Figure 2—figure supplements 3–8, Supplementary file 1).
We suggest that users of EVcomplex consider predicted contacts that lie below the threshold of 0.8 in the context of other biological knowledge, where available, or in comparison to other higher scoring contacts for the same complex. In this way additional true positive inter-residue contacts can be distinguished from false positives. For instance, the ethanolamine ammonia-lyase complex (complex 065) has only 3 predicted inter-protein residue pairs above the score threshold, but in fact has 5 additional correct pairs with EVcomplex scores slightly below the threshold of 0.8 which cluster with the 3 high-scoring contacts on the monomers, indicating that they are also correct.
Some of the high confidence inter-protein ECs in the test set are not close in 3D space when compared to their known 3D structures. These false positives may be a result of assumptions in the method that are not always correct. This includes (1) the assumption that the interaction between paired proteins is conserved across species and across paralogs, and (2) that truly co-evolved residues across proteins are indeed always close in 3D, which is not always the case. In addition, the complexes may also exist in alternative conformations that have not necessarily all been captured yet by crystal or NMR structures, for instance in the case of the large conformational changes of the BtuCDF complex (Hvorup et al., 2007).
Docking is accurate with few pairs of predicted contacts
To test whether the computed inter-protein ECs are sufficient for obtaining accurate 3D structures of the whole complex, we selected 15 diverse examples (with 5 or more inter-protein residue contacts) for docking (Table 1, Figure 3, Figure 3—figure supplement 1, Supplementary file 3) with HADDOCK (Dominguez et al., 2003; de Vries et al., 2007). The docking procedure is fast and generates 100 3D models of each complex using all residue pairs with EVcomplex scores above the selection threshold. We additionally dock negative controls to assess the amount of information added to the docking protocol by evolutionary couplings (500 models per run, no constraints other than center of mass, see 'Materials and methods'). The best models for all 15 complexes docked with evolutionary couplings have interface RMSDs under 6 Å, 12/15 have the best scoring model under 4 Å and the top ranked models for 11/15 are under 5 Å backbone interface RMSD compared to a crystal or NMR structure interface. Over 70% of the generated models are close to the experimental structures of the complexes (<4 Å backbone iRMSD), compared to less than 0.5% in the controls (and these were not high–ranked) (Figure 3—figure supplement 1, Supplementary file 3, 'Supplementary data'). Not surprisingly complexes that have the largest numbers of true positive predicted contacts perform the best when docking. For example, the ribosomal proteins RS3 and RS14 have 11 true positive inter-protein ECs and result in a top ranked model only 1.1 Å iRMSD from the reference structure. More surprisingly, other complexes with a lower proportion of true positive inter-protein contacts, such as Ubiquinol oxidase (6 out of 11) or the epsilon and gamma subunits of ATP synthase (8 out of 15) also produced accurate predicted complexes, with an iRMSD of 1.8 and 1.4 Å respectively. The docking experiments therefore demonstrate that inter-protein ECs, even in the presence of incorrect predictions, can be sufficient to give accurate 3D models of protein complexes, but more work will be needed to quantify the likelihood of successful docking from the predicted contacts.
Conserved residue networks provide evidence of functional constraints
The top 10 inter-EC pairs between MetI and MetN are accurate to within 8 Å in the MetNI complex (PDB: 3tui [Johnson et al., 2012]), resulting in an average 1.4 Å iRMSD from the crystal structure for all 100 computed 3D models (Table 1, Supplementary file 3 and 'Supplementary data'). The top 3 inter-EC residue pairs (K136-E108, A128-L105, and E74-R124, MetI-MetN respectively) constitute a residue network coupling the ATP-binding pocket of MetN to the membrane transporter MetI. This network calculated from the sequence alignment corresponds to residues identified experimentally that couple ATP hydrolysis to the open and closed conformations of the MetI dimer (Johnson et al., 2012) (Figure 4A). The vitamin B12 transporter (BtuC) belongs to a different structural class of ABC transporters, but also uses ATP hydrolysis via an interacting ATPase (BtuD). The top 5 inter-ECs co-locate the L-loop of BtuC close to the Q-loop ATP-binding domain of the ATPase, hence coupling the transporter with the ATP hydrolysis state in an analogous way to MetI-MetN. The identification of these coupled residues across the different subunits suggests that EVcomplex identifies not only residues close in space, but also particular pairs that are constrained by the transporter function of these complexes (Kadaba et al., 2008; Johnson et al., 2012).
The ATP synthase ε and γ subunit complex provides a challenge to our approach, since the ε subunit can take different positions relative to the γ subunit, executing the auto-inhibition of the enzyme by dramatic conformational changes (Cingolani and Duncan, 2011). In a real-world scenario, where we might not know this a priori, there may be conflicting constraints in the evolutionary record corresponding to the different positions of the flexible portion of ε subunit. EVcomplex accurately predicts 6 of the top 10 inter-EC pairs (within 8 Å in the crystal structure 1fs0 (Rodgers and Wilce, 2000) or 3oaa (Cingolani and Duncan, 2011)), with the top 2 inter-ECs εA45-γL215 and εA40-γL207 providing contact between the subunits along an inter-protein beta sheet. The location of the C-terminal helices of the ε subunit is significantly different across 3 crystal structures (PDB IDs: 1fs0 [Rodgers and Wilce, 2000], 1aqt [Uhlin et al., 1997], 3oaa [Cingolani and Duncan, 2011]). The top ranked intra-ECs support the conformation seen in 1aqt, with the C-terminal helices packed in an antiparallel manner and tucked against the N-terminal beta barrel (Figure 4B, green circles) and do not contain a high ranked evolutionary trace for the extended helical contact to the γ subunit seen in 1fs0 or 3oaa (Figure 4B, gray box). Docking with the top inter-ECs results in models with 1.4 Å backbone iRMSDs to the crystal structure for the interface between the N-terminal domain of the ε subunit and the γ subunit (Table 1, Supplementary file 3). εD82 and γR222 connect the ε-subunit via a network of 3 high-scoring intra-ECs between the N- and C-terminal helices to the core of the F1 ATP synthase. In summary, these examples suggest that inter-protein evolutionary couplings can provide residue relationships across the proteins that could aid identification of functional coupling pathways, in addition to obtaining 3D models of the complex.
De novo prediction of unknown complexes
Prediction of interactions for 32 protein pairs with high-scoring evolutionary couplings
A total of 82 protein complexes with unknown 3D structure of the interaction that satisfy the conditions for the current approach, i.e., have sufficient sequences and are close in all genomes, were predicted using EVcomplex (all residue–residue inter-protein evolutionary couplings scores are available in 'Supplementary data'). 32 of these have high EVcomplex scores with at least one predicted contact (Figure 5, Figure 5—figure supplement 1 and 2, and Supplementary file 4). Analysis of the inter-EC predictions for known 3D complex structures shows that protein pairs with more high-scoring ECs (EVcomplex score ≥0.8) have a higher proportion of true positives (Figure 2D). Hence, the protein complexes in the set of unknown structures with more high-scoring inter-ECs are most likely to have predicted ECs that indicate residue pairs close in 3D (column Q, Supplementary file 2, the exact pairs can be found in Supplementary file 4). Three examples of predictions with multiple high-scoring inter-ECs include MetQ-MetI, UmuD-UmuC, and DinJ–YafQ. The top 15 inter-ECs between MetQ and MetI are from one interface of MetQ to the MetI periplasmic loops, or the periplasmic end of the helices, consistent with the known binding of MetQ to MetI in the periplasm.
The UmuD and UmuC complex is induced in the stress/SOS response facilitating the cleavage of UmuD to UmuD' (between C24 and G25) to form UmuD'2 which then interacts with UmuC (DNA polymerase V) in order to copy damaged DNA (Beuning et al., 2006). The truncated dimer form (UmuD'2) has at least two contrasting conformations where the N-terminal arm is placed on opposite sides of the dimer in one conformation or in close proximity in the alternative (Figure 5—figure supplement 3). For 6/7 ECs above the score threshold, residues in UmuD predicted to interface with UmuC are co-located on one face of the dimer. Two residues (Y33, I38) are located in the N-terminal arm of UmuD that, after cleavage of the 24 N-terminal amino acids, may become available for binding UmuC. Since UmuD switches functions after this cleavage and can then bind UmuC, these inter-ECs may identify the critical residues for translesion synthesis function (Beuning et al., 2006). Although the ECs from this UmuD arm to UmuC involve residues in two separate domains of UmuC (S 415 and Y 74), intra-monomer evolutionary couplings predict that these residues are close in UmuC (Figure 5—figure supplement 3A, black rectangles). The relative positions of the contacting residues within each monomer therefore support the plausibility of the accuracy of the interaction interface.
Whilst this manuscript was in review, the 3D structure of the previously unsolved biofilm toxin/antitoxin DinJ–YafQ complex was published (PDB: 3mlo (Liang et al., 2014)), showing the intertwining of subunits in a heterotetrameric complex. 17/19 predicted EC residue pairs are within 8 Å in this 3D structure (Supplementary file 4 and 'Supplementary data'). In general, the agreement between our de novo predicted inter-protein ECs and available experimental data serves as a measure of confidence for the predicted residue pair interactions and suggests that EVcomplex can be used to reveal 3D structural details of yet unsolved protein complexes given sufficient evolutionary information.
EVcomplex predicts interacting protein pairs in a large complex
To investigate whether the EVcomplex score can also distinguish between interacting and non-interacting pairs of proteins, we use the E. coli ATP synthase complex as a test case. The ATP synthase structure is of wide biological interest (reviewed in Walker, 2013) with a remarkable 3D structural arrangement, but completion of all aspects of the 3D structure has remained experimentally challenging (Baker et al., 2012) (Figure 6A). As a demonstration exercise, we calculated evolutionary couplings for all 28 possible pair combinations of different ATP synthase subunits (centered around the E. coli ATP synthase) and transformed the ECs into EVcomplex scores for all inter-protein residue pairs (experimentally determined stoichiometry: α3β3γδεab2c10, Supplementary file 5 and 'Supplementary data'). Using the default EVcomplex score threshold of 0.8 to discriminate between interacting and non-interacting pairs of subunits, 24 of the 28 possible interactions between the subunits are correctly classified as interacting or non-interacting. The four incorrect predictions (namely: ε and c, γ and c, ε and β, b and β, for which there is some experimental evidence) are not identified as interacting using the 0.8 EVcomplex threshold. Choosing a threshold lower than 0.8 does identify 2 of these as interacting but also introduces new false positives. The ε and β interaction in the crystal structure 3oaa (Cingolani and Duncan, 2011) is a special case in that it involves a highly extended conformation of the last two helices of the ε subunit that reach up into the enzyme making contacts with the β subunit. The false negative EVcomplex score for this pair could be a result of the transience of their interaction or reflect a more general problem of lack of conservation of this interaction across the aligned proteins from different species. In total 80% of the interacting residue pairs in the known 3D structure parts of the synthase complex (7 pairs of subunits) are correctly predicted (threshold: 10 Å minimum atom distance between two residues). This exercise of predicting the presence or absence of interaction between any two proteins indicates the potential of the EVcomplex method in helping to elucidate protein–protein interaction networks from evolutionary sequence co-variation and identify interacting subunits of large macromolecular complexes.
EVcomplex predicts details of subunit interactions in ATP synthase
While much of the 3D structure of ATP synthase is known (Walker, 2013), the details of interactions between the a-, b-, and c-subunits have not yet been determined by crystallography. We analyse the details in these interactions, as the EVcomplex scores between these subunits are substantial (Figure 6B). We are fortunately able to provide a missing piece for this analysis, the unknown structure of the membrane-integral penta-helical a-subunit, using our previously described method for de novo 3D structure prediction of alpha-helical transmembrane proteins (Hopf et al., 2012). To our knowledge, there are no experimentally determined atomic resolution structures of the a-subunit of ATP synthase. A 3D model of the a-subunit is from 1999 (1c17(Rastogi and Girvin, 1999)) and was computed using five helical–helical interactions that were inferred from second suppressor mutation experiments, and then imposed as distance restraints for TMH2-5, revealing a four helical bundle (with no information for TMH1). Later, cross-linking experiments (Schwem and Fillingame, 2006) identified contacting residues from all pairs of helical combinations of TM2-TM5 (6 pairs), supporting the earlier 4 helical bundle topology. 7 of the 8 cross-linked pairs are either exactly the same pair (L120-I246) or adjacent to many pairs in the top L intra a-subunit evolutionary couplings (ECs).
In fact, the helix packing arrangement in the predicted structure of the a-subunit is consistent with the topology suggested on the basis of crosslinking studies (Long et al., 2002; DeLeon-Rangel et al., 2003; Fillingame and Steed, 2014), including the lack of contacts for transmembrane helix 1 with the other 4 helices ('Supplementary data').
The top inter-protein EC pair between subunits a and b, aK74–bE34, coincides with experimental crosslinking evidence of the interaction of aK74 with the b-subunit and the position of E34 of the b subunit emerging from the membrane on the cytoplasmic side (Long et al., 2002; DeLeon-Rangel et al., 2003). Indeed, 6 of the 13 high score ECs are in the same region as the experimental crosslinks, for instance between the cytoplasmic loop between the first two helices of the a-subunit and the b-subunit helix as it emerges from the membrane bilayer (DeLeon-Rangel et al., 2013), or between a239V in TM helix 5 and bL16 (Figure 6C, Figure 6—figure supplement 1, Supplementary file 6). Additionally, the top EC between the a- and c-subunits (aG213 – cM65) lies close to the functionally critical aR210–cD61 interaction (Dmitriev et al., 1999) on the same helical faces of the respective subunits (Figure 6C). This prediction of missing aspects of subunit interactions may help in the design of targeted experiments to complete the understanding of the intricate molecular mechanism of the ATP synthase complex.
A primary limitation of our current approach is its dependence on the availability of a large number of evolutionarily related sequences. If a protein interaction is conserved across enough sequenced genomes, using a single pair per genome can give accurate predictions of the interacting residues. However, if the protein pair is present in limited taxonomic branches, there may be insufficient sequences at any given time to make confident predictions. A solution to this could be to include multiple paralogs of the interacting proteins from each genome, but this requires correct pairing of the interaction partners, which is in general hard to ascertain. In addition, details of interactions may have diverged for paralogous pairs. Hence, in this current version of the method, we have imposed a genome distance requirement across all genomes for all homolog pairs in order to be less sensitive to these complications.
As the need to use genome proximity to pair sequences becomes less important with the increasing availability of genome sequences, there will be a dramatic increase in the number of interactions that can be inferred from evolutionary couplings, including those unique to eukaryotes. With currently available sequences (May 2014 release of the UniProt database), EVcomplex is able to provide information for about 1/10th of the known 3000 protein interactions in the E. coli genome. Once there are ∼10,000 bacterial genome sequences of sufficient diversity, one would have enough information to test each potentially interacting pair of homologs for evidence of interaction and, given sufficiently strong evolutionary couplings, infer the 3D structure of each protein–protein pair, as well as of complexes with more than two proteins. For any set of species, e.g., vertebrates or mammals, one can imagine guiding sequencing efforts to optimize species diversity to facilitate the extraction of evolutionary couplings. This can open the doors for more comprehensive and more rapid determination of approximate 3D structures of proteins and protein complexes, as well as for the elucidation in molecular detail of the most strongly evolutionarily constrained interactions, pointing to functional interactions.
Determining the three-dimensional models of complexes from the predicted contacts was successful in many of the tested instances. Using minimal computing resources and a small number of inter-EC-derived contacts, low interface positional RMSDs relative to experimental structures can be achieved. However, a significant number of proteins exist as homomultimers within larger complexes. To determine models of these complexes one must deconvolute homomultimeric inter-ECs from the intra-protein signal, which is an important technical challenge for future work.
The analysis of subunit interactions in ATP synthase in this work is a 'proof of principle' study showing that methods such as EVcomplex can determine which proteins interact with each other at the same time as specific residue pair couplings across the proteins (as also shown in the work by the Baker lab on ribosomal protein interactions (Ovchinnikov et al., 2014)). Understanding the networks of protein interactions is of critical interest in eukaryotic systems, such as networks of protein kinases, GPCRs, or PDZ domain proteins. An understanding of the distributions of interaction specificities is of high interest to many fields. Although we do not know how well our evolutionary coupling approach will handle less obligate interactions, results on the two-component signaling system (histidine kinase/response regulator) both here and in other work (Skerker et al., 2008; Weigt et al., 2009) suggest optimism.
The approximately scale-free EVcomplex score is a heuristic based on the distribution of raw EC scores from the statistical model, their dependence on sequence alignment depth and the length of the concatenated sequences. The score provides a simple way of accounting for these dependencies such that a uniform threshold, say 0.8, can be used for any protein pair with the expectation of reasonably accurate predictions. Since cutoff thresholds can be useful but overly sharp, we recommend investigating predicted contacts below the threshold used in this work, especially where there is independent biological knowledge to validate the predictions.
The work presented here is in anticipation of a genome-wide exploration and, as a proof of principle, shows the accurate prediction of inter-protein contacts in many cases and their utility for the computation of 3D structures across diverse complex interfaces. As with single protein (intra-EC) predictions, evolutionarily conserved conformational flexibility and oligomerization can result in more than one set of contacts that must be de-convoluted. Can evolutionary information help to predict the details and extent for each complex? A key challenge will be the development of algorithms that can disentangle evolutionary signals caused by alternative conformations of single complexes, alternative conformations of homologous complexes, and effectively deal with false positive signals. Taken together, these issues highlight fruitful areas for future development of evolutionary coupling methods.
Despite conditions for the successful de novo calculation of co-evolved residues, the method described here may accelerate the exploration of the protein–protein interaction world and the determination of protein complexes on a genome-wide scale at residue level resolution. The use of co-evolutionary analysis in computational models to determine protein specificity and promiscuity, co-evolutionary dynamics and functional drift will open up exciting future research questions.
Materials and methods
Selection of interacting protein pairs for co-evolution calculation
The candidate set of complexes for testing and de novo prediction was derived starting from a data set of binary protein–protein interactions in E. coli including yeast two-hybrid experiments, literature-curated interactions and 3D complex structures in the PDB (Rajagopala et al., 2014). Three complexes not contained in the list were added based on our analysis of other subunits in the same complex, namely BtuC/BtuF, MetI/MetQ, and the interaction between ATP synthase subunits a and b. Since our algorithm for concatenating multiple sequence pairs per species assumes the proximity of the interacting proteins on the respective genomes of each species (See below), we excluded any complex with a gene distance >20 from further analysis. The gene distance is calculated as the number of genes between the interacting partners based on an ordered list of genes in the E. coli genome obtained from the UniProt database. The resulting list of pairs (∼350) was then filtered for pseudo-homomultimeric complexes based on the identification of Pfam domains in the interacting proteins (330). All remaining complexes with a known 3D structure (as summarized in Rajagopala et al., 2014) or a homologous interacting 3D structure (93) (identified by intersecting the results of HMMER searches against the PDB for both monomers) were used for evaluating the method, while complexes without known structure (236) were assigned to the de novo prediction set (Figure 1—figure supplement 1). The set with protein complexes of known 3D structure was further filtered for structures that only cover fragments (<30 amino acids) of one or both of the monomers and structures with very low resolution (>5 Å), which led to the re-assignment of Ribonucleoside-diphosphate reductase 1 (complex_002), Type I restriction-modification enzyme EcoKI (complex_012), RpoC/RpoB (complex_041), RL11/Rl7 (complex_165), the ribosome with SecY (complex_226, complex_250, and complex_255), and RS3/RS (complex_254) to the set of unknown complexes. Large proteins were run with the specific interacting domains informed by the known 3D structure, when the full sequence was too large for the number of retrieved sequences (for domain annotation see 'Supplementary data').
This set could serve as a benchmark set for future development efforts in the community.
Multiple sequence alignments
Each protein from all pairs in our data set was used to generate a multiple sequence alignment (MSA) using jackhmmer (Johnson et al., 2010) to search the UniProt database (UniProt Consortium, 2014) with 5 iterations. To obtain alignments of consistent evolutionary depths across all the proteins, a bit score threshold of 0.5 * monomer sequence length was chosen as homolog inclusion criterion (-incdomT parameter), rather than a fixed E-value threshold which selects for different degrees of evolutionary divergence based on the length of the input sequence.
In order to calculate co-evolved residues across different proteins, the interacting pairs of sequences in each species need to be matched. Here, we assume that proteins in close proximity on the genome, e.g., on the same operon, are more likely to interact, as in the methods used previously matching histidine kinase and response regulator interacting pairs (Skerker et al., 2008; Weigt et al., 2009) ('Supplementary data'). We retrieved the genomic locations of proteins in the alignments and concatenated pairs following 2 rules: (i) the CDS of each concatenated protein pair must be located on the same genomic contig (using ENA (Pakseresht et al., 2014) for mapping) and (ii) each pair must be the closest to one another on the genome, when compared to all other possible pairings in the same species. The concatenated sequence pairs were filtered based on the distribution of genomic distances to exclude outlier pairs with high genomic distances of more than 10k nucleotides ('Supplementary data'). Alignment members were clustered together and reweighted if 80% or more of their residues were identical (thus implicitly removing duplicate sequences from the alignment). Supplementary file 1 and 2 report the total number of concatenated sequences, the lengths, and the effective number of sequences remaining after down-weighting in the evaluation and de novo prediction set, respectively.
Computation of evolutionary couplings
Inter- and intra-ECs were calculated on the alignment of concatenated sequences using a global probability model of sequence co-evolution, adapted from the method for single proteins (Marks et al., 2011; Morcos et al., 2011; Hopf et al., 2012) using a pseudo-likelihood maximization (PLM) (Balakrishnan et al., 2011; Ekeberg et al., 2013) rather than mean field approximation to calculate the coupling parameters. Columns in the alignment that contain more than 80% gaps were excluded and the weight of each sequence was adjusted to represent its cluster size in the alignment thus reducing the influence of identical or near-identical sequences in the calculation. For the evaluation set, we can then compare the predicted ECs for both within and between the proteins/domains to the crystal structures of the complexes (for contact maps and all EC scores, see 'Supplementary data').
Definition of a scale-free score for the assessment of interactions
In order to estimate the accuracy of the EC prediction, we evaluate the calculated inter-ECs based on the following observations: (1) most pairs of positions in an alignment are not coupled, i.e., have an EC score close to zero, and tend to be distant in the 3D structure; (2) the background distribution of EC scores between non-coupled positions is approximately symmetric around a zero mean; and (3) higher-scoring positive score outliers capture 3D proximity more accurately than lower-scoring outliers (See also Figure 2). The width of the (symmetric) background EC score distribution can be approximated using the absolute value of the minimal inter-EC score. The more a positive EC score exceeds the noise level of background coupling, the more likely it is to reflect true co-evolution between the coupled sites. For each inter-protein pair of sites i and j with pair coupling strength ECinter(i, j), we therefore calculate a raw reliability score ('pair coupling score ratio', Figure 2B) defined by
Since the accuracy of evolutionary couplings critically depends both on the number and diversity of sequences in the input alignment and the size of the statistical inference problem (Marks et al., 2011; Morcos et al., 2011; Jones et al., 2012), we incorporate a normalization factor to make the raw reliability score comparable across different protein pairs. The normalized EVcomplex score is defined as
where Neff is the effective number of sequences in the alignment after redundancy reduction, and L (total number of residues) is the length of the concatenated alignment. Previous work on single proteins has shown that the method requires a sufficient number of sequences in the alignment to be statistically meaningful. We thus filter for sequence sufficiency requiring Neff/L > 0.3 (Table 1, Supplementary files 1 and 2). Predictions of coupled residues in the evaluation set were evaluated against their residue distances in known structures of protein pairs (Rajagopala et al., 2014) (See Supplementary file 7) in order to determine the precision of the method.
To interpret the EVcomplex prediction of interaction between subunits a and b of the ATP synthase as well as UmuC and UmuD, individual monomer models were built de novo for the structurally unsolved a-subunit of ATP synthase and UmuC using the EVfold pipeline as previously published (Marks et al., 2011; Hopf et al., 2012). In both cases coupling parameters were calculated using PLM (Balakrishnan et al., 2011; Ekeberg et al., 2013) and sequences were clustered and weighted at 90% sequence identity (the resulting models are provided in 'Supplementary data').
Prediction of interactions in a set of subunits
Following this same protocol EVcomplex scores were calculated for all possible 28 combinations of the 8 E. coli ATP synthase F0 and F1 subunits. Since we want to compare the computational predictions to some 'ground truth', as with the complexes for the rest of the manuscript, we used known 3D structures of the ATP synthase complex to assign whether or not the subunits interact (PDB: 3oaa, 1fs0, 2a7u; Supplementary file 7). Since we are also determining whether the subunits interact, not necessarily knowing full atomic detail residue interactions, we included subunit interactions that have been inferred from cryo-EM, crosslinking or other experiments, but do not necessarily have a crystal structure. These are represented as solid blue boxes, if the interaction is well established (DeLeon-Rangel et al., 2013; Schulenberg et al., 1999; Brandt et al., 2013; McLachlin and Dunn, 2000), or crosshatched blue if there is a lack of consensus in the community (Figure 6B, left panel).
For each possible interaction the EVcomplex score of the highest ranked inter-EC was considered as a proxy for the likelihood of interaction. Pairs with scores above 0.8 are considered likely to interact, between 0.75 and 0.8 weakly predicted, while interactions with scores below 0.75 are rejected as possible complexes (blue boxes, blue crosshatched, and white respectively in right panel of Figure 6B, and 'Supplementary data').
Computation of 3D structure of complexes
A diverse set of 15 complexes was chosen from the 22 in the evaluation set that had at least 5 couplings above a complex score of 0.8 and was subsequently docked (Supplementary file 3). Proteins that have been crystallized together in a complex could bias the results of the docking, as they have complementary positions of the surface side chains. Therefore, where possible we used complexes that had a solved 3D structure of the unbound monomer, namely GcsH/GcsT, CyoA, FimC, DhaL, AtpE, PtqA/PtqB, RS10, and HK/RR, and in all other cases the side chains of the monomers were randomized either by using SCWRL4 (Krivov et al., 2009) or restrained minimization with Schrodinger Protein Preparation Wizard (Sastry et al., 2013) before docking. For ubiquinol oxidase (complex_054) the unbound structure of subunit 2 (CyoA) only covers the COX2 domain. In this case docking was performed using this unbound structure plus an additional run using the bound complex structure with perturbed side chains.
We used HADDOCK (Dominguez et al., 2003), a widely used docking program based on ARIA (Linge et al., 2003) and the CNS software (Brunger, 2007) (Crystallography and NMR System), to dock the monomers for each protein pair with all inter-ECs with an EVcomplex score of 0.8 or above implemented as distance restraints on the α-carbon atoms of the backbone.
Each docking calculation starts with a rigid-body energy minimization, followed by semi-flexible refinement in torsion angle space, and ends with further refinement of the models in explicit solvent (water). 500/100/100 models generated for each of the 3 steps, respectively. All other parameters were left as the default values in the HADDOCK protocol. Each protein complex was run using predicted ECs as unambiguous distance restraints on the Cα atoms (deff 5 Å, upper bound 2 Å, lower bound 2 Å; input files available in 'Supplementary data'). As a negative control, each protein complex was also docked using center of mass restraints alone (ab initio docking mode of HADDOCK) (de Vries et al., 2007) and in this case generating 10,000/500/500 models.
Each of the generated models is scored using a weighted sum of electrostatic (Eelec) and van der Waals (Evdw) energies complemented by an empirical desolvation energy term (Edesolv) (Fernandez-Recio et al., 2004). The distance restraint energy term was explicitly removed from the equation in the last iteration (Edist3 = 0.0) to enable comparison of the scores between the runs that used a different number of ECs as distance restraints.
Comparison of predicted to experimental structures
All computed models in the docked set were compared to the cognate crystal structures by the RMSD of all backbone atoms at the interface of the complex using ProFit v.3.1 (http://www.bioinf.org.uk/software/profit/). The interface is defined as the set of all residues that contain any atom <6 Å away from any atom of the complex partner. For the AtpE–AtpG complex, we excluded the 2 C-terminal helices of AtpE as these helices are mobile and take many different positions relative to other ATP synthase subunits (Cingolani and Duncan, 2011). Similarly, since the DHp domain of histidine kinases can take different positions relative to the CA domain, the HK-RR complex was compared over the interface between the DHp domain alone and the response regulator partner. In the case of the unbound ubiquinol oxidase docking results, only the interface between COX2 in subunit 2 and subunit 1 was considered. Accuracy of the computed models with EC restraints was compared with computed models with center of mass restraints alone (negative controls) (Figure 3—figure supplement 1, Supplementary file 3).
Data analysis was conducted primarily using IPython notebooks (Perez and Granger, 2007). A webserver and all data are available at EVcomplex.org and the Dryad Digital Repository (Hopf et al., 2014).
Supplementary Data
(Available at http://doi.org/10.5061/dryad.6t7b8 [Hopf et al., 2014])
Supplementary data 1: Concatenated alignments for complexes predicted in this work
Supplementary data 2: Genome distance distribution of concatenated sequences per alignment
Supplementary data 3: EVcomplex predictions for evaluation and de novo set
Supplementary data 4: Docking input files and top 10 predicted models for evaluation set
Supplementary data 5: ATP synthase predictions, ATP synthase subunit a model
Supplementary data 6: UmuC model
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Decision letter
John KuriyanReviewing Editor; Howard Hughes Medical Institute, University of California, Berkeley, United States
eLife posts the editorial decision letter and author response on a selection of the published articles (subject to the approval of the authors). An edited version of the letter sent to the authors after peer review is shown, indicating the substantive concerns or comments; minor concerns are not usually shown. Reviewers have the opportunity to discuss the decision before the letter is sent (see review process). Similarly, the author response typically shows only responses to the major concerns raised by the reviewers.
Thank you for sending your work entitled "Sequence co-evolution gives 3D contacts and structures of protein complexes""" for consideration at eLife. Your article has been favorably evaluated by John Kuriyan (Senior editor), working with a member of our Board of Reviewing Editors, and 3 reviewers.
The editors and the other reviewers discussed their comments before we reached this decision, and the Reviewing editor has assembled the following comments to help you prepare a revised submission.
This manuscript describes an approach to predict 3D residue-residue contacts at protein interfaces from an analysis of multiple sequence alignments. The described computational approach (using sequence co-evolution to predict contacts between interacting proteins and to generate three-dimensional models of complex structures) appears to be quite successful and is an important contribution that is of interest to a broad audience. The analyses, results, and conclusions are similar to work conducted during the same period by Baker & coworkers, published recently in eLife. Nevertheless, it is the consensus opinion of the reviewers that the present manuscript is potentially suitable for publication in eLife as well, provided that all of the comments raised by the reviewers can be addressed satisfactorily.
We have appended the comments from the three reviewers below. Although many individual points are raised, you will see that the principal concern of the reviewers is that the manuscript falls short of providing the reader with sufficient analysis to judge the validity of the conclusions. It should be possible for you to revise the manuscript to address these concerns without much in the way of new calculations, so we hope that it should be relatively straightforward to deal with these issues.
In addition, since the Baker paper came out recently, it should be given appropriate credit in a revised version. We recommend removing the word "new" from the manuscript for this reason, and instead simply state what was done.
Reviewer 1:
1) The Abstract claims that the authors' approach can discriminate between interacting and non-interacting proteins. However, as far as I can see, the rest of the paper concerns only calculating the interface residues of proteins which are known to interact. No evidence is presented to support the claim to be able to calculate non-interactors. The authors' claim should be corrected accordingly.
2) The authors claim that the co-evolutionary approach has not previously been applied to prediction of protein-protein interfaces. This is incorrect, as the authors seem to be unaware of the previous work of Raphael Guerois' group, which seems rather surprising. The authors should acknowledge the prior work of Faure et al (2012) and Andreani et al. (2013), and they should modify their claim to novelty accordingly. It would also be appropriate to cite properly the prior work of the Baker group (Ovchinnikov et al., 2014) in the same paragraph (the authors currently mention this work only as a "note added during submission"), and the recent review by Andreani and Guerois (2014).
3) In several places the authors say that their approach exploits the fact that interacting proteins are often coded close to each other in a genome. Why is this assumption necessary? According to the description given in Methods, any pair of sequences which are presumed to interact could be concatenated and then used in the authors method. Please clarify.
4) If I understand the authors' method correctly, they first concatenate the multiple sequences of the presumed interactors, and they then use their previous approach for detecting interacting pairs of columns in the multiple alignment. In this case, when concatenating the alignments for protein "A" with those of protein "B", the "intra-EC" pairs would appear as A-A and B-B pairs, while the "inter-EC" pairs would come from A-B or B-A interactions. My question is then, are there any observable differences in the inter and intra scores? One might expect that the conservation scores would be lower for inter interactions than intra interactions, as an interface might show more "plasticity" than the core of a domain. It would be very interesting if the authors could comment on this, preferably supported by numerical results.
5) It is incorrect for the authors to refer to their own data set as a "benchmark". Suggestion: remove all references to the term "benchmark". However, if the authors make all their data available in a convenient way on-line, it could be proposed as a new "benchmark".
6) It is wrong to claim that "Experimental evidence... agrees with EVcomplex predictions" (!). Please rewrite this to say that your predictions agree with the experimental evidence. But, if this is your claim, please also support it in some way. What is the experimental evidence? How to you calculate and quantify "agrees with"?
7) Same point in the main text. "The resulting model is consistent with the topologies from cross-linking". How do you quantify consistent? RMSD from the earlier models? Please provide supporting details.
Reviewer 2:
My main comments concern the way the data in the current manuscript are analyzed and presented. While the main Figures / Table 1 currently illustrate useful examples, they should also show overall analyses of the results over all cases in the benchmark datasets. In several cases this is essential to support the stated conclusions. It does not seem that this would require a substantial amount of new work or lengthy simulations, as most of the relevant data are in the Supplementary Materials (most data are in raw table format or with a separate Figure for each example, which makes it hard for the reader to gauge overall performance – this could be presented in a more easily digestible format in the main text). Specifically, I feel the paper could be substantially improved if the following points were addressed:
1) Please support the statement "Benchmark calculations here ... indicate that the number of sequences in the alignment is critical..." with a Figure showing overall performance versus # of sequences (Figure 3 shows dependency of accuracy on relative rank for only two examples).
2) Show a main Figure comparing predicted contacts against residue distances in the 30 known crystal structures of complexes that had a sufficient number of sequences (Figure 2 only shows contact plots for two examples, and structural pictures for several more, but no overall quantification). This type of analysis could also help to support the statement "For the top ranked benchmark complexes, the majority of the top 5 ECs is correct to within 8A..." with a more quantitative analysis (Table 1 only shows 7 of the 30 complexes in the dataset; Supplementary file 3 shows all individual contact maps, but no overall quantification).
3) "... these benchmarks show ... and demonstrate the criteria needed for successful prediction of unknown interactions". To support this statement, it would be useful to have a main Figure / Table for each important criterion, analyzed over an entire benchmark set (as for the number of sequences in point 1).
4) Please add metrics to support statements that ECs are "completely consistent" with crosslinking data, or that a coupling "coincides with experimental evidence". The comparison Table in Supplementary Figure 6 could be presented in more quantitative terms in the main text (for example, what does "crosslinking neighborhood" mean?).
5) What are the possible reasons for false-positive co-variation that does not correspond to contact?
6) The manuscript deals entirely with pairwise co-evolution, and seems to neglect the possibility of higher-order complexity in the sequence pattern.
Reviewer 3:
The manuscript presents an upbeat and optimistic view of the success of the method, with a relatively small amount of critical discussion and little if any investigation of what one can learn from cases in which it does not perform as expected. (For example, a list of possible reasons for false-positive co-variation that does not correspond to contact is given, but an analysis of actual instances is not.) Likewise, a set of methods is presented that is relatively particular, and the reason why these specific methods were chosen over other possibilities is not provided. Moreover, the manuscript deals entirely with pairwise co-evolution, and seems to neglect the possibility of higher-order complexity in the sequence pattern. The manuscript could be substantially strengthened by adding analyses and insights in these issues.https://doi.org/10.7554/eLife.03430.031
Article and author information
Author details
National Institute of General Medical Sciences (R01 GM106303)
- Thomas A Hopf
- Debora S Marks
- Chris Sander
Fulbright Commission
- Charlotta P I Schärfe
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Reviewing Editor
- John Kuriyan, Reviewing Editor, Howard Hughes Medical Institute, University of California, Berkeley, United States
Publication history
- Received: May 21, 2014
- Accepted: September 23, 2014
- Accepted Manuscript published: September 25, 2014 (version 1)
- Version of Record published: November 3, 2014 (version 2)
© 2014, Hopf et al.
This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.
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text:
Soy Batik Improvisation
I've just been notified that my art quilt, Stop Fear (Homage to Sister Corita Kent) has made it into the juried SAQA exhibit, Text Messages. Yippee! This piece was principally designed using a large one-color soy wax batik that I made a few years ago -- and i've had such a good time working with it, that I'm itching to get back into the studio next week for a few day-long soy wax sessions. I am also sending out soy batik lessons this week on Joggles, part of my MORE TEXT ON TEXTILES online workshop.
Sister Corita Kent has long been an inspiration to my work, and I can't think of using text in art without thinking of her. This batik and, even more importantly, its message, speak of her influence on my life and in my work. I was first introduced to a version of traditional batik using beeswax and parafin mixture and inkodyes when I was in my 20s -- it must have been the first surface design technique I learned and the teachers were visiting Sister Corita students who worked with us in our creative arts for kids programs one summer -- maybe 1967 or '68?
Inkodyes are still around -- and they are VERY light and water fast -- but I had heard that their toxicity is higher than MX dyes, so I've left them alone in the past several decades -- the colors, too are hard to mix, since -- kind of like ceramic glazes -- the color that goes onto the fabric is very different than the color that develops in sunlight as the dyes develop. However, this site with its app for making registered three color photo prints just might change my mind! This is what the site says about toxicity:
Our dyes and resist have been certified as having no chronic toxicity by an independent toxicologist. Our products conform to the Labeling of Hazardous Art Materials Act (Conforms with ASTM D-4236). We are committed to the health and safety of everyone who enjoys Inkodye, which is why we recommend using normal precaution to prevent unnecessary spilling, skin contact and ingestion. Children love to watch the magical color development, but should not use Inkodye unsupervised. Our dyes are water-based and only require household soap and water cleanup - we recommend that you wash your hands thoroughly after use and / or use gloves to prevent prolonged skin contact and staining! We recommend using the sun to develop your prints, but if you choose to iron wet Inkodye articles indoors please work in an environment with adequate ventilation!
At any rate, if you do use inkodyes with soy batik, you can wash out the wax after the dyes develop without using an iron -- bound to be better for the lungs and environment.
Basically, soy wax can be used as with traditional batik, but you can't immerse fabric without running the risk of it dissolving too much of your wax resist -- so the technique requires that you paint on your intense low-water type dye mix (including soda ash and urea to retard drying) and then batch under plastic, removing plastic to dry the fabric, then adding another layer of wax -- or you can wash out the soy wax between colors. -- hot water and detergent turns it into soy oil that washes down the drain. Alternatively use Inkodyes, according to directions.
(PS Thanks Nina-Marie for the invitation to add this link to your off-the-wall Friday posting!
Here are a few photos of the process:
You can also use an electric skillet. Be very careful with temperatures and keep a lid handy in case things get out of hand. Do not heat the wax so hot that it smokes. Fortunately soy wax fumes are not toxic or allergenic (as is the case with paraffin and bees wax fumes).
Another way to use soy wax is to use it with a thin paint, as I am doing here. Be sure the wax is hot enough that it makes the fabric a bit transparent looking-- the wax need to go all the way through the fabric.
You can take some of the wax out by ironing first -- if you use paint, you must iron it before you put it in the washer in order to set the paint, then put your fabric in hot soapy washer in the machine. Sometimes, with heavy wax applications, it may take two trips through the washer.

subset: commoncrawl


text:
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Amgen has developed a collection of online resources available to help you learn more about areas of interest.
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Refer to approved product information for complete indication.
Aranesp® is used for the treatment of anaemia associated with chronic renal failure, or resulting from concomitantly administered chemotherapy when treating a non-myeloid malignancy.
Aranesp® Product Information
BLINCYTO® is indicated for the treatment of relapsed or refractory B-cell precursor acute lymphoblastic leukaemia (ALL). BLINCYTO® is indicated for the treatment of minimal residual disease (MRD) positive B-cell precursor acute lymphoblastic leukaemia (ALL) in patients in complete haematological remission.
Note to indication: the indications in Philadelphia positive, MRD positive and paediatric patients were approved based on phase II, non-randomised evidence. An improvement in clinical outcomes by direct prospective comparison in a randomised setting relative to other standard-of-care salvage therapies has not been established.
BLINCYTO® Product Information
IMLYGIC™ is an oncolytic immunotherapy derived from recombinantly engineered herpes simplex virus type-1 and is indicated as a monotherapy for the treatment of melanoma in patients with unresectable cutaneous, subcutaneous or nodal lesions after initial surgery.
IMLYGIC™ Product Information
KYPROLIS®, as part of combination therapy with dexamethasone or lenalidomide and dexamethasone, is indicated for the treatment of patients with relapsed or refractory multiple myeloma who have received at least one prior therapy.
KYPROLIS® Product Information
A long acting leukocyte growth factor used to decrease the duration of severe neutropenia and so reduce the incidence of infection in patients with cancer.
Neulasta® Product Information
A leukocyte growth factor used to decrease the duration of neutropenia and incidence of infection in patients with cancer, and for use in patients with severe chronic neutropenia and HIV patients with neutropenia. It is also used in mobilisation of peripheral blood progenitor cells.
NEUPOGEN® Product Information
A thrombopoietin mimetic used to treat low blood platelet counts in adults with chronic immune thrombocytopenic purpura (ITP).
Nplate® Product Information
Prolia® Product Information
Repatha™ is indicated in adults with heterozygous familial hypercholesterolaemia (HeFH) or clinical atherosclerotic cardiovascular disease (DVC) in combination with a statin or statin with other lipid lowering therapies, or in combination with other lipid lowering therapies in patients who are statin-intolerant. Repatha™ is also indicated in adults and adolescents aged 12 years and over with homozygous familial hypercholesterolaemia in combination with other lipid lowering therapies.
Repatha™ Product Information
Sensipar® is used for the treatment of secondary hyperparathyroidism in dialysis patients with end stage renal disease, hypercalcaemia in patients with parathyroid carcinoma and primary hyperparathyroidism in patients for whom parathyroidectomy is not an option.
Sensipar® Product Information
An EGFR inhibitor used to treat patients with wild-type RAS colorectal cancer that has spread to other parts of the body in combination with chemotherapy or after failure of chemotherapy.
Vectibix® Product Information
XGEVA® is indicated for the prevention of skeletal related events in patients with bone metastases from solid tumours.
XGEVA® Product Information


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LOS ANGELES --A six-inch water main ruptured Tuesday in the Leimert Park area of Los Angeles, leaving nearly two dozen Department of Water and Power customers with low water pressure, a DWP spokeswoman said.The break was reported at about 3:30 am near 43rd Place and 8th Avenue.
DWP officials say a repair crew "pinched the main" to reduce run-off. No homes or businesses were left without water. Crews were working to restore full water pressure to the affected homes.
There were no street closures due to the broken main.


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SOURCE: Viztek, LLC
February 05, 2014 08:59 ET
Continued Innovation and Ability to Quickly Adapt to the Market Support Expanded Sales
Continued Innovation and Ability to Quickly Adapt to the Market Support Expanded Sales
RALEIGH, NC--(Marketwired - Feb 5, 2014) - (HIMSS Booth #4388) -- Viztek, the leading provider of complete digital software and hardware diagnostic imaging solutions, today announced 45 percent year-over-year revenue growth from the 2011-2013 calendar years. Strong DR and PACS sales supported this, with 20 percent growth in DR, and 15 percent growth in software. The PACS replacement market was particularly strong for the company, with 75 replacements in 2013, a year when Viztek also saw a significant jump in its customer base of hospitals with more than 200 beds.
"Our innovation and ability to move quickly are fueling our remarkable growth at a time when many, to the contrary of us, are struggling in this competitive market," explained Josip Cermin, Founder and President, Viztek. "With a new FDA approval at the start of 2014, combined with an expanded DR line-up and a brand new EHR, we are poised to repeat this successful growth again in fiscal year 2014. We have a talented and energetic team fueling our surging accomplishments."
End-to-End Hospital Solutions
A key focus for Viztek during 2013 was the delivery of end-to-end digital solutions for hospitals. Instrumental to successful expansion beyond the small market and into community hospitals is a robust DR line up including the Leggera Wireless DR detector. The industry's lightest ISO 4090 cassette size wireless DR panel leap-frogged other DR detectors with its advanced technologies for image storage and unrivaled productivity. With nearly 300 software sales, 75 of which were replacement PACS installations, Viztek also saw impressive demand for its Opal suite of products, replacing legacy PACS with Viztek's Opal-RAD PACS. A new sweet spot target for PACS in 2014 will be the 200-plus bed community hospital.
New EHR to Lead 2014 Line-Up
In early December Viztek introduced the first platform-agnostic EHR solution. Compatible with MAC, Android, Windows and more, the EHR fully functions at the browser-based level regardless of the operating system or device used. With a modular design to provide facilities with practice management, billing, order entry, PACS, RIS and more, the system is Meaningful Use certified and will relieve medical practices of the costly and complicated system integrations they use today.
"As Viztek looks to 2014, we plan to leverage our core competencies in imaging and IT, combined with the many advances available today in technologic tools and infrastructure, to continue expanding our product line-up and growing our install base in the hospital market. Imaging departments and the referring physician community need advanced and reliable technologies from an innovative partner, and Viztek is answering the call," proclaimed Cermin.
For more information on Viztek, visit the company's website, or see them at HIMSS, booth 4388.
About Viztek
Viztek is a leading provider of complete digital software and hardware imaging solutions, backed by comprehensive training and support for the hospital and private practice markets. The company provides state-of-the-art, web-based Opal-RAD PACS and RIS, delivering advanced connectivity and functionality at an affordable price. The PACS and RIS solutions are complimented by Viztek's comprehensive line of CR and DR solutions, with DR panels that come in multiple configurations ranging from retrofit packages to complete new radiology rooms and mobile solutions. For more information, visit www.viztek.net.
For media inquiries, contact
Brittny Quinn203-504-8230x134Email Contact
For media inquiries, contact
Brittny Quinn203-504-8230x134Email Contact
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Madrid forward, Karim Benzema says Chelsea midfielder, Eden Hazard will be a good signing for the La Liga table toppers.
The Belgium captain has been linked with a move to Real Madrid, but a true offer is yet to materialise.
Hazard has scored 14 goals and assisted five in the premier league so far this season and Benzema will welcome him to the Santiago Bernabeu.
"[Hazard] is a very good player, what can I say?" Benzema told Telefoot.
"I have not seen the rumours, but it's obvious that he would be a good signing."
Benzema's Madrid are top of La Liga following their come-from-behind 3-2 win at Sporting Gijon on Saturday.


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Shineray X2 Transformer
Check out Shineray's latest exercise in creative advertising. First at the ChongQing Motorcycle Expo / CIMTE 2008 we saw a full sized Shineray X2 turned into a 3D parts diagram mobile. Now Shineray brings us the Shineray X2 transformer. Seems the creative department at Shineray has "more than meets the eye". Hit the break to see the video.
The First Annual EICMA China To Be Held June 11-13
June 11 through 13 is the dates of the first annual EICMA China motorcycle show. EICMA has been holding a show in Milan for many years. Now with the Chinese motorcycle market growing so rapidly with both millions of consumers and hundreds of manufacturers they felt a China based show was justified.
The event was originally planned for Canton, China but will now be in Beijing at the China National Agricultural Exhibition Center. The facility is quite impressive and should make for some great photographs of Chinese motorcycles if anyone has the budget to fly out there.
EICMA China joins the CIMA show in a short list of major Chinese motorcycle shows.
See the EICMA press release after the break.
Bennche Relieves QLINK Of US Distribution Of Megelli
The latest Megelli motorcycles are some of the sharpest looking quarter-liter bikes to hit the beginner bike scene. In 2009 QLINK had all but secured US distribution of the bikes, announced rollout plans and retail price.
Former QLINK VP Johnny Tai left the company last year and started Texas based Bennche, Inc. He and his team put together a business plan and pitched it to UK design firm Megelli and their manufacturing partner Hisun. Obviously they were impressed and now Bennche has been granted exclusive distribution of the 250r, 250s and 250m to the USA market.
TF110 Reverse Trike ATV By Yongkang Easy Vehicle Co
Zhejiang Yongkang Easy Vehicle Company took the Bombardier Spyder reverse road trike concept and brought it to the dirt with the TF110. Being familiar with the Spyder I'm not sure why this reverse trike atv caught me off guard. It seems part of the benefit of having two wheels in the back when off road would be additional contact patch with loose dirt and rough terrain. Maybe in deep ruts the centered rear wheel would hook up on high, dry ground? The rider would be in a rather uncomfortable forward leaning position with the front wheels in ruts and rear wheel not.
The concept is interesting and there must be some situations where this 3 wheeled ATV shines. Anyone have any experience riding this Chinese creation? Have any thoughts on terrain it might handle well? Please leave a comment and let me know.
TF110 Specifications
|52.4x 49.5mm
|max speed
|mini ground clearance
|seat height
|reated loading capacity
|front tire
|rear tire
|Front brake
|Disc brake
|Rear brake
|Disc brake
|Chain drive
|engine type
|110cc air cooled,single cylinder,four-stroke
|start mode
|electric start
|tank capacity
|product dimension
Source: Yongkang Easy Vehicle Co.
Galaxy Motorcycles XTR250
Galaxy Motorcycles (Foshan Galaxy Vehicle Co.,Ltd) is based in southern China, near GuangZhou, a province of GuangDong. Galaxy is a division of Yinhe (Guangdong Yinhe Motorcycle Industry Co.,Ltd). As with most Chinese motorcycle manufacturers the lineup consists of small displacement machines, the largest being the Galaxy XTR250 dualsport. The 223cc air cooled four stroke appears to be well suited for dirt use but it has a headlight and turn signals.
The USD forks on the XTR250 is adjustable for compression and rebound have 10.5″ of travel. The rear shock has 10″ of travel and is linkless as most KTM's have been in the past. This is great for simplicity but without a quality rear shock dampening will not be as consistent as a suspension system with a linkage.
Nice features include electric start, red anodized hubs and two positions to mount the handlebars. ChinaV on MyChinaMoto.com mentions the fit and finish is not up to Japanese standards but above average for a Chinese motorcycle. He also took a video of the bike in action which you can see below.
In 2008 Galaxy entered two bikes in the Taklimakan Rally, the Dakar of China, and finished 5th overall and first in it's class.
If you are in the UK check out Southern MotorCycle (SMC) and in South Africa you can find them under the Puzey name.
Galaxy XTR250 #1 from China V on Vimeo.
|Galaxy XTR250 Specs:
|Engine Type
|Single cylinder, air cooled, four stroke
|Bore/ Stroke
|Max Power
|Max Torque
|Chain drive/ international 5 gears
|Electric/ kick
|Plunger type, PZ 30
|4T 15w/40
|Primary Ratio
|Final drive
|High tensile steel tube
|Handel Bar
|Aluminum handlebar ¢22mm
|Front suspension
|Upside down, Fully Adjustable, ¢41×840mm
|Rear Suspension
|Fully adjustable, with air bag
|Suspension travel front/ rear
|Front / rear disc
|Front/ rear rims
|"1.85-21;"2.15-18" Aluminum
|Front/rear tires
|"3.00-21″;"4.60-18" Off road tire
|Stainless steel syphon,Aluminum silencer copper
|Steering head angle
|Ground clearance
|Seat Height
|Fuel tank capacity
|Approximate 2.2 gallons
|Dry weight
|Max speed
Source: MyChinaMoto.com, Foshan Galaxy Sport Vehicle Company
Benelli Due 765 At CIMA
Benelli had the Due 765 on display at this years CIMA China International Motorcycle Trade Exhibition in Chongqing, China. It appears to be production ready and offer an inline 2 cylinder alternative to the Honda Hornet, Monster 696 and Kawasaki Z750.
A few years back Benelli was bought by a Chinese company Qianjiang World who has been putting new money into the company and developing new models. The Due 765 will most likely be a 2010 model.
Source: Motorcycle.com
Zongshen Cyclone Prototype At 2009 CIMA
Kevin Duke took a trip out to the 2009 CIMA Motor Show in Chongqing, China and just published the first of a Motorcycle.com 3 part series on the 27 motorcycle manufacturers in attendance. Footage of the Loncin, Lifan, Shineray, Qingqi, Haojue, Xgjao, CFMoto and others were highlighted in the first video clip.
Most interesting was a picture of a prototype Zongshen Cyclone that takes styling queues from the Suzuki Gladius 650 and Kawasaki ER6N. It features USD forks, wave rotors and a liquid cooled single cylinder engine I am guessing displaces 500cc. This would be high displacement for a Chinese motorcycle. However there has been a push for larger displacements in the Chinese market. It has some nice details including under seat exhaust and integrated turn signals. This is a big departure from the usual parts bin fair that usually adorns Chinese bikes.
I am looking forward to more information on this bike and the rest of the MO series on the CIMA show.
Source: Motorcycle.com
Shineray X4 Enduro
A picture of a Shineray X4 was posted on Shineray China's forum. I was not able to translate any details beyond the drawing with google translation tools. It looks to be based on the Shineray X2 XY250gy-2 but has more of a Honda Transalp setup.
Shineray seems to be intent on developing it's 250 single line up with a supermoto and sportbike version of the X2. It is possible we will see similar models in a 400cc version.
via: SRMC
Maxtra Racing Is Now Haojue Racing | 125cc MotoGP Preparation Progress
After some difficulties with rights to the Maxtra name, Maxtra Racing is now Haojue Racing. That explains why we have not been able to access their web site. Personally I like when Asian brands keep their name so I am glad to see them keep the name Haojue. Looks like the team is making great progress.
Here are some quotes from a recent email release from the team about their progress towards 125cc MotoGP competition…
The Chinese Haojue 125cc GP race team made its official test debut at the Jerez three day test, making steady progress refining machine set-up and testing more new parts.
The team received a great welcome from the rest of the MotoGP paddock with many positive comments on the amount of progress we have made with the machine in just eight months since turning a wheel for the first time.
The whole email is printed below. Please be sure to sign up for the Houjue email newsletter.
March 28 2009.
Pictured: Team Haojue riders Michael Ranseder and Matt Hoyle at the Jerez test.
The Chinese Haojue 125cc GP race team made its official test debut at the Jerez three day test, making steady progress refining machine set-up and testing more new parts.
"It has been something of a baptism of fire for the team in general and for our GP rookie Matt Hoyle in particular," said team director Garry Taylor.
"Most of the machines out there have a few seasons of race development, while we are still testing new ideas, parts and set-ups. We had a lot of work to do with various engine and chassis options in a limited amount of on-track time, but we have come away with a stack of information, data and experience which will help us continue to move forward.
"For Matt it was his first time on track with GP class riders and he coped really well. Michael worked really hard, riding smoothly and consistently to give great feedback to the team. He put in so many laps that the motor reached the end of its service life and he had to miss the last session.
"The team received a great welcome from the rest of the MotoGP paddock with many positive comments on the amount of progress we have made with the machine in just eight months since turning a wheel for the first time.
"Franco collated a vast amount of engine data which he will now take back to analyse with Jan in the ongoing development programme.
"The crew are now back to the workshop in the UK to prepare for the final pre-season tests at Qatar next week and the machines are already on their way."
Michael Ranseder; "Testing is always hard work, but it is very interesting to be part of the development programme for a complete new machine. I rode well within my limits at this test in order to give consistent feedback and I am happy that we are making progress all the time."
Matt Hoyle; "Well this was the first step on what I hope is a short ladder in the move up to the top level of 125 racing. The team is working really well and we are all learning all of the time. I'm loving it!"
Shineray XY250-5A Chief 250 Sportbike
Chinese motorcycle manufacturer Shineray developed a 250cc single powered fully faired sport bike called the XY250-5A Chief 250. It is powered by the same 250cc engine as their X2 MX bike. Styling feels familiar but I can't say it is a direct copy of any bike on the market. Shineray does not currently import to the United States. There is a guy in UK working on importing them. You can contact him on Shineray Forum
Have a look at the XY250-5A spec sheet. Looks like a fast little bike.
source: My China Moto


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Slow Cooker Meatball Stew is a tasty stew that is perfect for budget minded family that is fulfilling. A recipe from Seduction in the Kitchen.
So I did something different, I went to the Fabulous Food Show here in Cleveland. This show is 10 years old and I have always wanted to go. I either wasn't allowed (ex husband the first few years), had to work, or just simply did not have the money. This year, I actually forgot about it till I was at Giant Eagle, it is a grocery store here. I was buying a gift card there and I saw the sign about the show at the service center. Giant Eagle is a sponsor of this event, I realized I was off this weekend and I actually had money, and I really wanted to go. So I went over to desk and bought my ticket.
This is the first time I gone to a food expo like this. I took my biggest purse, for the goodies, packed it with my business cards for connecting with the vendors, and I went to my first food expo. I got to say from a food blogger point of view it was interesting.
Why I say that, as I was going from booth to booth trying the samples and talking to the vendors, I noticed how it interesting the way they acted to the words: "I have a food blog and I love creating recipes using products like yours and love to showcase them, here is my card I love to connect with you." Some were very excited when I gave them my card and they talk to me and had a good time. Others I would give my card and it was like talking to a wall. You see that glazed look in the eye, I'm not sure they even knew what a food blog really is. One vendor, who had pears, I could just tell he did not have a clue of what was saying to him. Of course he was also next to main stage and Buddy from the Cake Boss had just taken the stage, so possible he just could not hear.
Then there was a few love to socialize, but not about their product. I had a few vendors I would talk to their staff and they say, this the owner there, you should talk to him. Then I would wait, and wait, and wait. The owner was deep in conversation about say football with another person. Then when they finally came over to me, I felt like the employee was more excited than owner about the product and the owner would rather go back to talking about football.
Over all, I really did enjoyed myself and got some nice goodies too! I did post photos on Instagram and Facebook here is some of the highlights:
So the stage photos, the top one is Buddy from the Cake Boss doing a demonstration there. The other is Cleveland native Michael Symon. Funny think about Chef Symon, right there he is admitting his mom and dad are from Pittsburgh, his whole family roots for Pittsburgh because they are native Pittsburghers like me. He was raised in Cleveland so he is die-hard Cleveland fan, and he is only fan of Cleveland in his Pittsburgh family. I thought that was interesting, his dirty secret, he got Pittsburgh blood in him.
I really needed this outing, as I said it perked me up out my funk. It revived me. Funny how it literally only took food to do that. I'm excited I got goodies to play around with to create recipes with. One I'm going to do today is with that Chai Syrup, thinking Banana Cupcakes with Chai Buttercream.
So since this has been good stew weather lately, I love-making it in my slow cooker. I admit I have a tiny obsession with frozen meatballs. They are perfect budget friendly item I think.
I first heard of a meatball stew on a show I like to watch called Arranged on FYI network. My favorite couple is the young couple Christian and Maria, who are Romani Gypsy. They are fascinating to watch. Maria was making a meatball stew for family and it really intrigued me. So I decided I wanted to create a meatball stew in the slow cooker. It is perfect you put on low and go about your day, like me going to the Fabulous Food Show. Come home stew is done. The flavor in this came out so nice, I'm glad I made it.
Slow Cooker Meatball Stew
- Prep Time: 10 minutes
- Cook Time: 360 minutes
- Total Time: 370 minutes
- Yield: 6 servings
- Category: Slow Cooker
- Cuisine: American
A tasty stew that is perfect for budget minded family that is fulfilling. A recipe from Seduction in the Kitchen.
- 1 bag of frozen home style meatballs
- 1 cup of baby carrots
- 3 red potatoes (cut up)
- 1 small onion (chopped)
- 2 cloves garlic (minced)
- 2 celery stalks (chopped)
- 6 oz sliced mushrooms
- 2 cups beef broth
- 1 10 oz jar Bistro Au Jus gravy
- 1 tablespoon thyme
- Green onions (chopped for garnish)
- In a slow cooker, put everything in it.
- Give it a nice stir and put it on low.
- Let cook for about 6-8 hours
- Ladle some the stew into a bowl, garnish with the green onions and ENJOY!
- Calories: 350