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author:("gruen, Tim")
1.  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.
Knowing the structure of multi-subunit complexes is critical to understand basic cellular functions. However, when crystals of these complexes can be obtained they rarely diffract beyond 3 Å resolution, which complicates X-ray structure determination and refinement. The crystal structure of RNA polymerase I, an essential cellular machine that synthesizes the precursor of ribosomal RNA in the nucleolus of eukaryotic cells, has recently been solved. Here, the crucial steps that were undertaken to build the atomic model of this multi-subunit enzyme are reported, emphasizing how simple crystallographic experiments can be used to extract relevant biological information. In particular, this report discusses the combination of poor molecular replacement and experimental phases, the application of multi-crystal averaging and the use of anomalous scatterers as sequence markers to guide tracing and to locate the active site. The methods outlined here will likely serve as a reference for future structural determination of large complexes at low resolution.
doi:10.1107/S1399004714015788
PMCID: PMC4188003  PMID: 25286842
low-resolution structure determination; multi-subunit complexes; transcription; RNA polymerase I
2.  Refinement of macromolecular structures against neutron data with SHELXL2013  
Journal of Applied Crystallography  2013;47(Pt 1):462-466.
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.
Some of the improvements in SHELX2013 make SHELXL convenient to use for refinement of macromolecular structures against neutron data without the support of X-ray data. The new NEUT instruction adjusts the behaviour of the SFAC instruction as well as the default bond lengths of the AFIX instructions. This work presents a protocol on how to use SHELXL for refinement of protein structures against neutron data. It includes restraints extending the Engh & Huber [Acta Cryst. (1991), A47, 392–400] restraints to H atoms and discusses several of the features of SHELXL that make the program particularly useful for the investigation of H atoms with neutron diffraction. SHELXL2013 is already adequate for the refinement of small molecules against neutron data, but there is still room for improvement, like the introduction of chain IDs for the refinement of macromolecular structures.
doi:10.1107/S1600576713027659
PMCID: PMC3937812  PMID: 24587788
single-crystal neutron diffraction; macromolecular structure refinement; hydrogen restraints; SHELXL2013
3.  Integrated analysis of the conformation of a protein-linked spin label by crystallography, EPR and NMR spectroscopy 
Journal of Biomolecular Nmr  2011;49(2):111-119.
Long-range structural information derived from paramagnetic relaxation enhancement observed in the presence of a paramagnetic nitroxide radical is highly useful for structural characterization of globular, modular and intrinsically disordered proteins, as well as protein–protein and protein-DNA complexes. Here we characterized the conformation of a spin-label attached to the homodimeric protein CylR2 using a combination of X-ray crystallography, electron paramagnetic resonance (EPR) and NMR spectroscopy. Close agreement was found between the conformation of the spin label observed in the crystal structure with interspin distances measured by EPR and signal broadening in NMR spectra, suggesting that the conformation seen in the crystal structure is also preferred in solution. In contrast, conformations of the spin label observed in crystal structures of T4 lysozyme are not in agreement with the paramagnetic relaxation enhancement observed for spin-labeled CylR2 in solution. Our data demonstrate that accurate positioning of the paramagnetic center is essential for high-resolution structure determination.
Electronic supplementary material
The online version of this article (doi:10.1007/s10858-011-9471-y) contains supplementary material, which is available to authorized users.
doi:10.1007/s10858-011-9471-y
PMCID: PMC3042103  PMID: 21271275
Spin label; Crystal structure; MTSL; Protein; NMR
4.  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.
Medium- to high-resolution X-ray structures of DNA and RNA molecules were investigated to find geometric properties useful for automated model building in crystallographic electron-density maps. We describe a simple method, starting from a list of electron-density ‘blobs’, for identifying backbone phosphates and nucleic acid bases based on properties of the local electron-density distribution. This knowledge should be useful for the automated building of nucleic acid models into electron-density maps. We show that the distances and angles involving C1′ and the P atoms, using the pseudo-torsion angles and that describe the …P—C1′—P—C1′… chain, provide a promising basis for building the nucleic acid polymer. These quantities show reasonably narrow distributions with asymmetry that should allow the direction of the phosphate backbone to be established.
doi:10.1107/S0108767310039140
PMCID: PMC3006036  PMID: 21173468
nucleic acids; autobuilding; geometric properties; electron-density distribution
5.  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.
Experimental phasing is an essential technique for the solution of macromolecular structures. Since many heavy-atom ion soaks suffer from nonspecific binding, a novel class of compounds has been developed that combines heavy atoms with functional groups for binding to proteins. The phasing tool 5-amino-2,4,6-tribromoisophthalic acid (B3C) contains three functional groups (two carboxylate groups and one amino group) that interact with proteins via hydrogen bonds. Three Br atoms suitable for anomalous dispersion phasing are arranged in an equilateral triangle and are thus readily identified in the heavy-atom substructure. B3C was incorporated into proteinase K and a multiwavelength anomalous dispersion (MAD) experiment at the Br K edge was successfully carried out. Radiation damage to the bromine–carbon bond was investigated. A comparison with the phasing tool I3C that contains three I atoms for single-wavelength anomalous dispersion (SAD) phasing was also carried out.
doi:10.1107/S0907444909051609
PMCID: PMC2852301  PMID: 20382990
multi-wavelength anomalous dispersion; experimental phasing; heavy-atom derivatives

Results 1-5 (5)