The FUSION protein crystallization screen

Gorrec, F.; Bellini, Dom

doi:10.1107/s1600576722001765

Cited by 4 publications

(2 citation statements)

References 60 publications

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“…Drop ratios of 0.2 μl protein solution plus 0.2 μl reservoir solution were used for screening. The only useful dataset was collected from a crystal collected from the Fusion screen (Molecular Dimensions) 60 with following composition: 37.5% PEG 3350/PEG 1 K/MPD (1:1:1), 0.1 M Bicine/Trizma pH 8.5, 0.8% (w/v) Morpheus III Alkaloids and 0.12 M Morpheus Alcohols. Crystals were collected and flash frozen in liquid nitrogen.…”

Section: Methodsmentioning

confidence: 99%

Adding α,α-disubstituted and β-linked monomers to the genetic code of an organism

Dunkelmann,

Piedrafita,

Dickson

et al. 2024

Nature

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The genetic code of living cells has been reprogrammed to enable the site-specific incorporation of hundreds of non-canonical amino acids into proteins, and the encoded synthesis of non-canonical polymers and macrocyclic peptides and depsipeptides1–3. Current methods for engineering orthogonal aminoacyl-tRNA synthetases to acylate new monomers, as required for the expansion and reprogramming of the genetic code, rely on translational readouts and therefore require the monomers to be ribosomal substrates4–6. Orthogonal synthetases cannot be evolved to acylate orthogonal tRNAs with non-canonical monomers (ncMs) that are poor ribosomal substrates, and ribosomes cannot be evolved to polymerize ncMs that cannot be acylated onto orthogonal tRNAs—this co-dependence creates an evolutionary deadlock that has essentially restricted the scope of translation in living cells to α-l-amino acids and closely related hydroxy acids. Here we break this deadlock by developing tRNA display, which enables direct, rapid and scalable selection for orthogonal synthetases that selectively acylate their cognate orthogonal tRNAs with ncMs in Escherichia coli, independent of whether the ncMs are ribosomal substrates. Using tRNA display, we directly select orthogonal synthetases that specifically acylate their cognate orthogonal tRNA with eight non-canonical amino acids and eight ncMs, including several β-amino acids, α,α-disubstituted-amino acids and β-hydroxy acids. We build on these advances to demonstrate the genetically encoded, site-specific cellular incorporation of β-amino acids and α,α-disubstituted amino acids into a protein, and thereby expand the chemical scope of the genetic code to new classes of monomers.

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Section: Methodsmentioning

confidence: 99%

Adding α,α-disubstituted and β-linked monomers to the genetic code of an organism

Dunkelmann,

Piedrafita,

Dickson

et al. 2024

Nature

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“…To determine the structures of target proteins, researchers have developed crystallization tags. (11)(12)(13)(14) One promising method involves using porous protein crystals to immobilize target proteins. (7,(15)(16)(17) However, despite its potential, the versatility of this approach is limited.…”

Section: Introductionmentioning

confidence: 99%

High-throughput structure determination of an intrinsically disordered protein using cell-free protein crystallization

Kojima,

Abe,

Furuta

et al. 2023

Preprint

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Intrinsically disordered proteins (IDPs) play a crucial role in various biological phenomena, dynamically changing their conformations in response to external environmental cues. To gain a deeper understanding of these proteins, it is essential to identify the determinants that fix their structures at the atomic level. Here, we developed a pipeline for rapid crystal structure analysis of IDP using a cell-free protein crystallization (CFPC) method. Through this approach, we successfully demonstrated the determination of the structure of an IDP to uncover the key determinants that stabilize its conformation. Specifically, we focused on the 11-residue fragment of c-Myc, which forms an α-helix through dimerization with a binding partner protein. This fragment was strategically fused with an in-cell crystallizing protein and was expressed in a cell-free system. The resulting crystal structures of the c-Myc fragment were successfully determined at a resolution of 1.92 Å and we confirmed that they are identical to the structures of the complex with the native binding partner protein. This indicates that the environment of the scaffold crystal can fix the structure of c-Myc. Significantly, these crystals were obtained directly from a small reaction mixture (30 μL) incubated for only 72 hours. Analysis of 8 crystal structures derived from 22 mutants revealed two hydrophobic residues as the key determinants responsible for stabilizing the α-helical structure. These findings underscore the power of our CFPC screening method as a valuable tool for determining the structures of challenging target proteins and elucidating the essential molecular interactions that govern their stability.

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Structural and biophysical characterization of the cytoplasmic domains of HprS kinase and its interactions with the cognate regulator HprR

Koczurowska,

Carrillo,

Alai

et al. 2025

Archives of Biochemistry and Biophysics

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The FUSION protein crystallization screen

Cited by 4 publications

References 60 publications

Adding α,α-disubstituted and β-linked monomers to the genetic code of an organism

Adding α,α-disubstituted and β-linked monomers to the genetic code of an organism

High-throughput structure determination of an intrinsically disordered protein using cell-free protein crystallization

Structural and biophysical characterization of the cytoplasmic domains of HprS kinase and its interactions with the cognate regulator HprR

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