Refinement of Protein Structures into Low-Resolution Density Maps Using Rosetta

DiMaio, Frank; Tyka, Michael D.; Baker, Matthew L.; Chiu, Wah; Baker, David C.

doi:10.1016/j.jmb.2009.07.008

Cited by 274 publications

(295 citation statements)

References 27 publications

(31 reference statements)

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“…In X-ray crystallography, density maps determined at ∼4.0-Å resolution range are often considered marginal for determining the atomistic structures (32). However, recent studies have shown that it is indeed possible to reliably build a de novo Cα model directly from cryo-EM density map in this resolution range (28,33). It should also be noted that our density map (Fig.…”

Section: Validation Of Subunit Arrangement By Biochemical Near-neighbormentioning

confidence: 62%

4.0-Å resolution cryo-EM structure of the mammalian chaperonin TRiC/CCT reveals its unique subunit arrangement

Yao

Baker

Jakana

et al. 2010

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

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153

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The essential double-ring eukaryotic chaperonin TRiC/CCT (TCP1-ring complex or chaperonin containing TCP1) assists the folding of ∼5-10% of the cellular proteome. Many TRiC substrates cannot be folded by other chaperonins from prokaryotes or archaea. These unique folding properties are likely linked to TRiC's unique heterooligomeric subunit organization, whereby each ring consists of eight different paralogous subunits in an arrangement that remains uncertain. Using single particle cryo-EM without imposing symmetry, we determined the mammalian TRiC structure at 4.7-Å resolution. This revealed the existence of a 2-fold axis between its two rings resulting in two homotypic subunit interactions across the rings. A subsequent 2-fold symmetrized map yielded a 4.0-Å resolution structure that evinces the densities of a large fraction of side chains, loops, and insertions. These features permitted unambiguous identification of all eight individual subunits, despite their sequence similarity. Independent biochemical near-neighbor analysis supports our cryo-EM derived TRiC subunit arrangement. We obtained a Cα backbone model for each subunit from an initial homology model refined against the cryo-EM density. A subsequently optimized atomic model for a subunit showed ∼95% of the main chain dihedral angles in the allowable regions of the Ramachandran plot. The determination of the TRiC subunit arrangement opens the way to understand its unique function and mechanism. In particular, an unevenly distributed positively charged wall lining the closed folding chamber of TRiC differs strikingly from that of prokaryotic and archaeal chaperonins. These interior surface chemical properties likely play an important role in TRiC's cellular substrate specificity.asymmetric reconstruction | atomic model | subunit structure

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Section: Validation Of Subunit Arrangement By Biochemical Near-neighbormentioning

confidence: 62%

4.0-Å resolution cryo-EM structure of the mammalian chaperonin TRiC/CCT reveals its unique subunit arrangement

Yao

Baker

Jakana

et al. 2010

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

Self Cite

153

142

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“…The 5.5-Å resolution electron density maps allowed the X4 and Cernunnos main-chain traces to be positioned but were insufficient to determine the side-chain conformations. We next modeled the complex between the Cernunnos and X4 head domains using a module from the Rosetta software that allows modeling within the constraints of electron density maps (31). The model superimposed with an rmsd of 1.4 Å over 188 X4 and Cernunnos main-chain atoms of residues at the interface.…”

Section: Resultsmentioning

confidence: 99%

Structural characterization of filaments formed by human Xrcc4–Cernunnos/XLF complex involved in nonhomologous DNA end-joining

Ropars

Drevet

Legrand

et al. 2011

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

122

128

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Cernunnos/XLF is a core protein of the nonhomologous DNA end-joining (NHEJ) pathway that processes the majority of DNA double-strand breaks in mammals. Cernunnos stimulates the final ligation step catalyzed by the complex between DNA ligase IV and Xrcc4 (X4). Here we present the crystal structure of the X4 1-157 -Cernunnos 1-224 complex at 5.5-Å resolution and identify the relative positions of the two factors and their binding sites. The X-ray structure reveals a filament arrangement for X4 1-157 and Cernunnos 1-224 homodimers mediated by repeated interactions through their N-terminal head domains. A filament arrangement of the X4-Cernunnos complex was confirmed by transmission electron microscopy analyses both with truncated and full-length proteins. We further modeled the interface and used structure-based site-directed mutagenesis and calorimetry to characterize the roles of various residues at the X4-Cernunnos interface. We identified four X4 residues (Glu 55 , Asp 58 , Met 61 , and Phe 106 ) essential for the interaction with Cernunnos. These findings provide new insights into the molecular bases for stimulatory and bridging roles of Cernunnos in the final DNA ligation step.D NA double-strand breaks (DSBs) are the most toxic DNA lesions in the genome, and unrepaired DSBs can cause large-scale losses of genetic information through chromosome rearrangement (1, 2). These DNA damages result from exposure to exogenous damaging agents, such as ionizing radiation, radiomimetic compounds, and topoisomerase inhibitors. DSBs are also obligate intermediates in several recombination processes in vertebrates, including antigen receptor gene rearrangement, V(D)J recombination (3, 4). In higher eukaryotes, DSBs are repaired by several mechanisms, among which nonhomologous end-joining (NHEJ) represents the major pathway, particularly when sister chromatids are not available (5). Deficiency in the NHEJ machinery results in sensitivity to ionizing radiation and severe combined immune deficiencies in humans and mice due to abortive V(D)J recombination (6). NHEJ is orchestrated by at least seven proteins. The Ku70/Ku80 heterodimer adopts a preformed ring-shaped structure that recognizes and encircles the duplex DNA ends at the DSB (7). Ku70/Ku80 recruits a 469-kDa serine/threonine protein kinase, the DNA-PK catalytic subunit (DNA-PKcs), via a direct interaction and shifts about 10 bp inward so that DNA-PKcs acquires a position at the terminus through its large open-ring cradle structure (8). Upon association with Ku and DNA, DNA-PKcs is activated and phosphorylates several proteins including itself and Ku70/Ku80. The DNA-PK holoenzyme, constituted by Ku and DNA-PKcs, plays a central role in NHEJ. Among other functions, DNA-PK mediates the end-bridging of the DSB extremities (9), regulates access to the DNA ends by processing enzymes such as the DNA-PKcs-associated Artemis nuclease (10, 11), and recruits the Xrcc4-ligase IV complex to DNA ends for the ligation step (12). The Xrcc4-ligase IV complex carries out the final joinin...

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“…The atomic model of SelB with exception of tandem winged-helix motifs 3 and 4 was obtained by combining manual rebuilding and homology modelling with density-guided energy optimization, as implemented in the Rosetta package 69,70 , employing a template derived from Methanococcus maripaludis SelB (PDB ID: 4AC9) 9 and alignment provided by the HHPRED server 71 . SelB tandem winged-helix motifs (wh) 1 and 2 were modelled based on the crystal structure of the isolated domain 4 of SelB from Moorella thermoacetica (PDB ID: 1LVA) 72 ; and wh3 and wh4 with mRNA SECIS were modelled on the basis of the crystal structure of E. coli SECIS RNA bound to the domain of elongation factor SelB (PDB ID: 2PJP) 73 using Rosetta and Coot 74 .…”

Section: Atomic Model Refinement Pseudo-crystallographic Refinement mentioning

confidence: 99%

The pathway to GTPase activation of elongation factor SelB on the ribosome

Fischer

Neumann

Bock

et al. 2016

Nature

124

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Refinement of Protein Structures into Low-Resolution Density Maps Using Rosetta

Cited by 274 publications

References 27 publications

4.0-Å resolution cryo-EM structure of the mammalian chaperonin TRiC/CCT reveals its unique subunit arrangement

4.0-Å resolution cryo-EM structure of the mammalian chaperonin TRiC/CCT reveals its unique subunit arrangement

Structural characterization of filaments formed by human Xrcc4–Cernunnos/XLF complex involved in nonhomologous DNA end-joining

The pathway to GTPase activation of elongation factor SelB on the ribosome

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