Generalized Fragment Picking in Rosetta: Design, Protocols and Applications

Gront, Dominik; Kulp, Daniel W.; Vernon, Robert; Strauss, Charlie E. M.; Baker, David

doi:10.1371/journal.pone.0023294

Cited by 185 publications

(205 citation statements)

References 11 publications

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“…The 3D model of Kivd used for rational design was built using the Rosetta Comparative Modeling protocol. 36 Fragments sets 37 and three-dimensional evolutionary constraints 38 were generated using crystal structure PDB 2VBG. A total of 1000 models were generated, and the model with the lowest energy was used as the homology model of Kivd.…”

Section: Acs Synthetic Biologymentioning

confidence: 99%

Engineering a Thermostable Keto Acid Decarboxylase Using Directed Evolution and Computationally Directed Protein Design

Soh¹,

Mak

Lin³

et al. 2017

ACS Synth. Biol.

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Keto acid decarboxylase (Kdc) is a key enzyme in producing keto acid derived higher alcohols, like isobutanol. The most active Kdc's are found in mesophiles; the only reported Kdc activity in thermophiles is 2 orders of magnitude less active. Therefore, the thermostability of mesophilic Kdc limits isobutanol production temperature. Here, we report development of a thermostable 2-ketoisovalerate decarboxylase (Kivd) with 10.5-fold increased residual activity after 1h preincubation at 60 °C. Starting with mesophilic Lactococcus lactis Kivd, a library was generated using random mutagenesis and approximately 8,000 independent variants were screened. The top single-mutation variants were recombined. To further improve thermostability, 16 designs built using Rosetta Comparative Modeling were screened and the most active was recombined to form our best variant, LLM4. Compared to wild-type Kivd, a 13 °C increase in melting temperature and over 4-fold increase in half-life at 60 °C were observed. LLM4 will be useful for keto acid derived alcohol production in lignocellulosic thermophiles. KEYWORDS: thermostability, keto acid decarboxylase, high-throughput screening, protein design, directed evolution, isobutyl alcohol K eto acid decarboxylases (Kdc) are an important group of

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Section: Acs Synthetic Biologymentioning

confidence: 99%

Engineering a Thermostable Keto Acid Decarboxylase Using Directed Evolution and Computationally Directed Protein Design

Soh¹,

Mak

Lin³

et al. 2017

ACS Synth. Biol.

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show abstract

“…59 Templates were identified using the HMMER3 online server, 60 and the 10 best matches based on Evalue were used as templates for molecular modeling (PDB: 2kwc, 2li5, 3vh3, 3rui, 3vxw, 2kq7, 1eo6, 4co7, 1gnu, 2r2q ). Alignments were generated using Promals3D, 61 fragments sets were generated 62 and 3D evolutionary constraints were generated using the templates. 63 RosettaCM was used to generate 1000 decoys using the threaded templates as input.…”

Section: Structure-based Model Generationmentioning

confidence: 99%

Comparative analyses of ubiquitin-like ATG8 and cysteine protease ATG4 autophagy genes in the plant lineage and cross-kingdom processing of ATG8 by ATG4

et al. 2016

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Autophagy is important for degradation and recycling of intracellular components. In a diversity of genera and species, orthologs and paralogs of the yeast Atg4 and Atg8 proteins are crucial in the biogenesis of double-membrane autophagosomes that carry the cellular cargoes to vacuoles and lysosomes. Although many plant genome sequences are available, the ATG4 and ATG8 sequence analysis is limited to some model plants. We identified 28 ATG4 and 116 ATG8 genes from the available 18 different plant genome sequences. Gene structures and protein domain sequences of ATG4 and ATG8 are conserved in plant lineages. Phylogenetic analyses classified ATG8s into 3 subgroups suggesting divergence from the common ancestor. The ATG8 expansion in plants might be attributed to whole genome duplication, segmental and dispersed duplication, and purifying selection. Our results revealed that the yeast Atg4 processes Arabidopsis ATG8 but not human LC3A (HsLC3A). In contrast, HsATG4B can process yeast and plant ATG8s in vitro but yeast and plant ATG4s cannot process HsLC3A. Interestingly, in Nicotiana benthamiana plants the yeast Atg8 is processed compared to HsLC3A. However, HsLC3A is processed when coexpressed with HsATG4B in plants. Molecular modeling indicates that lack of processing of HsLC3A by plant and yeast ATG4 is not due to lack of interaction with HsLC3A. Our in-depth analyses of ATG4 and ATG8 in the plant lineage combined with results of cross-kingdom ATG8 processing by ATG4 further support the evolutionarily conserved maturation of ATG8. Broad ATG8 processing by HsATG4B and lack of processing of HsLC3A by yeast and plant ATG4s suggest that the cross-kingdom ATG8 processing is determined by ATG8 sequence rather than ATG4.

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“…For example, Rosetta (Gront et al, 2011) assembles the tertiary structure from fragments consisting of up to nine residues. This sequence is long enough to span part of a b-strand, the connecting loop and a turn of a subsequent a-helix and vice versa.…”

Section: Discussionmentioning

confidence: 99%

The unexpected structure of the designed protein Octarellin V.1 forms a challenge for protein structure prediction tools

Figueroa¹,

Sleutel²,

Vandevenne³

et al. 2016

Journal of Structural Biology

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a b s t r a c tDespite impressive successes in protein design, designing a well-folded protein of more 100 amino acids de novo remains a formidable challenge. Exploiting the promising biophysical features of the artificial protein Octarellin V, we improved this protein by directed evolution, thus creating a more stable and soluble protein: Octarellin V.1. Next, we obtained crystals of Octarellin V.1 in complex with crystallization chaperons and determined the tertiary structure. The experimental structure of Octarellin V.1 differs from its in silico design: the (aba) sandwich architecture bears some resemblance to a Rossman-like fold instead of the intended TIM-barrel fold. This surprising result gave us a unique and attractive opportunity to test the state of the art in protein structure prediction, using this artificial protein free of any natural selection. We tested 13 automated webservers for protein structure prediction and found none of them to predict the actual structure. More than 50% of them predicted a TIM-barrel fold, i.e. the structure we set out to design more than 10 years ago. In addition, local software runs that are human operated can sample a structure similar to the experimental one but fail in selecting it, suggesting that the scoring and ranking functions should be improved. We propose that artificial proteins could be used as tools to test the accuracy of protein structure prediction algorithms, because their lack of evolutionary pressure and unique sequences features.

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Generalized Fragment Picking in Rosetta: Design, Protocols and Applications

Cited by 185 publications

References 11 publications

Engineering a Thermostable Keto Acid Decarboxylase Using Directed Evolution and Computationally Directed Protein Design

Engineering a Thermostable Keto Acid Decarboxylase Using Directed Evolution and Computationally Directed Protein Design

Comparative analyses of ubiquitin-like ATG8 and cysteine protease ATG4 autophagy genes in the plant lineage and cross-kingdom processing of ATG8 by ATG4

The unexpected structure of the designed protein Octarellin V.1 forms a challenge for protein structure prediction tools

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