<i>Coot</i>: model-building tools for molecular graphics

Emsley, Paul; Cowtan, Kevin

doi:10.1107/s0907444904019158

Cited by 29,024 publications

(25,825 citation statements)

References 11 publications

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“…Individual domains of the Insulin receptor (L1, CR, L2, and FnIII-1, from PDB entry 4xzb) were positioned into the initial 4.6 Å resolution map using Molrep 45 and rigid-body refined using COOT 46 . The insulin and the α-CT helix were positioned by overlaying the microreceptor structure from PDB entry 3W11 9 onto the L1 domain of the complex and rigid body fitting them into the available density.…”

Section: Methodsmentioning

confidence: 99%

“…The sugars were extracted from PDB entry 4ZXB. All subsequent, lower resolution models were generated by manually positioning the higher resolution structure onto the available density and rigid body refining the individual domains to their final position using COOT 46 . Subsequently, the structures were subjected to 5 cycles of global real-space refinement with rotamer, Ramachandran plot and C-beta deviations restraints enabled in Phenix 47 .…”

Section: Methodsmentioning

confidence: 99%

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Structure of the insulin receptor–insulin complex by single-particle cryo-EM analysis

Scapin

Dandey

Zhang

et al. 2018

Nature

203

243

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Summary The insulin receptor (IR) is a dimeric protein that plays a crucial role in controlling glucose homeostasis, regulating lipid, protein and carbohydrate metabolism, and modulating brain neurotransmitter levels1,2. IR dysfunctions have been associated with many diseases, including diabetes, cancer, and Alzheimer’s1,3,4. The primary sequence has been known since the 1980s5, and is composed of an extracellular portion (ectodomain, ECD), a single transmembrane helix and an intracellular tyrosine kinase domain. Insulin binding to the dimeric ECD triggers kinase domain auto-phosphorylation and subsequent activation of downstream signaling molecules. Biochemical and mutagenesis data have identified two putative insulin binding sites (S1 and S2)6. While insulin bound to an ECD fragment containing S1 and the apo ectodomain have been characterized structurally7,8, details of insulin binding to the full receptor and the signal propagation mechanism are still not understood. Here we report single particle cryoEM reconstructions for the 1:2 (4.3 Å) and 1:1 (7.4 Å) IR ECD dimer:Insulin complexes. The symmetric 4.3 Å structure shows two insulin molecules per dimer, each bound between the Leucine-rich sub domain L1 of one monomer and the first fibronectin-like domain (FnIII-1) of the other monomer, and making extensive interactions with the α subunit C-terminal helix (α-CT helix). The 7.4 Å structure has only one similarly bound insulin per receptor dimer. The structures confirm the S1 binding interactions and define the full S2 binding site. These insulin receptor states suggest that recruitment of the α-CT helix upon binding of the first insulin changes the relative sub domain orientations and triggers downstream signal propagation.

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Structure of the insulin receptor–insulin complex by single-particle cryo-EM analysis

Scapin

Dandey

Zhang

et al. 2018

Nature

203

243

View full text Add to dashboard Cite

show abstract

“…Heavy‐atom search, density modification and initial model building were performed using Phenix AutoSol (Adams et al , 2010). Models were iteratively improved by manual building in Coot and refined using REFMAC5 and Phenix (Murshudov et al , 1997; Emsley & Cowtan, 2004). The stereochemistry of the final models was analysed with Procheck.…”

Section: Methodsmentioning

confidence: 99%

Functional role of TRIM E3 ligase oligomerization and regulation of catalytic activity

et al. 2016

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TRIM E3 ubiquitin ligases regulate a wide variety of cellular processes and are particularly important during innate immune signalling events. They are characterized by a conserved tripartite motif in their N‐terminal portion which comprises a canonical RING domain, one or two B‐box domains and a coiled‐coil region that mediates ligase dimerization. Self‐association via the coiled‐coil has been suggested to be crucial for catalytic activity of TRIMs; however, the precise molecular mechanism underlying this observation remains elusive. Here, we provide a detailed characterization of the TRIM ligases TRIM25 and TRIM32 and show how their oligomeric state is linked to catalytic activity. The crystal structure of a complex between the TRIM25 RING domain and an ubiquitin‐loaded E2 identifies the structural and mechanistic features that promote a closed E2~Ub conformation to activate the thioester for ubiquitin transfer allowing us to propose a model for the regulation of activity in the full‐length protein. Our data reveal an unexpected diversity in the self‐association mechanism of TRIMs that might be crucial for their biological function.

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“…The Zn‐ribbon was placed using the position of the TFIIB B‐ribbon in the Pol II PIC (PDB ID 5fyw) as a template, by alignment on the second largest subunit of Pol I (A135) and II (Rpb2). The orientation of the homology model for the Rrn7 Zn‐ribbon was manually adjusted in Coot (Emsley & Cowtan, 2004). The Rrn6 β‐propeller domain has been placed according to the fit to the density, but its exact orientation is uncertain.…”

Section: Methodsmentioning

confidence: 99%

Structural insights into transcription initiation by yeast RNA polymerase I

et al. 2017

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In eukaryotic cells, RNA polymerase I (Pol I) synthesizes precursor ribosomal RNA (pre‐rRNA) that is subsequently processed into mature rRNA. To initiate transcription, Pol I requires the assembly of a multi‐subunit pre‐initiation complex (PIC) at the ribosomal RNA promoter. In yeast, the minimal PIC includes Pol I, the transcription factor Rrn3, and Core Factor (CF) composed of subunits Rrn6, Rrn7, and Rrn11. Here, we present the cryo‐EM structure of the 18‐subunit yeast Pol I PIC bound to a transcription scaffold. The cryo‐EM map reveals an unexpected arrangement of the DNA and CF subunits relative to Pol I. The upstream DNA is positioned differently than in any previous structures of the Pol II PIC. Furthermore, the TFIIB‐related subunit Rrn7 also occupies a different location compared to the Pol II PIC although it uses similar interfaces as TFIIB to contact DNA. Our results show that although general features of eukaryotic transcription initiation are conserved, Pol I and Pol II use them differently in their respective transcription initiation complexes.

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Coot: model-building tools for molecular graphics

Abstract: CCP4mg is a project that aims to provide a general-purpose tool for structural biologists, providing tools for X-ray structure solution, structure comparison and analysis, and publication-quality graphics. The map-®tting tools are available as a stand-alone package, distributed as`Coot'.

Cited by 29,024 publications

References 11 publications

Structure of the insulin receptor–insulin complex by single-particle cryo-EM analysis

Structure of the insulin receptor–insulin complex by single-particle cryo-EM analysis

Functional role of TRIM E3 ligase oligomerization and regulation of catalytic activity

Structural insights into transcription initiation by yeast RNA polymerase I

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