High-Resolution Comparative Modeling with RosettaCM

Song, Yifan; DiMaio, Frank; Wang, Ray Yu-Ruei; Kim, David E.; Miles, Christopher E.; Brunette, TJ; Thompson, James; Baker, David

doi:10.1016/j.str.2013.08.005

Cited by 1,096 publications

(1,132 citation statements)

References 27 publications

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“…We used the RosettaCM pipeline (38) to generate ∼25 structural homology models for each domain. In each case, nonaligned residues were excluded, resulting in partial models.…”

Section: Resultsmentioning

confidence: 99%

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Unique double-ring structure of the peroxisomal Pex1/Pex6 ATPase complex revealed by cryo-electron microscopy

Blok

Tan

Wang

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

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Members of the AAA family of ATPases assemble into hexameric double rings and perform vital functions, yet their molecular mechanisms remain poorly understood. Here, we report structures of the Pex1/Pex6 complex; mutations in these proteins frequently cause peroxisomal diseases. The structures were determined in the presence of different nucleotides by cryo-electron microscopy. Models were generated using a computational approach that combines Monte Carlo placement of structurally homologous domains into density maps with energy minimization and refinement protocols. Pex1 and Pex6 alternate in an unprecedented hexameric double ring. Each protein has two N-terminal domains, N1 and N2, structurally related to the single N domains in p97 and N-ethylmaleimide sensitive factor (NSF); N1 of Pex1 is mobile, but the others are packed against the double ring. The N-terminal ATPase domains are inactive, forming a symmetric D1 ring, whereas the C-terminal domains are active, likely in different nucleotide states, and form an asymmetric D2 ring. These results suggest how subunit activity is coordinated and indicate striking similarities between Pex1/Pex6 and p97, supporting the hypothesis that the Pex1/Pex6 complex has a role in peroxisomal protein import analogous to p97 in ER-associated protein degradation.Pex1 | Pex6 | AAA ATPase | cryo-electron microscopy | peroxisome T he AAA (ATPases associated with diverse cellular activities) family of ATPases contains a large number of proteins that perform important functions in cells (1). Many members of this family form hexamers. These hexamers consist either of a single ring of ATPase domains or of stacked rings formed by tandem ATPase domains in a single polypeptide chain (type I and II ATPases, respectively). Both classes of AAA ATPases often use the energy of ATP hydrolysis to move macromolecules. Examples of single-ring ATPases include the F1 ATPase (2), which rotates a polypeptide inside its central pore, ClpX (3), which moves polypeptides into the proteolytic chamber of ClpP, and the helicase gp4 of T7 phage (4), which pulls a DNA strand through its center. Double-ring ATPases generally act on polypeptides. Prominent members of this family include N-ethylmaleimide sensitive factor (NSF), p97/VCP (valosin-containing protein), and Hsp104 in eukaryotes and ClpB in bacteria (5-7). NSF dissociates SNARE complexes generated during membrane fusion, p97/VCP plays a role in many processes, including ERassociated protein degradation (ERAD), and Hsp104 and ClpB disassemble protein aggregates.The molecular mechanism of many hexameric AAA ATPases is only poorly understood, particularly for those that move polypeptide chains. In addition, the mechanism appears to differ among the known examples. For the F1 ATPase, it has been established that ATPase domains hydrolyze ATP continuously in a strictly consecutive manner around the ring (2). In contrast, in another single-ring ATPase, ClpX, several ATPase domains hydrolyze ATP in sporadic bursts, either simultaneously or in short succession...

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“…We used the RosettaCM pipeline (38) to generate ∼25 structural homology models for each domain. In each case, nonaligned residues were excluded, resulting in partial models.…”

Section: Resultsmentioning

confidence: 99%

“…We were able to derive models for the Pex1/Pex6 complex in different nucleotide states, using a hybrid method that combines the placement of domains from structural homologs into the density map with model-building and refinement protocols implemented in Rosetta (38). Pex1 and Pex6 alternate around a double ring, forming an unprecedented arrangement.…”

mentioning

confidence: 99%

Unique double-ring structure of the peroxisomal Pex1/Pex6 ATPase complex revealed by cryo-electron microscopy

Blok

Tan

Wang

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

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“…The acidic loop (residues Glu-96 -Trp-111) was removed during the rigid-body docking step. Starting from the Patchdock rigid models with the incomplete acidic loop, we used the RosettaCM program (21) to build the acidic loop and simultaneously refine the rigid body orientations between UB K48R and Ube2g1 C90S . The backbone atoms of both proteins in the complex model were allowed to move with constraint to initial backbone during the flexible refinement, and 2000 refined models are generated.…”

Section: Methodsmentioning

confidence: 99%

Differential Ubiquitin Binding by the Acidic Loops of Ube2g1 and Ube2r1 Enzymes Distinguishes Their Lys-48-ubiquitylation Activities

Choi

Lee

et al. 2015

Journal of Biological Chemistry

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Background:The acidic loops of E2 enzymes are important for their Lys-48-ubiquitylation activity. Results: The presence of Tyr residues modulates the ubiquitin binding property of the acidic loop. Conclusion: A proper interaction of the acidic loop with the attached donor ubiquitin is important for Lys-48-ubiquitylation activity. Significance: One of molecular bases to study the mechanism of Lys-48-ubiquitylation is provided.

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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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High-Resolution Comparative Modeling with RosettaCM

Cited by 1,096 publications

References 27 publications

Unique double-ring structure of the peroxisomal Pex1/Pex6 ATPase complex revealed by cryo-electron microscopy

Unique double-ring structure of the peroxisomal Pex1/Pex6 ATPase complex revealed by cryo-electron microscopy

Differential Ubiquitin Binding by the Acidic Loops of Ube2g1 and Ube2r1 Enzymes Distinguishes Their Lys-48-ubiquitylation Activities

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

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