Coot is a tool widely used for model building, refinement, and validation of macromolecular structures. It has been extensively used for crystallography and, more recently, improvements have been introduced to aid in cryo‐EM model building and refinement, as cryo‐EM structures with resolution ranging 2.5–4 A are now routinely available. Model building into these maps can be time‐consuming and requires experience in both biochemistry and building into low‐resolution maps. To simplify and expedite the model building task, and minimize the needed expertise, new tools are being added in Coot. Some examples include morphing, Geman‐McClure restraints, full‐chain refinement, and Fourier‐model based residue‐type‐specific Ramachandran restraints. Here, we present the current state‐of‐the‐art in Coot usage.

Newly transcribed eukaryotic precursor messenger RNAs (pre-mRNAs) are processed at their 3′ ends by the ~1-megadalton multiprotein cleavage and polyadenylation factor (CPF). CPF cleaves pre-mRNAs, adds a polyadenylate tail, and triggers transcription termination, but it is unclear how its various enzymes are coordinated and assembled. Here, we show that the nuclease, polymerase, and phosphatase activities of yeast CPF are organized into three modules. Using electron cryomicroscopy, we determined a 3.5-angstrom-resolution structure of the ~200-kilodalton polymerase module. This revealed four β propellers, in an assembly markedly similar to those of other protein complexes that bind nucleic acid. Combined with in vitro reconstitution experiments, our data show that the polymerase module brings together factors required for specific and efficient polyadenylation, to help coordinate mRNA 3′-end processing.

Summary Cleavage and polyadenylation factor (CPF/CPSF) is a multi-protein complex essential for formation of eukaryotic mRNA 3ʹ ends. CPF cleaves pre-mRNAs at a specific site and adds a poly(A) tail. The cleavage reaction defines the 3ʹ end of the mature mRNA, and thus the activity of the endonuclease is highly regulated. Here, we show that reconstitution of specific pre-mRNA cleavage with recombinant yeast proteins requires incorporation of the Ysh1 endonuclease into an eight-subunit “CPF core ” complex. Cleavage also requires the accessory cleavage factors IA and IB, which bind substrate pre-mRNAs and CPF, likely facilitating assembly of an active complex. Using X-ray crystallography, electron microscopy, and mass spectrometry, we determine the structure of Ysh1 bound to Mpe1 and the arrangement of subunits within CPF core . Together, our data suggest that the active mRNA 3ʹ end processing machinery is a dynamic assembly that is licensed to cleave only when all protein factors come together at the polyadenylation site.

Background: Suppression of Fra a gene expression leads to down-regulation of color-producing flavonoid biosynthesis in strawberry.Results: Fra proteins can bind natural flavonoids, which induce conformational changes in conserved loop regions.Conclusion: Fra a proteins control flavonoid biosynthesis through binding to metabolic intermediates.Significance: PR-10 proteins may play a role in the control of secondary metabolism through binding of metabolites to their ligand-binding cavities.