Experimental phasing with SHELXC/D/E: combining chain tracing with density modification

Sheldrick, George M.

doi:10.1107/s0907444909038360

Cited by 1,083 publications

(946 citation statements)

References 42 publications

(48 reference statements)

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“…After performing a fluorescence scan, three data sets were collected from a single crystal at the peak, inflection, and low-energy remote wavelengths (Extended Data Table 2b). From the peak data set, the positions of 14 zinc ions (7 in each Pol I complex) were determined with SHELX C and D 53 . The occupancies calculated by SHELX were between 100% and 58% for the first 14 sites, followed by a drop to ,25% for additional peaks that were not considered.…”

Section: Research Article Methodsmentioning

confidence: 99%

RNA polymerase I structure and transcription regulation

Engel

Sainsbury

Cheung

et al. 2013

Nature

199

305

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Transcription of ribosomal RNA by RNA polymerase (Pol) I initiates ribosome biogenesis and regulates eukaryotic cell growth. The crystal structure of Pol I fromthe yeast Saccharomyces cerevisiae at 2.8A˚ resolution reveals all 14 subunits of the 590-kilodalton enzyme, and shows differences to Pol II. An ‘expander’ element occupies the DNA template site and stabilizes an expanded active centre cleft with an unwound bridge helix. A ‘connector’ element invades the cleft of an adjacent polymerase and stabilizes an inactive polymerase dimer. The connector and expander must detach during Pol I activation to enable transcription initiation and cleft contraction by convergent movement of the polymerase ‘core’ and ‘shelf’ modules. Conversion between an inactive expanded and an active contracted polymerase state may generally underlie transcription. Regulatory factors can modulate the core–shelf interface that includes a ‘composite’ active site for RNA chain initiation, elongation, proofreading and termination

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Section: Research Article Methodsmentioning

confidence: 99%

RNA polymerase I structure and transcription regulation

Engel

Sainsbury

Cheung

et al. 2013

Nature

199

305

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“…Initial automated heavy atom substructure determination, phasing, and density modification were done using autoSHARP (36). A total of 31 bromide sites were found by ShelxD (37) in the single monomer present in the asymmetric unit and used in the phasing process. BUCCANEER (38) was able to build an incomplete model.…”

Section: Methodsmentioning

confidence: 99%

Recognition of protein-linked glycans as a determinant of peptidase activity

Noach

Ficko-Blean

Pluvinage

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

137

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The vast majority of proteins are posttranslationally altered, with the addition of covalently linked sugars (glycosylation) being one of the most abundant modifications. However, despite the hydrolysis of protein peptide bonds by peptidases being a process essential to all life on Earth, the fundamental details of how peptidases accommodate posttranslational modifications, including glycosylation, has not been addressed. Through biochemical analyses and X-ray crystallographic structures we show that to hydrolyze their substrates, three structurally related metallopeptidases require the specific recognition of O-linked glycan modifications via carbohydrate-specific subsites immediately adjacent to their peptidase catalytic machinery. The three peptidases showed selectivity for different glycans, revealing protein-specific adaptations to particular glycan modifications, yet always cleaved the peptide bond immediately preceding the glycosylated residue. This insight builds upon the paradigm of how peptidases recognize substrates and provides a molecular understanding of glycoprotein degradation.

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“…Data were indexed, integrated, and scaled with the program HKL2000 (56). Phases were determined using the program SHELX (57). Refinement, density modification, and model building were done using the program package PHENIX (58).…”

Section: Methodsmentioning

confidence: 99%

Functional architecture of the Reb1-Ter complex of Schizosaccharomyces pombe

Jaiswal

Choudhury

Zaman

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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Reb1 of Schizosaccharomyces pombe represents a family of multifunctional proteins that bind to specific terminator sites (Ter) and cause polar termination of transcription catalyzed by RNA polymerase I (pol I) and arrest of replication forks approaching the Ter sites from the opposite direction. However, it remains to be investigated whether the same mechanism causes arrest of both DNA transactions. Here, we present the structure of Reb1 as a complex with a Ter site at a resolution of 2.7 Å. Structure-guided molecular genetic analyses revealed that it has distinct and well-defined DNA binding and transcription termination (TTD) domains. The region of the protein involved in replication termination is distinct from the TTD. Mechanistically, the data support the conclusion that transcription termination is not caused by just high affinity Reb1-Ter protein-DNA interactions. Rather, protein-protein interactions between the TTD with the Rpa12 subunit of RNA pol I seem to be an integral part of the mechanism. This conclusion is further supported by the observation that double mutations in TTD that abolished its interaction with Rpa12 also greatly reduced transcription termination thereby revealing a conduit for functional communications between RNA pol I and the terminator protein.crystal structure | RNA polymerase I | transcription termination | protein-DNA interaction | replication termination E ukaryotic rDNAs are found as multiple tandem copies encoding pre-rRNA and upstream and downstream regulatory elements (1-3) including DNA sequences (Ter) that promote site-specific termination of transcription catalyzed by RNA polymerase I (pol I) from yeast to humans (4-14). Specialized transcription terminator proteins bind to Ter sites and not only arrest transcription by RNA polymerase I (pol I) in a polar mode but also replication forks approaching from the opposite direction. Several studies have suggested that pol I transcription termination is a multistep process that requires (i) pausing of chain elongation by the terminator protein and (ii) dissociation and release of pol I and the primary transcript from the template. In mice, dissociation of pol I and release of the transcript require the release factor PTRF, a 44-kDa protein that interacts with the largest subunit of pol I (15). In yeast, additional factors for the processing of the end include the endonuclease Rnt1, the 5′-3′ Rat1 exonuclease, Sen1 helicase, and the kinase Grc3. They are part of an alternate pathway for termination by transcriptional coprocessing (4,(16)(17)(18)(19). In addition, in vivo analyses of Saccharomyces cerevisiae have shown a requirement for Rpa12, a component of pol I necessary for transcription termination (20). Whether the polar arrest of transcription is caused by an interaction between Rpa12 with the terminator protein is unknown.The terminator proteins that mediate transcription termination have been identified in multiple organisms and include Reb1 and Nsi1 [also called yeast transcription terminator (Ytt1)] of S. cerevisiae (...

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Experimental phasing with SHELXC/D/E: combining chain tracing with density modification

Cited by 1,083 publications

References 42 publications

RNA polymerase I structure and transcription regulation

RNA polymerase I structure and transcription regulation

Recognition of protein-linked glycans as a determinant of peptidase activity

Functional architecture of the Reb1-Ter complex of Schizosaccharomyces pombe

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