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Cited by 27Bioinformatics and Genomic NetworksProtein Structure and DynamicsGenomics and Phylogenetic Studies

Functions of Essential Genes and a Scale-Free Protein Interaction Network Revealed by Structure-Based Function and Interaction Prediction for a Minimal Genome

Open access
Zhang, Chengxin, Zheng, Wei, Cheng, Micah, Omenn, Gilbert S., Freddolino, Peter L., Zhang, Yang·2021·Journal of Proteome Research
DOI 10.1021/acs.jproteome.0c00359PMC PMC7867644
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Abstract

When the JCVI-syn3.0 genome was designed and implemented in 2016 as the minimal genome of a free-living organism, approximately one-third of the 438 protein-coding genes had no known function. Subsequent refinement into JCVI-syn3A led to inclusion of 16 additional protein-coding genes, including several unknown functions, resulting in an improved growth phenotype. Here, we seek to unveil the biological roles and protein-protein interaction (PPI) networks for these poorly characterized proteins using state-of-the-art deep learning contact-assisted structure prediction, followed by structure-based annotation of functions and PPI predictions. Our pipeline is able to confidently assign functions for many previously unannotated proteins such as putative vitamin transporters, which suggest the importance of nutrient uptake even in a minimized genome. Remarkably, despite the artificial selection of genes in the minimal syn3 genome, our reconstructed PPI network still shows a power law distribution of node degrees typical of naturally evolved bacterial PPI networks. Making use of our framework for combined structure/function/interaction modeling, we are able to identify both fundamental aspects of network biology that are retained in a minimal proteome and additional essential functions not yet recognized among the poorly annotated components of the syn3.0 and syn3A proteomes.

AI snapshot

Written from the paper’s full text.

TL;DR

This study applied contact-assisted structure prediction and structure-based function/interaction annotation to the JCVI-syn3.0 minimal genome, assigning Gene Ontology functions to many previously unannotated proteins. It also reconstructed a predicted protein–protein interaction network that retained a scale-free degree distribution despite the genome’s artificial reduction.

Key findings

  • The C-I-TASSER/COFACTOR pipeline confidently assigned molecular function and biological process GO terms to 373 and 382 syn3.0 proteins, corresponding to 86% and 88% of the proteome; among proteins classified as unknown by TIGRfam, it annotated 49% with MF terms and 45% with BP terms, versus 5% for each category in UniProt.
  • Contact-assisted models had an average estimated TM-score of 0.76, 8.6% higher than the 0.70 average for I-TASSER models without contact prediction; 328 of 434 proteins (76%) were estimated to have better models under C-I-TASSER.
  • Previously unannotated proteins were enriched for transporter activity and phosphatase activity (MF) and for response to other organism and dephosphorylation (BP). Case studies included MMSYN1_0877 as a probable riboflavin/vitamin transporter (MF C-score 0.82; structural similarity to RibU with TM-score 0.72) and MMSYN1_0440 as a hyaluronic acid binding protein (MF C-score 0.91; BP “response to other organism” C-score 0.57).
  • The predicted PPI network was sparse, with 2,483 of 95,703 protein pairs (2.6%) predicted to interact, yet its node-degree distribution followed a power law with τ = 1.40, unlike a random network with the same number of pairs; the authors compare this to E. coli’s scale-free network with τ = 1.3.

Methods

  • Structures for all 438 syn3.0 protein-coding genes were predicted with C-I-TASSER, which combines deep-learning residue–residue contact maps with I-TASSER assembly; a control set used I-TASSER without contact prediction. Function annotation used COFACTOR, integrating structure-based, sequence-based, and PPI-based submodules, with C-score > 0.5 treated as confident; GO enrichment was tested by a rate-ratio approach after excluding overly generic terms.
  • PPI prediction used SPRING dimer threading, calling an interaction when the SPRING-score exceeded 2 and both monomer threading Z-scores exceeded −2; only heterodimeric interactions were considered. Cutoffs were trained on 1,732 structurally characterized interacting pairs and 4,117 noninteracting pairs from DIP.
  • Input data came from NCBI accession CP014940.1, covering 438 protein-coding genes; proteins were mapped to UniProt 2019_09 M. mycoides entries, with GO annotations from UniProt-GOA release 2019-09-17. The 16 additional protein-coding genes of JCVI-syn3A were included on the webserver, but the main analysis focused on syn3.0.

Limitations

  • The paper states that 14% of syn3.0 proteins for MF and 12% for BP could not be annotated with specific terms, partly because many targets are highly transmembrane; native structures were unavailable, so model quality was estimated rather than measured directly; only heterodimeric PPIs were considered; and the main analysis centers on syn3.0, with syn3A additions only displayed online.
  • Evident from the text: all functions and interactions are computational predictions rather than experimental validations, and the approach depends on existing sequence, structure, and interaction databases, which may limit sensitivity for proteins without suitable templates. The case-study ligand assignments are supported by docking and structural comparisons, but the paper does not report direct biochemical assays for those specific predictions.

Model-written summary. Check it against the paper before relying on it.

Integrity signals

Registry

Crossref · 10.1021/acs.jproteome.0c00359

Cites

70 of 70 references verified0 citation errors · 0 not checkable

Statistics

Nothing to recompute.

Claims the paper makes

0 of 35 cited sources checked · 0 supported · 0 flagged

  • Findingown data

    Deep learning contact-map information improves both the coverage and accuracy of protein structure and function models.

    “while the utilization of deep neural-network contact-map information shows significant enhancements of both coverage and accuracy of protein structure and functional models.”
    • Also stated in: § Contact-Assisted Protein Structure Prediction and Structure-Based Function Prediction Increase the Coverage of Function Annotation · plain
      “the confidence score of COFACTOR GO term prediction is consistently improved by using structure models from contact-assisted C-I-TASSER over the traditional I-TASSER approach for all three aspects of GO terms (Figure 1F–H).”
    Quote in the paperSubstantial claim
  • Findingown data

    For all five original annotation-confidence classes, C-I-TASSER/COFACTOR assigns GO terms to more syn3.0 proteins than UniProt does.

    “for all five classes, the numbers of proteins for which GO terms can be assigned by the structure-based function annotation pipeline C-I-TASSER/COFACTOR are consistently greater than those in UniProt.”
    Quote in the paperSubstantial claim
  • Findingown data

    The reconstructed protein interaction network of syn3.0 has a scale-free degree distribution like naturally evolved bacterial PPI networks.

    “our reconstructed PPI network still shows a power law distribution of node degrees typical of naturally evolved bacterial PPI networks.”
    • Also stated in: § INTRODUCTION · plain
      “the PPI network of syn3.0 follows the scale-free network architecture often noted in natural PPIs but rare in randomly formed networks”
    Quote in the paperSubstantial claim
    • Rests on an assumption: The SPRING-predicted protein interaction network for syn3.0 reflects the organism's actual protein-protein interactions closely enough to infer network architecture.
  • Findingown data

    Functions involving nutrient acquisition, microbe-host interactions, and nucleotide metabolism are enriched among syn3.0 genes that were previously unannotated.

    “Functions related to nutrient acquisition, microbe–host interactions, and nucleotide metabolism are enriched among the set of previously unannotated genes”
    • Also stated in: § Functions Enriched in Uncharacterized Proteins Highlight the Dependency of syn3.0 on the Environment · plain
      “UniProt unannotated proteins are enriched for “transporter activity” and “phosphatase activity” for MF and “response to other organism” and “dephosphorylation” for BP (Figure 2).”
    Quote in the paperSubstantial claim
  • Findingown data

    112 of the 438 syn3.0 proteins (26%) are unannotated according to UniProt-GOA entries.

    “112 (26%) of the 438 proteins in syn3.0 are unannotated based on their UniProt entries.”
    Quote in the paperSubstantial claim
  • May combine two claims. 26
  • Classified as probably not a substantial claim. 13

  • May combine two claims.

    Findingown data

    The C-I-TASSER/COFACTOR pipeline provides high-confidence molecular function and biological process GO annotations for 86% and 88% of the syn3.0 proteome, respectively.

    “We found that high-confidence molecular function (MF) and biological process (BP) annotations from gene ontology (GO) can be provided for 86 and 88% of the syn3.0 proteome, respectively”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    C-I-TASSER/COFACTOR annotates 49% of unknown syn3.0 proteins with specific MF terms and 45% with specific BP terms.

    “C-I-TASSER/COFACTOR can annotate 49 and 45% of all unknown proteins with specific MF and BP terms, respectively”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    COACHD docking identified riboflavin, dATP, and imidazole as potential ligands for MMSYN1_0877, with X-SCORE binding energies of -8.08, -6.73, and -5.17 kcal/mol, suggesting riboflavin is the most likely ligand.

    “we ran COACHD,63 which identified three potential ligands: riboflavin (RBF), dATP (DTP), and imidazole (IMD). The ligand–protein binding energies for the docked poses, as estimated by X-SCORE,64 were −8.08, −6.73, and −5.17 kcal/mol, respectively, suggesting that riboflavin is the most likely ligand for this protein.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    UniProt-unannotated syn3.0 proteins are enriched for transporter activity and phosphatase activity among MF terms, and for response to other organism and dephosphorylation among BP terms.

    “UniProt unannotated proteins are enriched for “transporter activity” and “phosphatase activity” for MF and “response to other organism” and “dephosphorylation” for BP (Figure 2).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    14% of syn3.0 proteins cannot be assigned specific MF terms and 12% cannot be assigned specific BP terms.

    “there are still 14 and 12% of the syn3.0 proteins that cannot be annotated with specific MF and BP terms, respectively”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    The C-I-TASSER model of MMSYN1_0877 is a multipass transmembrane helix bundle with estimated TM-score 0.59 and a recognized riboflavin ligand.

    “The C-I-TASSER structure model exhibits a multipass transmembrane helix bundle with an estimated TM-score of 0.59 (indicating correct topological fold39), with a riboflavin (i.e., vitamin B2) ligand recognized by COFACTOR.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    In the alignment of MMSYN1_0877 with riboflavin transporter structures, the position corresponding to a residue in 3P5N is conserved in five of the compared crystal structures, with the only mismatch being glutamate in MMSYN1_0877 and aspartate in the two crystallized riboflavin transporters.

    “but no other crystal structures in the alignment, the residue in the equivalent position for MMSYN1_0877 is identical for five of them, and one mismatch is glutamate in MMSYN1_0877 but aspartate in the two crystallized riboflavin transporters”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    At multiple substrate-contacting alignment positions, MMSYN1_0877 is more similar to riboflavin transporters than to other structures; at position 71, MMSYN1_0877 and 5KBW have lysine while 3P5N has threonine.

    “MMSYN1_0877 also resembled the riboflavin transporters more closely at several other such substrate-contacting positions, for example, position 71 in the alignment (K in MMSYN1_0877 and 5KBW but T in 3P5N).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    The annotation work produced confident MF and BP models for 373 and 382 syn3.0 proteins, respectively, corresponding to 86% and 88% of the previously unannotated proteome.

    “providing confident MF and BP models for 373 and 382 syn3.0 proteins, which represent, respectively, 86 and 88% of the proteome that were previously unannotated.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    The power-law fit for the syn3.0 PPI network has τ = 1.40, with reduced χ-squared near 0 and coefficient of determination near 1.

    “A high goodness-of-fit is achieved with the parameter τ = 1.40, resulting in the reduced χ-squared statistics and the coefficient of determination and approaching 0 and 1, respectively (Figure 4B,C).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    The syn3.0 PPI network is scale-free, with the fraction of proteins having k partners following a power-law distribution.

    “However, we found that the PPI network is actually scale-free: P(k), the fraction of proteins in the network having k partners, follows a power law distribution”
    • Also stated in: § DISCUSSION AND CONCLUSIONS · plain
      “An unexpected discovery of this study is that the artificially reduced minimal syn3.0 genome retains a scale-free PPI network, similar to other naturally occurring PPI networks such as that of E. coli.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    The syn3.0 PPI network differs significantly from a random network with the same numbers of positive (2483) and total (95,703) protein pairs, whose degree distribution fits a power law poorly, with reduced χ-squared above 1.5 and coefficient of determination below 0.

    “This is significantly different from a randomly generated PPI network with the same number of positive (2483) and total (95,703) protein pairs (Figure S3), where the number of PPI partners per protein fits poorly to the power law with the reduced χ-squared statistics and the coefficient of determination consistently greater than 1.5 and less than 0, respectively.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation

    JCVI-syn3A includes 16 additional protein-coding genes not in syn3.0; these genes are nonessential but make the cell less fragile and give more stable morphology.

    “a new version of the minimal genome, JCVI-syn3A (NCBI accession CP016816.2), was published,18 which includes 16 additional protein coding genes not included in the JCVI-syn3.0 genome. Although these new genes are not essential for the survival of the cell, they make the cell less fragile and cause it to have a more stable cellular morphology.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [18] source

      Not checked in the free run.

  • Classified as probably not a substantial claim.

    Premisecitation

    About one-third of syn3.0 protein-coding genes could not be annotated by sequence homologues from characterized protein domain families.

    “Upon the initial construction of syn3.0, researchers noted that ~1/3 of the protein coding genes in its genome could not be annotated by sequence homologues from characterized protein domain families;17”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [17] source

      Not checked in the free run.

  • May combine two claims.

    Interpretationreasoning

    In syn3.0, the ribC gene is absent, so riboflavin import via MMSYN1_0877 followed by RibF processing is the likely sole route for riboflavin-containing compound synthesis.

    “In the case of syn3.0, the ribC gene is also absent, apparently leaving riboflavin import via MMSYN1_0877 followed by RibF processing as the likely sole path for synthesis of riboflavin-containing compounds.”
    Quote in the paperSubstantial claim
    • Rests on an assumption: M. mycoides has no identifiable de novo riboflavin biosynthesis pathway.

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation

    The syn3 proteome was expanded by 19 genes, including 16 protein-coding genes, which improved growth phenotype; 11 of the new protein-coding genes are poorly annotated.

    “The syn3 proteome was also recently expanded through the addition of 19 genes, including 16 protein-coding genes, which while nonessential resulted in an improved growth phenotype; the resulting organism was labeled JCVI-syn3A;18 11 of the new protein-coding genes in JCVI-syn3A are also poorly annotated.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [18] source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Deep neural network-based contact prediction algorithms have greatly enhanced protein structure assembly accuracy.

    “the recent development of sequence-derived residue–residue contact prediction algorithms based on deep neural networks27,28 has greatly enhanced the accuracy of protein structure assembly”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites [27] source

      Not checked in the free run.

    • cites [28] source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    The original syn3.0 study classified proteins into five annotation-confidence classes based on TIGRfam matches.

    “which were categorized by the original syn3.0 study17 into five classes (Unknown, Generic, Putative, Probable, and Equivalog) in ascending order of function annotation confidence, based on a protein’s match to the TIGRfam protein family database.52”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites [17] source

      Not checked in the free run.

    • cites [52] source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation

    Antczak and colleagues added functional information for 66 syn3.0 proteins of unknown function, with an abundance of putative transporters and transmembrane proteins.

    “Antczak and colleagues applied a multipipeline approach to provide consensus predictions that added functional information for 66 of the proteins of unknown function in syn3.0, demonstrating a particular abundance of putative transporters and other transmembrane proteins.21”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [21] source

      Not checked in the free run.

  • May combine two claims.

    Premisecitation

    The I-TASSER/COFACTOR-based protocol accurately assigns functions for many microbial and human proteins and is among top predictors in CAFA3 and CAFA PI.

    “This pipeline has been shown to accurately assign functions for many proteins in microbes22 and in humans25 and is among the top predictors in the most recent Critical Assessment of Function Annotation round 3 (CAFA3) and CAFA PI competitions.26”
    Quote in the paperSubstantial claim0 of 3 sources checked
    • cites [22] source

      Not checked in the free run.

    • cites [25] source

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    • cites [26] source

      Not checked in the free run.

  • Classified as probably not a substantial claim. May combine two claims.

    Premiseown data

    The syn3.0 genome has 473 genes, of which 438 encode proteins and 35 encode noncoding RNAs with known functions.

    “While the genome consists of 473 genes, this study only considered the 438 protein coding genes, as the other 35 genes encode noncoding RNAs with well-known functions such as tRNAs and rRNAs.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation

    Yang and Tsui attempted to annotate syn3.0 proteins by secondary structure matching, which recognizes similar structure folds but not necessarily related functions.

    “Later, Yang and Tsui attempted to annotate syn3.0 proteins by secondary structure matching,20 which was developed to recognize templates with a similar structure fold but not necessarily of a related function.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [20] source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    TM-score >0.5 indicates correct global topology for structure models.

    “TM-score ranges between 0 and 1, with TM-score > 0.5 meaning structure models of correct global topology.39”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [39] source

      Not checked in the free run.

  • May combine two claims.

    Interpretationreasoning

    This cellular component pattern is partly due to syn3.0's simple cellular structure, with a single membrane and no cell wall or membrane-bound organelles, so most proteins localize to cytoplasm or plasma membrane.

    “This is partly due to the simple cellular structure of syn3.0 (which has a single cell membrane and no cell wall or membrane-bound organelles), where most proteins localize to the cytoplasm or plasma membrane instead of more specific subcellular locations.”
    Quote in the paperNot a substantial claim

  • Classified as probably not a substantial claim.

    Premisecitation

    A C-scoreFunc >0.5 corresponds to a confident function prediction.

    “Each predicted function has a confidence score (C-scoreFunc) ranging from 0 to 1, with C-scoreFunc > 0.5 corresponding to a confident function prediction.22,25”
    Quote in the paperSubstantial claim0 of 2 sources checked
    • cites [22] source

      Not checked in the free run.

    • cites [25] source

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  • Classified as probably not a substantial claim.

    Study design and conductreasoning

    GO terms associated with more than 10% of annotated proteins were discarded as too generic in all analysis steps.

    “we discard any GO terms associated with >10% of annotated proteins in all steps of our analysis, including the definition of previously unannotated/annotated proteins and the rate ratio test of GO term enrichment.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisereasoning

    The original JCVI-syn3.0 genome still represents the most minimal genome achieved in the series.

    “our main analysis focuses on the original JCVI-syn3.0 genome, which still represents the most “minimal” genome achieved in the series.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    E. coli has a scale-free PPI network with τ = 1.3, as estimated in the authors' recent work.

    “E. coli, for example, also has a scale-free PPI network69 with τ = 1.3 as estimated by our recent work.70”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites [69] source

      Not checked in the free run.

    • cites [70] Gong W., 2020, Integrating Multimeric Threading with High-Throughput Experiments for Structural Interactome of Escherichia Coli

      Not checked in the free run.

  • May combine two claims.

    Interpretationown data

    The combined evidence indicates that riboflavin is probably the primary target of MMSYN1_0877, although other vitamins might also bind it.

    “Taken together, these data suggest that, while other vitamins could potentially bind to this protein, riboflavin is most likely the main target.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationreasoning

    MMSYN1_0440 might maintain membrane integrity or cell-cell contacts in syn3.0 and probably binds hyaluronic acid polymers in the medium.

    “MMSYN1_0440 may play an architectural role in maintaining membrane integrity or cell–cell contacts in syn3.0, likely interacting with hyaluronic acid polymers present in the media.”
    Quote in the paperNot a substantial claim
    • Rests on an assumption: Animal-derived hyaluronic acid is present in the culture medium.

  • May combine two claims.

    Interpretationown data

    Combining deep neural network contact maps with structural assembly simulations is essential for improving structure model quality and for increasing the coverage and reliability of functional predictions.

    “In particular, the integration of deep neural network-based contact maps with the structural assembly simulations plays an essential role for not only improving the quality of structure models but also for increasing the coverage and reliability of functional predictions.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationcitation

    In agreement with Antczak et al., the newly annotated proteins' functions emphasize nutrient acquisition and reveal a broad range of uptake and metabolic pathways that were not previously recognized.

    “Consistent with the findings of Antczak et al.,21 the spectrum of function annotations for these newly annotated proteins (Figure 2) places a strong emphasis on the importance of nutrient acquisition, demonstrating a broad range of uptake and metabolic pathways that had previously not been appreciated.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [21] source

      Not checked in the free run.

  • May combine two claims.

    Interpretationnone given

    These newly predicted phosphatases might function in nutrient acquisition, removal of toxic metabolic byproducts, or signaling and regulation.

    “and may play roles in nutrient acquisition, removal of toxic metabolic byproducts, or signaling/regulation.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Interpretationreasoning

    Proteins that detect host-derived growth stimulators are likely dispensable if their downstream signaling pathways can be identified, whereas proteins needed for simple metabolite uptake are likely not dispensable.

    “it is likely that the latter class of proteins may be dispensable if the downstream signaling paths can be elucidated, whereas the former likely cannot.”
    Quote in the paperSubstantial claim
34 of 34

  • The paper relies on this without support.

    The SPRING-predicted protein interaction network for syn3.0 reflects the organism's actual protein-protein interactions closely enough to infer network architecture.

    “The PPI network of syn3.0 was predicted using the SPRING48 dimer threading program.”

  • The paper relies on this without support.

    M. mycoides has no identifiable de novo riboflavin biosynthesis pathway.

    “M. mycoides lacks an identifiable pathway for de novo riboflavin biosynthesis”

  • The paper relies on this without support.

    syn3.0 can be grown in vitro without needing to interact with other organisms.

    “Considering that syn3.0 can be cultured in vitro without the need to interact with other organisms”

  • The paper relies on this without support.

    The ancestral M. mycoides is an obligate parasite of animal hosts.

    “the ancestral M. mycoides is an obligate parasite of animal hosts”

  • The paper relies on this without support.

    Genes are retained in the syn3.0 genome solely because they are essential, without explicit consideration of how many potential PPI partners they have.

    “genes are retained in the syn3.0 genome based solely on their essentiality without explicit consideration for the number of potential PPI.”

  • The paper relies on this without support.

    Animal-derived hyaluronic acid is present in the culture medium.

    “animal-derived hyaluronic acid present in the culture media”
6 of 6

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?

  • Previous bioinformatic work by Zhang et al. via COFACTOR predicted peptidase ester or lipase cosubstrate binding sites at residues 96 and 166-168 of /0520.

    “This catalytic site conservation is additionally corroborated by previous bioinformatic work by Zhang et al.19 via the COFACTOR analysis pipeline where peptidase ester or lipase cosubstrate binding sites are predicted at residues 96 and 166–168 especially.”

    From Toward the Complete Functional Characterization of a Minimal Bacterial Proteome (2022) · cites this paper as [19]

    The Materials and Methods section (‘Function Annotation and Enrichment Analysis’; page unavailable) confirms that Zhang et al. used COFACTOR to predict ligand-binding sites. Our copy does not report peptidase ester or lipase cosubstrate binding sites for /0520 at residues 96 or 166–168; searches for the protein identifier, site terms, and residue numbers did not find that specific prediction. (LLM, conf=0.80)

    Experimental — accuracy not yet measured

  • Gene /0520 codes for a yqkD-like serine peptidase/esterase/lipase of the α-β hydrolase superfamily, likely a membrane lipase/aminopeptidase involved in cell growth and morphology.

    “yqkD/0520 | cell growth and morphology | membrane lipase/aminopeptidase”

    From Toward the Complete Functional Characterization of a Minimal Bacterial Proteome (2022) · cites this paper as [19]

    The Results passages discuss predicted functions of other syn3.0 proteins, including MMSYN1_0440 and MMSYN1_0877, but do not address gene /0520. Searches for yqkD, MMSYN1_0520, and the claimed peptidase, lipase, and cell-morphology functions found no passage that verifies this annotation. (LLM, conf=0.72)

    Experimental — accuracy not yet measured

  • Gene /0520 was previously reported as a member of the α-β hydrolase family.

    “Previously, Pelletier et al. and others19,26 reported this gene as being a member of the α–β hydrolase family.”

    From Toward the Complete Functional Characterization of a Minimal Bacterial Proteome (2022) · cites this paper as [19]

    The Results passages discuss annotations for MMSYN1_0440 and other syn3.0 proteins, but do not identify gene /0520 as an α–β hydrolase. Searches of our copy for 0520, hydrolase, and Pelletier found no passage reporting that classification; Pelletier appears in a References entry, without the claimed annotation. (LLM, conf=0.75)

    Experimental — accuracy not yet measured

  • Gene /0878 is gabP, an amino acid transporter involved in nutrient uptake.

    “gabP/0878 | nutrient uptake | amino acid transporter”

    From Toward the Complete Functional Characterization of a Minimal Bacterial Proteome (2022) · cites this paper as [19]

    The Results section on MMSYN1_0877 identifies that different gene as a predicted riboflavin transporter. The Discussion describes nutrient uptake broadly, but searches for MMSYN1_0878 and gabP found no passage identifying /0878 as an amino acid transporter. (LLM, conf=0.74)

    Experimental — accuracy not yet measured

  • More recent work has determined that /0878 could have likely amino acid binding and transport functionality.

    “More recent work has determined that this protein could have likely amino acid binding and transport functionality.3,19”

    From Toward the Complete Functional Characterization of a Minimal Bacterial Proteome (2022) · cites this paper as [19]

    The Results section identifies MMSYN1_0877 as a predicted riboflavin transporter, not MMSYN1_0878 as an amino acid-binding or -transport protein. Searches for MMSYN1_0878 and amino acid binding and transport found no passage in our copy addressing the claimed function of /0878; the paper’s general discussion of transporter activity does not establish it. (LLM, conf=0.74)

    Experimental — accuracy not yet measured

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.

  • Predicted-model counts exceed the previously unannotated group

    Minor

    The discussion describes 373 and 382 modeled proteins as previously unannotated, but the results identify only 112 proteins as unannotated under the paper's UniProt-based definition.

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Authors
Zhang, C., Zheng, W., Cheng, M., Omenn, G. S., Freddolino, P. L., & Zhang, Y.
Affiliations
University of Michigan (US)
Corresponding author
  • Freddolino, Peter L.
  • Zhang, Yang
ORCID
Venue
Journal of Proteome Research
Publisher
American Chemical Society (ACS)
Published
2021-01-04
Volume
20
Issue
2
Pages
1178-1189
ISSN
  • 1535-3893 (ISSN-L)
  • 1535-3907
DOI
10.1021/acs.jproteome.0c00359
PMC
PMC7867644
OpenAlex
W3119833134
Cited by
27
References
70
Citations by year
2026: 2 · 2025: 1 · 2024: 2 · 2023: 6 · 2022: 7 · 2021: 9
FWCI
1.88
Type
journal article
Language
en
Open access
Licence
Locations
  • Journal of Proteome Research (publishedVersion)
  • PubMed (publishedVersion)
  • Europe PMC (PubMed Central) (acceptedVersion)
  • PubMed Central (acceptedVersion)
Funders
  • National Institute of Environmental Health Sciences (P30ES017885)
  • National Cancer Institute (U24CA210967)
  • NIH Office of the Director (OD026825)
  • National Institute of Allergy and Infectious Diseases (AI134678)
  • National Institute of General Medical Sciences (GM083107, GM128637, GM136422)
  • National Science Foundation (IIS1901191, MTM2025426)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Bioinformatics and Genomic Networks
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Protein Structure and Dynamics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Genomics and Phylogenetic Studies
Keywords
minimal genome, protein-protein interaction network, scale-free networks, protein function prediction, protein structure prediction, essential genes, nutrient uptake, synthetic biology, protein-protein interaction prediction
MeSH terms
Computational Biology, Genes, Essential, Proteome, Protein Interaction Maps

About these checks

Registry, retraction, Cites and statistics results are computed, not judged, with one exception: a printed reference that no index lists is read once by a model before it counts as a citation error. Claims are extracted by a model and kept only when their quote appears verbatim in the text; each cited source is then checked, with a model’s help, for whether it says what the claim relies on. Those verdicts can be wrong — read the claim’s quote and the cited source before relying on one.

Claims read by deepseek-v4.1-flash under definition version 3 (extract-v3) on 2026-10-05.

Classified by jev-1.13-20260917 (gold-guideline-v0.2) on 2026-10-05.

Statistics and arithmetic are recomputed, not judged.

Confidence bands are shown only for fields calibrated on our annotated test set. None is calibrated yet, so no band is shown.

What counts as a claim ›How we measure confidence ›How we check this ›

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