Automated model building and protein identification in cryo-EM maps

Jamali, Kiarash; Käll, Lukas; Zhang, Rui; Brown, Alan; Kimanius, Dari; Scheres, S.H.W.

doi:10.1101/2023.05.16.541002

Cited by 71 publications

(55 citation statements)

References 62 publications

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“…Positioning the ligand in BD2 was compatible with electron density, whereas positioning in BD1 caused a clash with DCAF16 due to the rigid linker. DCAF16 was built using a combination of models from ColabFold 69,70 (version 1.3), ModelAngelo 71 (version 0.2.2) and manual building in Coot. ColabFold correctly predicted the α5 and α6 helices that bind the DDB1 central cavity while ModelAngelo correctly built the 4-helical bundle of α3, 4, 7 and 8, as well as α6 in the DDB1 cavity.…”

Section: Methodsmentioning

confidence: 99%

Targeted protein degradation via intramolecular bivalent glues

Hsia

Hinterndorfer

Cowan

et al. 2023

Preprint

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Targeted protein degradation is a drug modality represented by compounds that recruit a target to an E3 ubiquitin ligase to promote target ubiquitination and proteasomal degradation. Historically, the field distinguishes monovalent degraders from bifunctional degraders (PROTACs) that connect target and ligase via separate binding ligands joined via a linker1-4. Here, we elucidate the mechanism of action of a PROTAC-like degrader of the transcriptional coactivator BRD4, composed of a BRD4 ligand linked to a ligand for the E3 ligase CRL4DCAF15. Using orthogonal CRISPR/Cas9 screens we identify the degrader activity is independent of DCAF15, and relies on a different CRL4 substrate receptor, DCAF16. We demonstrate an intrinsic affinity between BRD4 and DCAF16, which is dependent on the tandem bromodomains of BRD4 and further increased by the degrader without physically engaging DCAF16 in isolation. Structural characterization of the resulting ternary complex reveals both BRD4 bromodomains are bivalently engaged in cis by the degrader and are bound to DCAF16 through several interfacial BRD4-DCAF16 and degrader-DCAF16 contacts. Our findings demonstrate that intramolecularly bridging domains can confer glue-type stabilization of intrinsic target-E3 interactions, and we propose this as a general strategy to modulate the surface topology of target proteins to nucleate co-opting of E3 ligases or other cellular effector proteins for effective proximity-based pharmacology.

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Section: Methodsmentioning

confidence: 99%

Targeted protein degradation via intramolecular bivalent glues

Hsia

Hinterndorfer

Cowan

et al. 2023

Preprint

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“…The dome‐shaped reconstruction that was retrieved (Figure 2F) could not be built with current refinement tools; due to the resolution of ∼8 Å, AI tools like FindMySequence [42], DeepTracer [43], or ModelAngelo [44] are not applicable. To address this issue, the 150 most abundant proteins were selected, ribosomal proteins were excluded, and modeling was focused on less abundant protein signatures.…”

Section: Resultsmentioning

confidence: 99%

Enabling cryo‐EM density interpretation from yeast native cell extracts by proteomics data and AlphaFold structures

et al. 2023

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In the cellular context, proteins participate in communities to perform their function. The detection and identification of these communities as well as in‐community interactions has long been the subject of investigation, mainly through proteomics analysis with mass spectrometry. With the advent of cryogenic electron microscopy and the “resolution revolution,” their visualization has recently been made possible, even in complex, native samples. The advances in both fields have resulted in the generation of large amounts of data, whose analysis requires advanced computation, often employing machine learning approaches to reach the desired outcome. In this work, we first performed a robust proteomics analysis of mass spectrometry (MS) data derived from a yeast native cell extract and used this information to identify protein communities and inter‐protein interactions. Cryo‐EM analysis of the cell extract provided a reconstruction of a biomolecule at medium resolution (∼8 Å (FSC = 0.143)). Utilizing MS‐derived proteomics data and systematic fitting of AlphaFold‐predicted atomic models, this density was assigned to the 2.6 MDa complex of yeast fatty acid synthase. Our proposed workflow identifies protein complexes in native cell extracts from Saccharomyces cerevisiae by combining proteomics, cryo‐EM, and AI‐guided protein structure prediction.

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“…This suggests that the parallel ridge of unidentified densities outside this segment is only present in some of the fibrils. Model building using Model Angelo 49 suggests that these unidentified densities may correspond to R2 residues K281-Q288, with a correlation coefficient of 0.3 between the map and the model ( Fig. S8 ).…”

Section: Resultsmentioning

confidence: 99%

Structures of AT8 and PHF1 Phospho-Mimetic Tau: Insights Into the Posttranslational Modification Code of Tau Amyloid Formation

Mammeri,

Dregni,

Duan

et al. 2023

Preprint

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The microtubule-associated protein tau aggregates into amyloid fibrils in Alzheimer's disease and other neurodegenerative diseases. In these tauopathies, tau is hyperphosphorylated, suggesting that this posttranslational modification may induce pathological tau aggregation. Tau is also phosphorylated in normal developing brains. To investigate how tau phosphorylation induces amyloid fibrils, here we report the atomic structures of two phospho-mimetic full-length tau fibrils assembled without anionic cofactors. One set of phospho-mimetic mutations is targeted by the antibody AT8, while the other set is targeted by the antibody PHF1. Solid-state NMR and cryo-electron microscopy data reveal that AT8 tau forms a unique triangular fibril core that encompasses the entire C-terminal third of the protein, whereas PHF1 tau forms a triple-stranded core. These results demonstrate that specific post-translational modifications induce structurally specific tau aggregates. We propose that these aggregates may evolve into pathological filaments under suitable cellular conditions or remain as transient species in normal brains.

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Automated model building and protein identification in cryo-EM maps

Cited by 71 publications

References 62 publications

Targeted protein degradation via intramolecular bivalent glues

Targeted protein degradation via intramolecular bivalent glues

Enabling cryo‐EM density interpretation from yeast native cell extracts by proteomics data and AlphaFold structures

Structures of AT8 and PHF1 Phospho-Mimetic Tau: Insights Into the Posttranslational Modification Code of Tau Amyloid Formation

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