Protein identification from electron cryomicroscopy maps by automated model building and side-chain matching

Terwilliger, Thomas C.; Sobolev, Oleg V.; Afonine, Pavel V.; Adams, Paul D.; Ho, Chi Min; Li, Xiaorun; Zhou, Z. Hong

doi:10.1107/s2059798321001765

Cited by 11 publications

(11 citation statements)

References 22 publications

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“…Polyalanine models were built manually in COOT [ 7 ] in both flipped and unflipped maps in the forward and reverse directions, resulting in four polyalanine models. Phenix.sequence_from_map [ 44 ] was run with each of the maps and corresponding polyalanine models against all annotated sequences (8172 sequences) from human brain tissue. The LLG scores from phenix.sequence_from_map were plotted as a function of the sequence id and top-ranking sequence was identified as major prion protein (Uniprot ID: P04156), residues 80–141.…”

Section: Methodsmentioning

confidence: 99%

Cryo-EM structures of prion protein filaments from Gerstmann–Sträussler–Scheinker disease

et al. 2022

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Prion protein (PrP) aggregation and formation of PrP amyloid (APrP) are central events in the pathogenesis of prion diseases. In the dominantly inherited prion protein amyloidosis known as Gerstmann–Sträussler–Scheinker (GSS) disease, plaques made of PrP amyloid are present throughout the brain. The c.593t > c mutation in the prion protein gene (PRNP) results in a phenylalanine to serine amino acid substitution at PrP residue 198 (F198S) and causes the most severe amyloidosis among GSS variants. It has been shown that neurodegeneration in this disease is associated with the presence of extracellular APrP plaques and neuronal intracytoplasmic Tau inclusions, that have been shown to contain paired helical filaments identical to those found in Alzheimer disease. Using cryogenic electron microscopy (cryo-EM), we determined for the first time the structures of filaments of human APrP, isolated post-mortem from the brain of two symptomatic PRNP F198S mutation carriers. We report that in GSS (F198S) APrP filaments are composed of dimeric, trimeric and tetrameric left-handed protofilaments with their protomers sharing a common protein fold. The protomers in the cross-β spines consist of 62 amino acids and span from glycine 80 to phenylalanine 141, adopting a previously unseen spiral fold with a thicker outer layer and a thinner inner layer. Each protomer comprises nine short β-strands, with the β1 and β8 strands, as well as the β4 and β9 strands, forming a steric zipper. The data obtained by cryo-EM provide insights into the structural complexity of the PrP filament in a dominantly inherited human PrP amyloidosis. The novel findings highlight the urgency of extending our knowledge of the filaments' structures that may underlie distinct clinical and pathologic phenotypes of human neurodegenerative diseases.

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Section: Methodsmentioning

confidence: 99%

Cryo-EM structures of prion protein filaments from Gerstmann–Sträussler–Scheinker disease

et al. 2022

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“…It also requires manual curation and selection of the most reliable fragments, presumably due to the fact that by default the method strongly penalizes gaps in BLAST alignments and requires reliable continuous main-chain traces on input. In a recent update (published when our article was in preparation) CryoID authors modified their protocol, which instead of BLAST can also use a dynamic programming sequencealignment procedure that accounts for tracing errors in the models (Terwilliger et al, 2021). Although the alignments are obtained by default using a simplified six-letter sequence, they are scored using individual residue probabilities derived from a rotamer-template-matching procedure implemented in phenix.sequence_from_map.…”

Section: Introductionmentioning

confidence: 99%

findMySequence: a neural-network-based approach for identification of unknown proteins in X-ray crystallography and cryo-EM

Chojnowski

Simpkin

Leonardo

et al. 2021

Int Union Crystallogr J

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Although experimental protein-structure determination usually targets known proteins, chains of unknown sequence are often encountered. They can be purified from natural sources, appear as an unexpected fragment of a well characterized protein or appear as a contaminant. Regardless of the source of the problem, the unknown protein always requires characterization. Here, an automated pipeline is presented for the identification of protein sequences from cryo-EM reconstructions and crystallographic data. The method's application to characterize the crystal structure of an unknown protein purified from a snake venom is presented. It is also shown that the approach can be successfully applied to the identification of protein sequences and validation of sequence assignments in cryo-EM protein structures.

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“…Using the data truncated to 2.70 Å, 8 possible selenium sites were located and refined with the Autosol program in the PHENIX package (36), resulting in an overall figure of merit of 0.31. The resulting electron density map was used to construct an initial model with the AutoBuild program in PHENIX (37).…”

Section: Methodsmentioning

confidence: 99%

“…The structures of Sso2081:cA 4 and Sso2081 S11A : A 4 >P complexes were solved by molecular replacement with the program Phaser-MR of PHENIX (38), using the structure of apo Sso2081 as template. The resulting solution was used to construct the initial model with the program AutoBuild in PHENIX (37). Iterative model building and refinement were carried out with COOT (39), PHENIX (36) and REFMAC5 (40).…”

Section: Methodsmentioning

confidence: 99%

Molecular basis of stepwise cyclic tetra-adenylate cleavage by the type III CRISPR ring nuclease Crn1/Sso2081

Luo²,

Lin

2022

Preprint

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The cyclic oligoadenylates (cOAs) act as second messengers of type III CRISPR immunity system through activating the auxiliary nucleases for indiscriminate RNA degradation. The cOA-degrading nucleases (ring nucleases) provide an off-switch regulation of the signaling, thereby preventing cell dormancy or cell death. Here, we describe the crystal structures of the CRISPR-associated ring nuclease 1 (Crn1) from Saccharolobus solfataricus (Sso) 2081 in its apo or bound to cA4 in both pre-cleavage and transient intermediate states. Sso2081 harbors a unique helical insert that encloses cA4 in the central cavity. Two free phosphates symmetrically bind the catalytic site of apo Sso2081 and overlap with the two scissile phosphates of cA4, supporting a bilaterally symmetrical cleavage. The structure of transient intermediate state captured by Ser11Ala mutation immediately illustrates a stepwise cleavage of cA4 by Sso2081. Our study establishes atomic mechanisms of cA4 recognition and degradation by the type III CRISPR ring nuclease Crn1/Sso2081.

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Protein identification from electron cryomicroscopy maps by automated model building and side-chain matching

Cited by 11 publications

References 22 publications

Cryo-EM structures of prion protein filaments from Gerstmann–Sträussler–Scheinker disease

Cryo-EM structures of prion protein filaments from Gerstmann–Sträussler–Scheinker disease

findMySequence: a neural-network-based approach for identification of unknown proteins in X-ray crystallography and cryo-EM

Molecular basis of stepwise cyclic tetra-adenylate cleavage by the type III CRISPR ring nuclease Crn1/Sso2081

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