Current developments in <i>Coot</i> for macromolecular model building of Electron Cryo‐microscopy and Crystallographic Data

Casañal, Ana; Lohkamp, Bernhard; Emsley, Paul

doi:10.1002/pro.3791

Cited by 597 publications

(500 citation statements)

References 49 publications

(91 reference statements)

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“…Based on symmetry considerations, we also concluded that ZP-C(I) and EGF IV/ZP-N(I) belong to two distinct additional copies of UMOD (chains B and C, respectively; corresponding to UMOD 2 and UMOD 4 in Figure 2), both of which interact with chain A as well as with each other. The resulting initial set of coordinates, consisting of one chain encompassing the whole polymerization region of UMOD (A) and two half chains (B, C), was subjected to molecular dynamics (MD) flexible fitting in Namdinator (Kidmose et al, 2019) and manually rebuilt using Coot (Casañal et al, 2019) as implemented in CCP-EM (Burnley et al, 2017) . After further improvement by Cryo_fit (Kim et al, 2019b) , as well as additional rebuilding in Coot (whose carbohydrate-building tool (Emsley and Crispin, 2018) was used to add the N-glycan chains attached to EGF IV N322, ZP-N N396 and ZP-C N513) and ISOLDE (Croll, 2018) , the model was real-space refined against the UMOD fl data in PHENIX (Afonine et al, 2018a) using a data/restraint weight of 0.8, a non-bonded weight of 250.0 and restraints generated using the starting coordinates as a reference.…”

Section: Model Building Refinement Validation and Analysismentioning

confidence: 99%

Cryo-EM Structure of Native Human Uromodulin, a Zona Pellucida Module Polymer

Stsiapanava

Brunati

et al. 2020

Preprint

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SUMMARYAssembly of extracellular filaments and matrices mediating fundamental biological processes such as morphogenesis, hearing, fertilization and antibacterial defense is driven by a ubiquitous polymerization module known as zona pellucida (ZP) “domain”. Despite the conservation of this element from hydra to human, no information is available on the filamentous conformation of any ZP module protein. Here we report the cryo-electron microscopy structure of uromodulin (UMOD)/Tamm-Horsfall protein, the most abundant protein in human urine and an archetypal ZP module-containing molecule, in its mature homopolymeric state. UMOD forms a one-start helix with an unprecedented 180-degree twist between subunits enfolded by interdomain linkers that have completely reorganized as a result of propeptide dissociation. Lateral interaction between filaments in the urine generates sheets exposing a checkerboard of binding sites to capture uropathogenic bacteria, and UMOD-based models of mammalian and avian heteromeric egg coat filaments identify a common sperm-binding region at the interface between subunits.

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Section: Model Building Refinement Validation and Analysismentioning

confidence: 99%

Cryo-EM Structure of Native Human Uromodulin, a Zona Pellucida Module Polymer

Stsiapanava

Brunati

et al. 2020

Preprint

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“…Protein and rRNA chains were visually inspected in Coot (Casañal et al, 2020) and manually adjusted where residues did not fit well into the density. Focused-refined maps on smaller regions were used to make further manual adjustments to the model, alternating with PHENIX RSR.…”

Section: Modelingmentioning

confidence: 99%

Structure of the Bacterial Ribosome at 2 Å Resolution

Watson

Ward

Méheust

et al. 2020

Preprint

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Continuing advances in cryo-electron microscopy (cryo-EM) demonstrate the promise it holds for revealing biological structures at chemical resolution, in which noncovalent interactions, RNA and protein modifications, and solvation can be modeled accurately. At present, the best cryo-EM-derived models of the bacterial ribosome are of the large (50S) ribosomal subunit with effective global resolutions of 2.4-2.5 Å, based on map-to-model Fourier shell correlation (FSC). Here we present a model of the E. coli 70S ribosome with an effective global resolution of 2.0 Å, based on maps showcasing unambiguous positioning of residues, their detailed chemical interactions, and chemical modifications. These modifications include the first examples of isopeptide and thioamide backbone substitutions in ribosomal proteins, the former of which is likely conserved in all domains of life. The model also defines extensive solvation of the small (30S) ribosomal subunit for the first time, as well as interactions with A-site and P-site tRNAs, mRNA, and the antibiotic paromomycin. The high quality of the maps now allows a deeper phylogenetic analysis of ribosomal components, and identification of structural conservation to the level of solvation. The maps and models of the bacterial ribosome presented here should enable future structural analysis of the chemical basis for translation, and the development of robust tools for cryo-EM structure modeling and refinement.

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“…Recent developments (Frenz et al, 2017; Igaev et al, 2019) in molecular dynamics programs have provided methods for fitting atomic models to electron density maps using methods independent of classical crystallographic programs such as COOT (Casanal et al, 2019; Emsley and Cowtan, 2004) or PHENIX (Adams et al, 2010), which have themselves added cryo-EM optimised functions, and which would appear to work well for “mid-to-low” resolution cryo-EM maps which have previously been difficult to interpret with the crystallographic packages. We have not used these here, although are investigating their use for other protein complexes.…”

Section: Discussionmentioning

confidence: 99%

Sub-3 Å resolution structure of apoferritin using a multi-purpose TEM with a side-entry cryo-holder

Kayama

Burton‐Smith

Song

et al. 2020

Preprint

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15The structural analysis of protein complexes by cryo-electron microscopy (cryo-EM) single 16 particle analysis (SPA) has had great impact as a biophysical method in recent years. Many results 17 of cryo-EM SPA are based on state-of-the-art cryo-electron microscopes customized for SPA. 18These are currently only available in limited locations around the world, where securing machine 19 time is highly competitive. One potential solution for this time-competitive situation is to reuse 20 existing multi-purpose equipment. Here, we used a multi-purpose TEM with a side entry cryo-21 holder at our facility to evaluate the potential of high-resolution SPA. We report a 3 Å resolution 22 map of apoferritin with local resolution extending to 2.6 Å. The map clearly showed two positions 23 of an aromatic side chain. We also verified the optimal imaging conditions depending on different 24 electron microscope and camera combinations. This study demonstrates the possibilities of more 25 widely available and established electron microscopes, and their applications for cryo-EM SPA. 26 27 Keywords 28 apoferritin; benchmarking; cryo-electron microscopy; multi-purpose TEM; single particle 29 analysis; side-entry cryo-holder 30

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Current developments in Coot for macromolecular model building of Electron Cryo‐microscopy and Crystallographic Data

Cited by 597 publications

References 49 publications

Cryo-EM Structure of Native Human Uromodulin, a Zona Pellucida Module Polymer

Cryo-EM Structure of Native Human Uromodulin, a Zona Pellucida Module Polymer

Structure of the Bacterial Ribosome at 2 Å Resolution

Sub-3 Å resolution structure of apoferritin using a multi-purpose TEM with a side-entry cryo-holder

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