Tim Gruene

Crystal structure of the 14-subunit RNA polymerase I

Protein biosynthesis depends on the availability of ribosomes, which in turn relies on ribosomal RNA production. In eukaryotes, this process is carried out by RNA polymerase I (Pol I), a 14-subunit enzyme, the activity of which is a major determinant of cell growth. Here we present the crystal structure of Pol I from Saccharomyces cerevisiae at 3.0 Å resolution. The Pol I structure shows a compact core with a wide DNA-binding cleft and a tightly anchored stalk. An extended loop mimics the DNA backbone in the cleft and may be involved in regulating Pol I transcription. Subunit A12.2 extends from the A190 jaw to the active site and inserts a transcription elongation factor TFIIS-like zinc ribbon into the nucleotide triphosphate entry pore, providing insight into the role of A12.2 in RNA cleavage and Pol I insensitivity to α-amanitin. The A49–A34.5 heterodimer embraces subunit A135 through extended arms, thereby contacting and potentially regulating subunit A12.2.

Refinement of macromolecular structures against neutron data with SHELXL2013

SHELXL2013 contains improvements over the previous versions that facilitate the refinement of macromolecular structures against neutron data. This article highlights several features of particular interest for this purpose and includes a list of restraints for H-atom refinement.

A magic triangle for experimental phasing of macromolecules

Obtaining phase information for the solution of macromolecular structures is still one of the bottlenecks in X-ray crystallography. 5-Amino-2,4,6-triiodoisophthalic acid (I3C), in which three covalently bound iodines form an equilateral triangle, was incorporated into proteins in order to obtain phases by single-wavelength anomalous dispersion (SAD). An improved binding capability compared with simple heavy-metal ions, ready availability, improved recognition of potential heavy-atom sites and low toxicity make I3C particularly suitable for experimental phasing.

Structure of Tripeptidyl-peptidase I Provides Insight into the Molecular Basis of Late Infantile Neuronal Ceroid Lipofuscinosis

Late infantile neuronal ceroid lipofuscinosis, a fatal neurodegenerative disease of childhood, is caused by mutations in the TPP1 gene that encodes tripeptidyl-peptidase I. We show that purified TPP1 requires at least partial glycosylation for in vitro autoprocessing and proteolytic activity. We crystallized the fully glycosylated TPP1 precursor under conditions that implied partial autocatalytic cleavage between the prosegment and the catalytic domain. X-ray crystallographic analysis at 2.35 Å resolution reveals a globular structure with a subtilisin-like fold, a Ser475-Glu272-Asp360 catalytic triad, and an octahedrally coordinated Ca2+-binding site that are characteristic features of the S53 sedolisin family of peptidases. In contrast to other S53 peptidases, the TPP1 structure revealed steric constraints on the P4 substrate pocket explaining its preferential cleavage of tripeptides from the unsubstituted N terminus of proteins. Two alternative conformations of the catalytic Asp276 are associated with the activation status of TPP1. 28 disease-causing missense mutations are analyzed in the light of the TPP1 structure providing insight into the molecular basis of late infantile neuronal ceroid lipofuscinosis.

Structures of viscotoxins A1 and B2 from European mistletoe solved using native data alone.

Crystals of the cytotoxic thionin proteins viscotoxins A1 and B2 extracted from mistletoe diffracted to high resolution (1.25 and 1.05 A, respectively) and are excellent candidates for testing crystallographic methods. Ab initio direct methods were only successful in solving the viscotoxin B2 structure, which with 861 unique non-H atoms is one of the largest unknown structures without an atom heavier than sulfur to be solved in this way, but sulfur-SAD phasing provided a convincing solution for viscotoxin A1. Both proteins form dimers in the crystal and viscotoxin B2 (net charge +4 per monomer), but not viscotoxin A1 (net charge +6), is coordinated by sulfate or phosphate anions. The viscotoxin A1 crystal has a higher solvent content than the viscotoxin B2 crystal (49% as opposed to 28%) with solvent channels along the crystallographic 4(3) axes.

The magic triangle goes MAD: experimental phasing with a bromine derivative

5-Amino-2,4,6-tribromoisophthalic acid is used as a phasing tool for protein structure determination by MAD phasing. It is the second representative of a novel class of compounds for heavy-atom derivatization that combine heavy atoms with amino and carboxyl groups for binding to proteins.

New method to compute Rcomplete enables maximum likelihood refinement for small datasets

Significance Modern crystallographic structure determination uses maximum likelihood methods. They rely on error estimates between the work model and the unknown target based on a small fraction of the data. This can introduce a large uncertainty and, even worse, restricts the method to projects where sufficient data are available. We investigate the Rcomplete method. It enables the use of all data for error estimation. It reduces the uncertainty associated with the conventional Rfree approach for small datasets. We show that our approach reduces the effect of overfitting. This enables maximum likelihood methods to be extended to a much wider field of applications, including free electron laser experiments, high-pressure crystallography, and low-resolution structures. The crystallographic reliability index Rcomplete is based on a method proposed more than two decades ago. Because its calculation is computationally expensive its use did not spread into the crystallographic community in favor of the cross-validation method known as Rfree. The importance of Rfree has grown beyond a pure validation tool. However, its application requires a sufficiently large dataset. In this work we assess the reliability of Rcomplete and we compare it with k-fold cross-validation, bootstrapping, and jackknifing. As opposed to proper cross-validation as realized with Rfree, Rcomplete relies on a method of reducing bias from the structural model. We compare two different methods reducing model bias and question the widely spread notion that random parameter shifts are required for this purpose. We show that Rcomplete has as little statistical bias as Rfree with the benefit of a much smaller variance. Because the calculation of Rcomplete is based on the entire dataset instead of a small subset, it allows the estimation of maximum likelihood parameters even for small datasets. Rcomplete enables maximum likelihood-based refinement to be extended to virtually all areas of crystallographic structure determination including high-pressure studies, neutron diffraction studies, and datasets from free electron lasers.

Solving the RNA polymerase I structural puzzle.

Details of the RNA polymerase I crystal structure determination provide a framework for solution of the structures of other multi-subunit complexes. Simple crystallographic experiments are described to extract relevant biological information such as the location of the enzyme active site.

Geometric properties of nucleic acids with potential for autobuilding.

Algorithms and geometrical properties are described for the automated building of nucleic acids in experimental electron density.

mrtailor: a tool for PDB-file preparation for the generation of external restraints.

Model building starting from, for example, a molecular-replacement solution with low sequence similarity introduces model bias, which can be difficult to detect, especially at low resolution. The program mrtailor removes low-similarity regions from a template PDB file according to sequence similarity between the target sequence and the template sequence and maps the target sequence onto the PDB file. The modified PDB file can be used to generate external restraints for low-resolution refinement with reduced model bias and can be used as a starting point for model building and refinement. The program can call ProSMART [Nicholls et al. (2012), Acta Cryst. D68, 404-417] directly in order to create external restraints suitable for REFMAC5 [Murshudov et al. (2011), Acta Cryst. D67, 355-367]. Both a command-line version and a GUI exist.