De novo protein design by deep network hallucination
Open accessPreprint — published as 10.1038/s41586-021-04184-w · Paper page
Abstract
There has been considerable recent progress in protein structure prediction using deep neural networks to infer distance constraints from amino acid residue co-evolution 1–3 . We investigated whether the information captured by such networks is sufficiently rich to generate new folded proteins with sequences unrelated to those of the naturally occuring proteins used in training the models. We generated random amino acid sequences, and input them into the trRosetta structure prediction network to predict starting distance maps, which as expected are quite featureless. We then carried out Monte Carlo sampling in amino acid sequence space, optimizing the contrast (KL-divergence) between the distance distributions predicted by the network and the background distribution. Optimization from different random starting points resulted in a wide range of proteins with diverse sequences and all alpha, all beta sheet, and mixed alpha-beta structures. We obtained synthetic genes encoding 129 of these network hallucinated sequences, expressed and purified the proteins in E coli, and found that 27 folded to monomeric stable structures with circular dichroism spectra consistent with the hallucinated structures. Thus deep networks trained to predict native protein structures from their sequences can be inverted to design new proteins, and such networks and methods should contribute, alongside traditional physically based models, to the de novo design of proteins with new functions.
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TL;DR
The authors invert the trRosetta structure-prediction network by Monte Carlo sampling in amino acid sequence space to “hallucinate” new 100-residue proteins with sequences unrelated to native proteins. Of 129 selected designs expressed in E. coli, 27 folded to monodisperse species with CD spectra matching the hallucinated structures, and three experimentally solved structures closely matched the models.
Key findings
- Hallucinated sequences had very low similarity to native proteins, with best BLAST E-values to UniProt of ~0.1, while their predicted structures had TM-align scores of 0.6–0.9 to native PDB structures.
- Of 129 hallucinated proteins expressed and purified from E. coli, 27 yielded SEC peaks corresponding to monomeric or small oligomeric species and CD spectra consistent with the target structures; 21 were highly thermostable with apparent melting temperatures above 70 °C, and alpha-beta designs showed no unfolding transitions up to 95 °C.
- In orthogonal Rosetta folding simulations, 82 of 129 designs had lowest-energy structures close to the hallucinated structures with Cα-RMSDs <3.0 Å, and for all 129 the design model was lower in energy than any structure obtained from an extended chain.
- Experimental structures matched hallucinated models: NMR structure of design 0515 had ~1.84 Å Cα RMSD to the model; X-ray structure of 0217 at 2.9 Å resolution had 2.53 Å backbone RMSD over all 100 residues; X-ray structure of 0738_mod at 2.4 Å resolution had 3.68 Å Cα RMSD over 96 residues, with N- and C-terminal halves aligning at 1.32 Å over 57 residues and 2.17 Å over 43 residues.
Methods
- The trRosetta network predicts inter-residue distance and orientation distributions from sequence; a background network with similar architecture but no amino acid sequence identity information provided a reference distribution. The objective combined KL-divergence of trRosetta predictions from the background with negative KL-divergence of amino acid composition from PDB frequencies, optimized by simulated annealing in sequence space with 40,000 attempted moves, starting temperature 0.1 halved every 5,000 steps, cysteines excluded, and length L = 100.
- Two thousand designs were generated, clustered by TM-score into 95 clusters at a 0.75 threshold; 129 sequences from 27 structural clusters were selected for experimental testing, expressed in E. coli BL21(DE3), purified by IMAC and size-exclusion chromatography, and characterized by circular dichroism, NMR, and X-ray crystallography.
- Structural comparisons used TM-align, Theseus superposition, and Rosetta ab initio folding simulations; crystallographic data were processed with HKL2000/Scalepack and structures solved by molecular replacement.
Limitations
- The paper states that many hallucinated proteins form oligomers in solution, likely because the network incorporated sequence features from native protein-protein interfaces; surface hydrophobic substitutions were needed to produce monomeric species in some cases.
- The paper states that design 0738_mod showed multiple monomeric conformations in slow conformational exchange, and suggests that incorporating an explicit sidechain representation in the hallucination method could reduce such structural heterogeneity.
- Evident from the text: only 27 of 129 expressed designs folded to monodisperse species, only three structures were determined experimentally, one of those required surface modification, and the study did not test designed functions.
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- Findingown data
The overall distributions of hallucinated sequences and structures are very different from those of naturally occurring proteins of the same 100-residue length used during trRosetta training.
“The overall distributions of hallucinated sequences and structures are very different from those of naturally occurring proteins of the same (100 residue) length which were used during trRosetta training (Extended Data Fig. 1a–e).”
Quote in the paperSubstantial claim - Findingown data
For all 129 hallucinated proteins, the lowest energy structure sampled starting from the design model was lower in energy than any other structure obtained starting from an extended chain.
“for all 129 the lowest energy structure sampled starting from the design model was lower in energy than any other structure obtained starting from an extended chain.”
Quote in the paperSubstantial claim - Findingown data
Hallucinated proteins resemble one another more closely than they resemble natural proteins.
“The hallucinated proteins are more similar to each other (blue lines) than they are to natural proteins (grey lines).”
Quote in the paperSubstantial claim - Findingown data
The hallucinated designs are more ideal than natural proteins in having fewer loops but longer secondary structure elements.
“Hallucinations are more ideal than natural proteins in having less loops but longer secondary structure elements.”
Quote in the paperNot a substantial claim - Findingcitation
The trRosetta deep network, trained with multiple sequence information, can consistently predict structure accurately for de novo designed proteins from a single sequence, without co-evolution information.
“we found that the trRosetta deep neural network trained using multiple sequence information could consistently predict structure quite accurately for de novo designed proteins from just a single sequence -- i.e., in the complete absence of co-evolution information3.”
Quote in the paperSubstantial claim0 of 1 sources checked- cites [3] Yang J Improved protein structure prediction using predicted interresidue orientations. Proc. Natl. Acad. Sci. U. S. A 117, 1496– 1503 (2020). 31896580 PMC6983395 source
Not checked in the free run.
- Findingown data
Structural superposition of the 0738 model and the 0738_mod crystal structure revealed a 3.68 Å Cα RMSD over 96 residues.
“Structural superposition of the 0738 model and 0738_mod crystal structure revealed a 3.68 Å Cɑ RMSD over 96 residues”
Quote in the paperSubstantial claim - Findingown data
Substitution of surface hydrophobic residues with polar residues in a subset of oligomeric hallucinations resulted in monomeric species by SEC.
“we found that substitution of surface hydrophobic residues with polar residues in a subset of the hallucinations that formed oligomers resulted in monomeric species by SEC”
Quote in the paperSubstantial claim - Findingown data
Of 129 expressed and purified hallucinated proteins, 27 yielded size exclusion chromatography peaks corresponding to monomeric or small oligomeric species.
“Of these, 27 yielded size exclusion chromatography (SEC) peaks corresponding to monomeric or small oligomeric species”
Quote in the paperSubstantial claim - Findingown data
The experimentally validated proteins span a wide range of topologies.
“The experimentally validated proteins span a wide range of topologies”
Quote in the paperSubstantial claim - Findingown data
Trajectories started from different random sequences resulted in very different sequences and structures.
“trajectories started from different random sequences resulted in very different sequences and structures (Fig 1e).”
Quote in the paperNot a substantial claim - Findingcitation
The trRosetta model predicted effects of amino acid substitutions on folding that were consistent with biophysical expectation.
“The trRosetta model also predicted effects of amino acid substitutions on folding consistent with biophysical expectation3.”
Quote in the paperSubstantial claim0 of 1 sources checked- cites [3] Yang J Improved protein structure prediction using predicted interresidue orientations. Proc. Natl. Acad. Sci. U. S. A 117, 1496– 1503 (2020). 31896580 PMC6983395 source
Not checked in the free run.
- Findingown data
The trRosetta-based design procedure shows no clear tendency to transfer sequence fragments from training-set proteins into the hallucinated designs.
“There is no apparent tendency for the trRosetta-based design procedure to “copy over” sequence fragments from the proteins in the training set into the hallucinated designs.”
Quote in the paperSubstantial claim - Findingown data
The backbone structures of hallucinated proteins resemble ideal proteins generated by de novo protein design more than native proteins.
“A striking feature of these structures is that their backbone structures resemble the “ideal” proteins generated by de novo protein design more than native proteins, despite the fact that the network was trained on the latter.”
Quote in the paperSubstantial claim - Findingown data
Superposition of the hallucinated model and the NMR medoid structure of 0515 reveals a 1.85 Å backbone RMSD over 100 residues.
“Superposition of hallucinated model (blue) and NMR medoid structure (gray) of 0515 reveal 1.85 Å backbone RMSD over 100 residues”
Quote in the paperSubstantial claim - Findingown data
The alpha-beta designs in Fig. 4 are particularly stable, with none undergoing unfolding transitions up to 95 °C.
“the alpha-beta designs in Fig. 4 are particularly stable as none undergo unfolding transitions up to 95 °C”
Quote in the paperNot a substantial claim - Findingown data
The sequences of the three hallucinated proteins whose structures were solved all have BLAST E-values worse than 0.021.
“the sequences of the three hallucinated proteins whose structures we solved here all have E-values worse than 0.021 (Suppl. Table 1).”
Quote in the paperSubstantial claim - Findingown data
Additional examples of thermostable hallucinations have CD spectra consistent with the target structure.
“Additional examples of thermostable hallucinations with CD spectra consistent with the target structure.”
Quote in the paperNot a substantial claim - Findingown data
The predicted distance maps become progressively sharper during the simulations.
“The predicted distance maps become progressively sharper during the course of the simulations”
Quote in the paperSubstantial claim - Findingown data
Different Monte Carlo trajectories starting from different random seeds converge on different sequence-structure pairs.
“different Monte Carlo trajectories starting from different random number seeds converge on different sequence-structure pairs (Fig. 2a and b).”
Quote in the paperSubstantial claim - Findingown data
After about 20,000 Monte Carlo steps, the resulting distance maps were at least as featured as those predicted for naturally occurring sequences and structurally confirmed de novo proteins designed using Rosetta.
“in almost all cases after ~20,000 Monte Carlo steps the resulting distance maps were at least as featured (non-uniform) as those predicted for naturally occurring sequences and structurally confirmed de novo proteins designed using Rosetta.”
Quote in the paperSubstantial claim - Findingown data
The similarity of hallucinated sequences to native protein sequences was very low, with best BLAST E-values to Uniprot of about 0.1.
“The similarity of the hallucinated sequences to native protein sequences was very low, with best Blast19 E-values to the Uniprot database of ~0.1 (Fig. 1f).”
Quote in the paperSubstantial claim0 of 1 sources checked- cites [19] Altschul SF, Gish W, Miller W, Myers EW & Lipman DJ Basic local alignment search tool. J. Mol. Biol 215, 403– 410 (1990). 2231712 source
Not checked in the free run.
- Findingown data
The sequence and structure subspaces spanned by the hallucinated proteins and by natural proteins of similar length (100 ± 10 aa) are quite distinct.
“These scatter plots show that subspaces spanning by hallucinated proteins and natural proteins of similar size (100 ± 10 aa) are quite distinct”
Quote in the paperSubstantial claim - Findingown data
Two thousand new proteins were generated with sequences predicted by trRosetta to fold into well-defined structures.
“We used this approach to generate two thousand new proteins with sequences predicted by the trRosetta network to fold into well-defined structures”
Quote in the paperSubstantial claim - Findingown data
Structural superposition of the two chains observed in the asymmetric unit of the 0217 crystal structure reveals a 2.8 Å backbone RMSD over 91 residues.
“Structural superposition of chains observed in the asymmetric unit reveal a 2.8 Å backbone RMSD over 91 residues.”
Quote in the paperNot a substantial claim - Findingown data
The most similar structure in the PDB to the 0738_mod structure is the de novo designed protein Top7.
“the most similar structure in the PDB to the 0738_mod structure is the de novo designed protein Top7 (Suppl. Fig. 4).”
Quote in the paperSubstantial claim - Findingown data
Twenty-one of the proteins were highly thermostable with apparent melting temperatures above 70 °C.
“Twenty-one of the proteins were highly thermostable with apparent melting temperatures above 70 °C”
Quote in the paperSubstantial claim - Findingown data
For design 0738_mod, solution data indicate multiple monomeric conformations in slow conformational exchange.
“For design 0738_mod the solution data indicate multiple monomeric conformations in solution in slow conformational exchange”
Quote in the paperSubstantial claim - Findingown data
For 0417, size-exclusion chromatography shows a minor peak for a larger oligomer, supporting the NMR result.
“Size-exclusion chromatography trace of 0417 displays a small additional peak corresponding to a larger oligomeric species which corroborates the NMR analysis.”
Quote in the paperNot a substantial claim - Findingown data
For 82 of 129 hallucinated proteins, the lowest energy structures found in Rosetta folding simulations were close to the corresponding hallucinated structures with Cα-RMSDs below 3.0 Å.
“For 82 out of 129, the lowest energy structures found in the simulations were close to the corresponding hallucinated structures with Cα-RMSDs <3.0 Å”
Quote in the paperSubstantial claim - Findingreasoning
Random 100-amino-acid sequences fed to trRosetta produce diffuse, featureless distance distributions.
“As expected for random sequences, which have a vanishingly small probability of folding to a defined structure, the distance distributions were diffuse and much less featured than those obtained with actual protein sequences.”
Quote in the paperSubstantial claim- Rests on an assumption: Random amino acid sequences have a vanishingly small probability of folding to a defined structure.
- Interpretationown data
Each of the three designs displays distinctive dynamic behavior in solution.
“These results show that the three designs have characteristic dynamics in solution.”
Quote in the paperNot a substantial claim - Interpretationreasoning
During training, the trRosetta network learned fundamental relationships between protein sequence and structure beyond co-evolution information.
“These results suggested that during training the trRosetta network was going beyond exploiting co-evolution information and learning fundamental relationships between protein sequence and structure.”
Quote in the paperSubstantial claim - Study design and conductown data
The resulting protein samples were generally more than 95% homogeneous on SDS-PAGE gels.
“The resulting samples were generally >95% homogeneous on SDS-PAGE gels.”
Quote in the paperNot a substantial claim - Premisenone given
The trRosetta network predicts distributions of distances and orientations between all pairs of residues in a set of aligned protein sequences for a protein family.
“The trRosetta network predicts distributions of distances and orientations between all pairs of residues in a set of aligned protein sequences for a protein family (Fig. 1a)”
Quote in the paperSubstantial claim - Interpretationown data
The trRosetta network does not merely reproduce natural proteins of the same length.
“the network is not simply recapitulating native proteins of the same length.”
Quote in the paperSubstantial claim - Interpretationown data
The close agreement between experimental NMR and crystal structures and the corresponding hallucinated design models for the three characterized proteins suggests that many of these proteins fold into the predicted hallucinated structures.
“The close agreement between experimental solution NMR and crystal structures with the corresponding hallucinated design models for the three proteins that we characterized in detail suggest that many of these proteins fold into the predicted hallucinated structures.”
Quote in the paperSubstantial claim - Interpretationown data
The high similarity of the NMR and crystal structures to the hallucinated models demonstrates that the hallucination process solves the classic de novo protein design problem, despite having no explicit knowledge of protein folding physics.
“The high similarity of the NMR and crystal structures to the hallucinated structure models demonstrate that the hallucination process solves the classic de novo protein design problem, despite having no explicit knowledge of the physics of protein folding.”
Quote in the paperSubstantial claim - Study design and conductnone given
Rosetta was not used in any way for either sequence generation or selection for experimental characterization.
“we did not use Rosetta in any way for either sequence generation or selection for experimental characterization”
Quote in the paperSubstantial claim - Interpretationown data
The network-hallucinated proteins can fold into a wide range of stable structures with the predicted secondary structures.
“Taken together, these data indicate that the network hallucinated proteins can fold into a wide range of stable structures with the predicted secondary structures.”
Quote in the paperSubstantial claim - Interpretationown data
For 0417, the NMR data indicate a folded structure with both alpha and beta secondary structure.
“The NMR data are consistent with a folded structure containing a mix of alpha and beta secondary structure.”
Quote in the paperSubstantial claim - Interpretationown data
The agreement between the experimental and hallucinated structures suggests the network can accurately generate protein backbones and sequences that encode them.
“The agreement observed between these two experimental and hallucinated structures suggests the network can accurately generate protein backbones and sequences that encode them.”
Quote in the paperSubstantial claim - Interpretationown data
According to the Rosetta physically-based energy model, the network-generated sequences encode the corresponding structures.
“Thus according to the Rosetta physically-based energy model, the network generated sequences do indeed encode the corresponding structures.”
Quote in the paperSubstantial claim - Interpretationown data
A deep network trained only on natural sequences and structures can generalize to create new proteins with sequences unrelated to natural proteins that fold into stable structures.
“Our results demonstrate that a deep neural network trained exclusively on native sequences and structures can generalize to create new proteins, with sequences unrelated to those of native proteins, that fold into stable structures.”
Quote in the paperSubstantial claim - Interpretationreasoning
During training on large numbers of irregular native protein structures, the deep network learned to encode ideal protein structure properties similar to expert protein designers using traditional approaches.
“During training on large numbers of (irregular) native protein structures, the deep neural network evidently learned to encode ideal protein structure properties very similar to expert protein designers using more traditional scientific approaches”
Quote in the paperSubstantial claim
- May combine two claims. 30
- The quote only partly states this claim. 2
- Classified as probably not a substantial claim. 5
May combine two claims.
Findingown dataFor the 27 proteins, circular dichroism spectra were consistent with the target structures, with characteristic profiles of all-alpha helical proteins for all-alpha designs and of alpha-beta proteins for alpha-beta designs.
“In all cases, the CD spectra were consistent with the target structures (Figs. 3e, 4e and Extended Data Fig. 3e,k), with the characteristic profiles of all alpha helical proteins for the all alpha helical designs (Fig. 3e and Extended Data Fig. 3e), and of alpha-beta proteins for the alpha-beta designs”
Quote in the paperSubstantial claimThe quote only partly states this claim.
Findingown dataAll experimentally validated protein sequences are predicted by Rosetta large-scale energy calculations to have funneled landscapes leading into the target structure.
“all of the sequences are predicted by Rosetta large scale energy calculations to have funneled landscapes leading into the target structure”
Quote in the paperNot a substantial claimMay combine two claims.
Findingown dataThe surface-modified hallucination 0738_mod yielded crystals, and its 2.4 Å resolution structure was determined.
“One of these surface-modified hallucinations, 0738_mod, yielded crystals and the 2.4 Å resolution structure was determined.”
Quote in the paperSubstantial claimMay combine two claims.
Findingown dataBoth de novo designed proteins and the hallucinated proteins have regular alpha helices and beta sheets and lack long loops and other idiosyncrasies of native protein structures.
“Both de novo designed proteins and the hallucinated proteins generated here have regular alpha helices and beta sheets, and lack the long loops and other idiosyncrasies of native protein structures”
Quote in the paperSubstantial claimMay combine two claims.
Findingown dataThe 2.9 Å resolution crystal structure of design 0217 revealed a 3-helix bundle with an overall fold similar to the hallucinated model, with a backbone RMSD of 2.53 Å over all 100 residues.
“We also succeeded in determining a 2.9 Å resolution crystal structure of design 0217, which revealed a 3-helix bundle with an overall fold similar to the hallucinated model; the backbone RMSD between model and crystal structure is 2.53 Å over all 100 residues”
Quote in the paperSubstantial claimMay combine two claims.
Findingown dataThe homodimer 0217 has a lower average heteronuclear NOE than 0515 and 0738_mod, and it shows fewer peaks than expected because of exchange broadening.
“The average hetNOE for the homodimer 0217 is lower than for 0515 and 0738_mod, and it has fewer peaks than expected due to exchange broadening.”
Quote in the paperNot a substantial claimMay combine two claims.
Findingown dataDespite register shifts, the N- and C-terminal halves of the 0738_mod crystal structure align remarkably well to the corresponding regions in the hallucinated model, with backbone RMSDs of 1.32 Å over 57 residues and 2.17 Å over 43 residues, respectively.
“the N- and C-terminal halves of the crystal structure align remarkably well to the corresponding regions in the hallucinated model with backbone RMSDs of 1.32 Å over 57 residues, and 2.17 Å over 43 residues for the N-terminal and C-terminal half, respectively”
Quote in the paperSubstantial claimMay combine two claims.
Findingown dataNMR data on design 0417 are consistent with the alpha/beta fold of the hallucinated model, and SEC and NMR relaxation measurements indicate it is primarily monomeric with some transient self-association in solution.
“NMR data on design 0417 are consistent with the alpha/beta fold of the hallucinated model, and both SEC and NMR relaxation measurements (15N T1 ~730 ms, 15N T2 ~77 ms, and τc ~10.4 ns at 25 °C, Extended Data Fig. 6) indicate that it is primarily monomeric but with some transient self-association in solution.”
Quote in the paperNot a substantial claimMay combine two claims.
Findingown dataDesign 0217 forms a dimer in the crystal structure, consistent with SEC-MALS analysis and NMR rotational correlation time measurements.
“For example, 0217 forms a dimer in the crystal structure (Extended Data Fig. 5), consistent with SEC-MALS analysis and NMR rotational correlation time measurements”
Quote in the paperNot a substantial claimMay combine two claims.
Findingown dataThe predicted structures resemble but are not identical to native structures in the PDB, with TM-align scores of 0.6-0.9.
“the predicted structures resemble but are not identical to native structures in the PDB, with TM-align scores of 0.6–0.9 (Fig. 1g).”
Quote in the paperSubstantial claimThe quote only partly states this claim.
Findingown dataMonte Carlo optimization of KL divergence between trRosetta-predicted distance distributions and background distributions yields novel proteins spanning a wide range of sequences and predicted structures.
“Optimization from different random starting points results in novel proteins spanning a very wide range of sequences and predicted structures.”
Quote in the paperSubstantial claimMay combine two claims.
Findingown dataThe 0217 model displays amphipathic sequence patterning across the 3-helix topology, with numerous solvent-exposed hydrophobic residues that mediate the dimer contacts in the crystal structure.
“the 0217 model displays clear amphipathic sequence patterning across the 3-helix topology, with numerous solvent-exposed hydrophobic residues (Suppl. Fig. 2) that mediate the dimer contacts in the crystal structure.”
Quote in the paperSubstantial claimMay combine two claims.
Findingown dataSolution data for designs 0515 and 0217 suggest well-ordered structures in solution, with internal dynamics typical of small natural proteins.
“The solution data for designs 0515 and 0217 (dimer) suggest well-ordered structures in solution, with internal dynamics typical of small natural proteins.”
Quote in the paperSubstantial claimMay combine two claims.
Findingown dataA single molecular replacement solution for 0217 was obtained in space group I 2 3 using one ab initio model; it had two copies per asymmetric unit and a TFZ score of 13.3, while no other model scored above 8.
“A single solution was found in I 2 3 from one of the ab initio models with two copies in the asymmetric unit and a TFZ score of 13.3 (no other model yielded a TFZ score >8).”
Quote in the paperSubstantial claimMay combine two claims.
Findingown dataSEC data demonstrate that the proteins are monodisperse in solution, with 0515 and 0738_mod predominantly monomeric and 0217 predominantly dimeric.
“SEC data demonstrating monodispersity of these proteins in solution, with predominantly monomer for 0515 and 0738_mod and predominantly dimer for 0217.”
Quote in the paperSubstantial claimMay combine two claims.
Findingown dataThe accuracy of the 0738_mod structure does not reflect PDB memorization; the closest BLAST hits in the PDB for the N- and C-terminal halves have E-values of 0.29 and 0.63, respectively.
“The accuracy does not reflect PDB memorization; the closest BLAST hits in the PDB for the N and C terminal halves have E-values of 0.29 and 0.63, respectively.”
Quote in the paperSubstantial claimMay combine two claims.
Findingown dataTwo thousand proteins were generated using the hallucination procedure and compared structurally using TM-score; average-linkage hierarchical clustering yielded 95 clusters with an average inter-cluster similarity of TM-score 0.75.
“Two thousand proteins were generated using the hallucination procedure described above, and structurally compared to each other using the template modeling score (TM-score)29. Average-linkage hierarchical clustering yielded 95 clusters with an average inter-cluster similarity of TM-score = 0.75.”
Quote in the paperSubstantial claim0 of 1 sources checked- cites [29] Zhang Y & Skolnick J TM-align: a protein structure alignment algorithm based on the TM-score. Nucleic Acids Res. 33, 2302– 2309 (2005). 15849316 PMC1084323 source
Not checked in the free run.
Probably not a substantial claim: may not pass S3 (load-bearing).
Findingnone givenOne hundred twenty-nine hallucinated sequences from the remaining 27 structural clusters were selected for experimental testing.
“One-hundred twenty-nine hallucinated sequences from the remaining 27 structural clusters (no more than 10 designs per cluster) were selected for experimental testing.”
Quote in the paperNot a substantial claimMay combine two claims.
Findingown dataThe hallucinated structures span all-alpha, all-beta, and mixed alpha-beta fold classes, with 95 different sub-folds at a TM-score clustering threshold of 0.75.
“The structures span all alpha, all beta and mixed alpha-beta fold classes, with 95 different sub-folds at a TM-score clustering threshold of 0.75.”
Quote in the paperSubstantial claimMay combine two claims.
Findingown dataThe solution NMR structure of design 0515 is a monomeric antiparallel four-helix bundle, and the ensemble of 20 structures has a Cα RMSD to the hallucinated model of about 1.84 Å.
“We determined the solution NMR structure for design 0515 to be a monomeric antiparallel four-helix bundle (1D estimated 15N T1 ~ 780 ms, 15N T2 ~ 77 ms, and τc ~ 9.6 ns at 25 °C); the ensemble of 20 structures had a Cɑ root mean square deviation (RMSD) to the hallucinated model of ~1.84 Å”
Quote in the paperSubstantial claimMay combine two claims.
Findingown dataFor 0217, molecular replacement with trRosetta and classical Rosetta ab initio models both produced converged ensembles on one topology, but the ab initio set showed substantial internal diversity.
“While both sets of predictions yielded converged ensembles on a single topology, the classical ab initio models had significant diversity within that ensemble.”
Quote in the paperNot a substantial claimMay combine two claims.
Findingown data0738_mod has a heteronuclear NOE distribution similar to that of monomeric 0515, but it shows over twice the expected number of peaks, which suggests at least two folded conformations in solution undergoing slow exchange on the NMR timescale.
“Although 0738_mod has a similar hetNOE distribution as monomeric 0515, it has more than double the expected number of peaks, indicating at least two folded conformations (for all or parts of the protein) in solution that are in slow conformational exchange on the NMR time-scale.”
Quote in the paperNot a substantial claimMay combine two claims.
Study design and conductown dataSDS-PAGE data show that each of the three designs is more than 95% homogeneous, which together with MALDI-TOF mass spectrometry indicates that the observed spectral heterogeneity is not due to chemical heterogeneity.
“SDS-PAGE data (not shown) show that each is >95% homogeneous, which together with MALDI-TOF mass spectrometry indicate that the spectral heterogeneity observed is not due to chemical heterogeneity.”
Quote in the paperSubstantial claimMay combine two claims.
PremisecitationRandom sequences give diffuse trRosetta predictions, while existing de novo designs produce peaked distributions with low variance.
“Random sequences give diffuse predictions, while existing de novo designs produce peaked distributions with low variance3.”
Quote in the paperSubstantial claim0 of 1 sources checked- cites [3] Yang J Improved protein structure prediction using predicted interresidue orientations. Proc. Natl. Acad. Sci. U. S. A 117, 1496– 1503 (2020). 31896580 PMC6983395 source
Not checked in the free run.
May combine two claims.
Study design and conductreasoningThe Rosetta folding test is completely orthogonal because the network was trained exclusively on native protein structures and has no access to the Rosetta energy function.
“This is a completely orthogonal test as the network was trained exclusively on native protein structures, and has no access to the Rosetta energy function.”
Quote in the paperSubstantial claimProbably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.
Study design and conductown dataFor the 0738_mod crystal structure, the final Ramachandran favored and outlier percentages were 96% and 0%, respectively.
“For 0738_mod refinement, final Ramachandran favored and outliers were 96% and 0%, respectively.”
Quote in the paperNot a substantial claimProbably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.
Study design and conductown dataFor the 0217 crystal structure, the final Ramachandran favored and outlier percentages were 99% and 0%, respectively.
“For 0217 final Ramachandran favored and outliers were 99% and 0%, respectively.”
Quote in the paperNot a substantial claimMay combine two claims.
Interpretationown dataMany hallucinated proteins are monomeric, stable, have the expected secondary structure, and are strongly predicted by Rosetta to fold to the target structure in orthogonal calculations.
“Many of the hallucinated proteins are monomeric, stable, have the expected secondary structure, and are strongly predicted to fold to the target structure by Rosetta in completely orthogonal calculations”
Quote in the paperSubstantial claimMay combine two claims.
Interpretationown dataThe network appears to incorporate sequence features associated with protein-protein interfaces of native oligomeric proteins in the PDB training set, likely explaining why many hallucinated proteins self-associate.
“The network appears to incorporate sequence features associated with the protein-protein interfaces of the native oligomeric proteins included in the PDB training set, likely explaining why many of the network hallucinated proteins self associate.”
Quote in the paperNot a substantial claimMay combine two claims.
Interpretationown dataEven under optimized conditions, 0417 shows evidence of exchange broadening, temperature- and glycerol-dependent resonances, and doubled resonances, all indicating transient self-association.
“Even under optimized conditions, there is still evidence of exchange broadening (e.g. Trp side chain NεHs are weak), resonances that appear only at high temperature and high glycerol concentrations, and some resonances that are doubled; all indications of transient self-association.”
Quote in the paperNot a substantial claimMay combine two claims.
Study design and conductown dataThe 0515 sample used for structure determination was over 98% pure by SDS-PAGE and over 95% labeled with isotopes according to MALDI-TOF mass spectrometry.
“This sample was >98% homogeneous by SDS-PAGE analysis, and >95% isotope enriched based on MALDI-TOF mass spectrometry.”
Quote in the paperNot a substantial claimClassified as probably not a substantial claim.
PremisecitationIn benchmark tests, the trRosetta network outperformed other methods.
“in benchmark tests this network outperformed other methods3.”
Quote in the paperSubstantial claim0 of 1 sources checked- cites [3] Yang J Improved protein structure prediction using predicted interresidue orientations. Proc. Natl. Acad. Sci. U. S. A 117, 1496– 1503 (2020). 31896580 PMC6983395 source
Not checked in the free run.
May combine two claims.
PremisecitationThe trRosetta network is a 2D residual-convolutional neural network that takes 1- and 2-site features from a multiple sequence alignment or a single sequence and outputs probabilistic distance and orientation distributions for all residue pairs.
“The trRosetta protein structure prediction network, described in detail elsewhere3, is a 2D residual-convolutional neural network which takes 1- and 2-site features derived from a multiple sequence alignment or a single sequence as an input and produces a 2D output describing distances and orientations for all residue pairs in a protein in a probabilistic manner”
Quote in the paperSubstantial claim0 of 1 sources checked- cites [3] Yang J Improved protein structure prediction using predicted interresidue orientations. Proc. Natl. Acad. Sci. U. S. A 117, 1496– 1503 (2020). 31896580 PMC6983395 source
Not checked in the free run.
May combine two claims.
Study design and conductown dataFor 0738_mod, the two-conformation interpretation was supported by new peaks at 288 K, altered peaks at 308 and 318 K, and by 15N ZZ-exchange cross peaks detected at 318 K using 600 and 750 ms mixing times.
“This was further validated by the appearance of new peaks in spectra at lower temperature (288K), and different peaks at higher temperatures (308 and 318K), and confirmed by detection of 15N ZZ-exchange cross peaks at 318K with 600 and 750 ms mixing times (Bruker pulse sequence hsqcetexf3gp, data not shown)58.”
Quote in the paperSubstantial claim0 of 1 sources checked- cites [58] Montelione GT & Wagner G 2D Chemical exchange NMR spectroscopy by proton-detected heteronuclear correlation. Journal of the American Chemical Society vol. 111 3096– 3098 (1989).
Not checked in the free run.
Probably not a substantial claim: may not pass S3 (load-bearing).
Study design and conductown dataThe NMR samples were each about 98% or more homogeneous based on SDS-PAGE.
“These samples were each >~98% homogeneous, based on SDS-PAGE.”
Quote in the paperNot a substantial claim
The paper relies on this without support.
Random amino acid sequences have a vanishingly small probability of folding to a defined structure.
“As expected for random sequences, which have a vanishingly small probability of folding to a defined structure”
The authors state this as an assumption.
The background network's predictions can be viewed as representing a generic molten globule state.
“this can loosely be viewed as representing a generic “molten globule” state”
Claims other papers make about this one
Each is checked against this paper’s own text: does it say what the citing paper says it does?
Deep learning methods such as AlphaFold and trRosetta, which extract information from the PDB, outperform traditional approaches that explicitly model the folding process.
“The bi-annual Critical Assessment of Structure (CASP) meetings have demonstrated that deep learning methods such as AlphaFold (1, 2) and trRosetta (3), that extract information from the large database of known protein structures in the PDB, outperform more traditional approaches that explicitly model the folding process.”
From Accurate prediction of protein structures and interactions using a 3-track network (2021) · cites this paper as [2]
The Introduction (page not provided) says deep networks improved protein-structure prediction accuracy, notes an AlphaFold observation at CASP13, and reports that trRosetta outperformed other methods in benchmark tests. The Conclusion says the network learned from native protein structures and contrasts it with physically based approaches. The paper does not establish the claimed CASP comparison in which both AlphaFold and trRosetta outperform approaches that explicitly model folding. (LLM, conf=0.82)
Experimental — accuracy not yet measuredCrystal and NMR structures confirm that hallucinated sequences fold to the hallucinated structures.
“crystal and NMR structures confirm that the hallucinated sequences fold to the hallucinated structures (12).”
From Scaffolding protein functional sites using deep learning (2022) · cites this paper as [12]
The trRosetta structure-prediction neural network can be used to generate new proteins by maximizing the trRosetta output probability that a sequence folds to some unspecified three-dimensional structure during Monte Carlo sampling in sequence space.
“We previously demonstrated that the trRosetta structure-prediction neural network (11) can be used to generate new proteins by maximizing the trRosetta output probability that a sequence folds to some (unspecified) three dimensional structure during Monte Carlo sampling in sequence space (12).”
From Scaffolding protein functional sites using deep learning (2022) · cites this paper as [12]
There has been recent success in applying the hallucination technique to general protein design.
“There has also been recent success in applying the hallucination technique to general protein design [72].”
From Computational and artificial intelligence-based methods for antibody development (2023) · cites this paper as [72]
The RoseTTAFold approach has allowed for de novo design through network hallucination by training a similar model specially for inpainting portions of proteins given context.
“The competing RoseTTAFold approach has allowed for de novo design through network hallucination by training a similar model specially for inpainting portions of proteins given context [72,83].”
From Computational and artificial intelligence-based methods for antibody development (2023) · cites this paper as [72]
Self-check
Percentages, sums and reported differences re-computed from the paper’s own numbers.
Each contradiction between two passages listed here was confirmed by an independent check.
The reported resolution for 0217 differs from its table entry
MinorThe text reports a 2.9 Å crystal structure for 0217, but the crystallographic table gives its data-collection resolution as 3.0 Å.
“We also succeeded in determining a 2.9 Å resolution crystal structure of design 0217”
“| Resolution (Å) | 47.8–3.0 (3.1–3.0)b | 50.0–2.4 (2.49–2.40) |”
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- Authors
- Anishchenko, I., Chidyausiku, T. M., Ovchinnikov, S., Pellock, S. J., & Baker, D.
- Affiliations
- University of Washington (US)
- Howard Hughes Medical Institute (US)
- Corresponding author
- Baker, David
- Venue
- Nature
- Publisher
- openRxiv
- Published
- 2020-07-23
- Volume
- 600
- Issue
- 7889
- Pages
- 547-552
- ISSN
- 0028-0836 (ISSN-L)
- PMC
- PMC9293396
- OpenAlex
- W3216341763
- Cited by
- 629
- References
- 61
- Citations by year
- 2026: 102 · 2025: 120 · 2024: 139 · 2023: 122 · 2022: 110 · 2021: 29 · 2020: 6
- FWCI
- 31.36
- Type
- journal article
- Language
- en
- Open access
- green
- doi.org
- Licence
- Locations
- Nature (publishedVersion)
- bioRxiv (Cold Spring Harbor Laboratory) (acceptedVersion, cc-by-nc-nd)
- PubMed (publishedVersion)
- PubMed Central (acceptedVersion)
- RePEc: Research Papers in Economics (submittedVersion)
- Funders
- Howard Hughes Medical Institute
- Novo Nordisk Fonden (NNF18OC0030446)
- National Institutes of Health (1s10od021527-01, DP5OD026389)
- National Institute of General Medical Sciences (R01GM120574, R35GM141818, P30GM124165)
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Protein Structure and Dynamics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Machine Learning in Bioinformatics
- Physical Sciences › Computer Science › Computational Theory and Mathematics › Computational Drug Discovery Methods
- Keywords
- de novo protein design, deep neural network, hallucinations, protein structure prediction, Kullback-Leibler divergence, synthetic proteins, protein folding, circular dichroism, X-ray crystallography, nuclear magnetic resonance
- MeSH terms
- Amino Acid Sequence, Hallucinations, Humans, Protein Conformation, Proteins, Neural Networks, Computer, Crystallography, X-Ray
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