(A) An overview of the effect of Spc110p deletions on assembly and viability, summarizing previously published data from Lyon et al., 2016. Assembly data was generated using Spc110p-GCN4 tetramer …
(A) Spc110p N-terminal region secondary structure prediction, showing lack of predicted secondary structure for the first 111 residues. Also shown are Spc110pCM1(117-146) and the Spc110pNCC(164-208) …
XL-MS datasets for γ-tubulin small complex (γTuSC) crosslinked to Spc110p1-220-GCN4 dimer (A, B) or Spc110p1-401-GST (C, D) with either EDC (A, C) or DSS (B, D). Each colored rectangle represents a …
The modeling results shown are based on the γTuSC-Spc110p1-220 GCN4 crosslinks; similar results were obtained in all cases using γTuSC-Spc110p1-401-GST crosslinks (see Appendix 1). (A) Monomer of …
This schematic describes the integrative structure modeling procedures used in this paper. The first row details the information to be used in modeling. The background color of each information …
(A) Results of test 1, convergence of the model score, for the 2840 good-scoring models; the scores do not continue to improve as more models are computed essentially independently. The error bar …
(A, B) Segmented density of (A) open γTuRCWT and (B) closed γTuRCSS. γTuRC subunits are colored as in the figure inset. Density was segmented within 4.5 Å of the atomic model, showing one Spc110p …
(A) Classification scheme for γTuRCWT processing. Images are projection images of the 3D classes obtained. (B) Fourier shell correlation FSC (blue) and map to model FSC (orange) for the closed γTuRCW…
(A) Classification scheme for γTuRCSS processing. Images are projection images of the 3D classes obtained. (B) FSC (blue) and map to model FSC (orange) for the γTuRCSS. γTuRC: γ-tubulin ring complex.
(A) γTuRCSS local resolution map. The color scale is shown between panels (A) and (B). (B) γTuRCWT local resolution map. The color scale is shown between panels (A) and (B). (C) Local resolution map …
Helices are numbered from 1, whereas sheets are labeled from (A). Turns are labeled by type. Plots were generated using the pdbsum server (Laskowski, 2009).
(A) An alignment of γ-tubulin (γ-tubulin bound to Spc98p, khaki, this work; human free γ-tubulin, sky blue, PDB ID 1Z5W; yeast straight β-tubulin, forest green, PDB ID 5W3F) using their N-terminal …
(A) Filtered segmented difference map between experimental density and the fitted atomic model without Spc110p overlaid on a γTuRCSS surface lacking Spc110p. The difference map was segmented to show …
(A) View of the Spc110pCM1 motif binding site with Spc98p with the H19-S3 region, colored as in Figure 4. Spc110pCM1:Spc98p hydrogen bond interactions are indicated in red. All of the hydrogen bonds …
(A) Overview of a dimer of γ-tubulin small complex (γTuSC) colored as in Figure 2, with the central Spc97p/98p colored according to their conservation. (B) View of the Spc110pCM1 binding site at the …
Top and side views of open γTuRCWT (A, C) and closed γTuRCSS (B, D). Panels (E) and (F) show a bottom view of an assembled γTuRCSS. The arrow indicates the seventh Spc110pNCC binding site in the …
(A) A filtered micrograph showing raw particles shows well-dispersed single particles and a mixture of γ-tubulin small complex (γTuSC) monomers and dimers. (B) Representative examples of classes …
(A) Classification scheme for WT γTuSC monomer and dimer processing. (B) FSC (blue) and map to model FSC for the closed WT γTuSC monomer. (C) FSC (blue) and map to model FSC for the open WT γTuSC …
(A) Approximately 3.7 Å reconstruction γTuSC showing Spc97p (sky blue), Spc98p (dodger blue), and γ-tubulin (khaki). (B) Approximately 4.5 Å reconstruction of a dimer of γTuSC colored as in panel (A)…
(A) Views of the monomeric γTuSC aligned with an open γTuRCWT filament monomer using the N-terminal helical bundles of Spc97/98p. Monomeric γTuSC is colored as in Figure 2, and the γTuSCWT filament …
Morph of the monomeric γTuSC conformation to the conformation adopted by a γTuRCWT filament monomer in the open state. The γTuSC is colored with Tub4p in khaki, Spc97p in light blue, and Spc98p in …
Morph of a γTuRCWT filament monomer in the open conformation to the conformation adopted by a γTuRCSS filament monomer in the closed state. The γTuSC is colored with Tub4p in khaki, Spc97p in light …
Morph of a γTuRCSS filament monomer in the closed state to the conformation adopted by a γTuRCWT filament monomer in the closed state. The γTuSC is colored with Tub4p in khaki, Spc97p in light blue, …
(A) γTuSCSS dimer, colored as in Figure 2, with phosphorylation sites from Fong et al., 2018 marked with red balls (no known phenotype) or purple balls (phenotype previously reported). Boxes are …
(A) Yeast closed (this work), (B) Xenopus (PDB ID 6TF9), and (C) human γTuRC (PDB ID 6V6S) structures placed adjacent to a microtubule to illustrate the motions required to properly template …
(A) Human γTuRC density (EMDB ID 21068) is fitted with the C-terminal region of yeast GCP2, γ-tubulin, and CM1 helix (this work), showing that a CM1 helix binds between GCP2 and GCP6 in human γTuRC. …
(A, B) Overlay of the two γ-tubulins adjacent to the CM1 binding site observed in the human γTuRC structure for (A) Xenopus (no CM1 bound) and (B) human (CM1-bound) highlighting the large motion …
Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
---|---|---|---|---|
Software, algorithm | IMP (Integrative Modeling Platform) | https://integrativemodeling.org;https://doi.org/10.1371/journal.pbio.1001244 | RRID:SCR_002982 | Version 2.8 |
Software, algorithm | UCSF Chimera | https://www.cgl.ucsf.edu/chimera/ https://doi.org/10.1002/jcc.20084 | RRID:SCR_004097 | |
Strain, strain background (Escherichia coli) | BL21(DE3) CodonPlus-RIL | Agilent | Part No.:230245 | |
Genetic reagent (Homo sapiens, Saccharomyces cerevisiae) | pET28a-3C-Xrcc4-Spc110(164-207) | This paper | Uniprot:Q13426 (Xrcc4); Uniprot:32380 (Spc110) | Construct contains residues 2–132 of H. sapiens Xrcc4 fused with residues 164–207 of S. cerevisiae Spc110 |
Software, algorithm | XDS | Kabsch, 2010; DOI: https://doi.org/10.1107/S0907444909047337 | RRID:SCR_015652 | Version: October 15, 2015 |
Software, algorithm | Phenix | Adams et al., 2010; DOI: https://doi.org/10.1107/S0907444909052925; McCoy et al., 2007; DOI: https://doi.org/10.1107/S0021889807021206; Terwilliger et al., 2008; DOI: https://doi.org/10.1107/S090744490705024X Afonine et al., 2012; DOI:https://doi.org/10.1107/S0907444912001308 | RRID:SCR_014224 | Version 1.10.1_2155 |
Software, algorithm | Coot | Emsley et al., 2010; DOI: https://doi.org/10.1107/S0907444910007493 | RRID:SCR_014222 | Version 0.8.3 |
Software, algorithm | Kojak, XL identification algorithm | http://www.kojak-ms.org/ | RRID:SCR_021028 | Versions 1.4.1 and 1.4.3 |
Software, algorithm | ProXL, protein XL data visualization | https://proxl-ms.org/ | RRID:SCR_021027 | |
Chemical compound, drug | DSS | Thermo Fisher Scientific | 21655 | |
Chemical compound, drug | EDC | Thermo Fisher Scientific | A35391 | |
Chemical compound, drug | Sulfo-NHS | Thermo Fisher Scientific | A39269 | |
Software, algorithm | cisTEM | Grant et al., 2018.DOI:10.7554/eLife.35383 | RRID:SCR_016502 | Version 1.0 beta |
Software, algorithm | Relion | Scheres, 2012. PMID:23000701; DOI: 10.1016/j.jsb.2012.09.006 | RRID:SCR_016274 | |
Genetic reagent (S. cerevisiae) | pFastBac-Tub4p | Vinh et al., 2002. doi: 10.1091/mbc.02-01-0607 | ||
Genetic reagent (S. cerevisiae) | pFastBac-Spc97p | Vinh et al., 2002. doi: 10.1091/mbc.02-01-0607 | ||
Genetic reagent (S. cerevisiae) | pFastBac-Spc98p | Vinh et al., 2002. doi: 10.1091/mbc.02-01-0607 | ||
Genetic reagent (S. cerevisiae) | pFastBac-GST-Spc110p1-220 | Vinh et al., 2002. doi: 10.1091/mbc.02-01-0607 | ||
Genetic reagent (S. cerevisiae) | pFastBac-Tub4pS58C/G288C | Kollman et al., 2015. DOI: 10.1038/nsmb.2953 |
X-ray data collection and refinement statistics.
Cryo-EM data collection, processing, and modeling statistics.