Automated harvesting and processing of protein crystals through laser photoablation

New methods for crystal mounting, soaking and cryocooling contribute to bridging the automation gap between crystallization and X-ray data collection.

Novel mode of inhibition by D-tagatose 6-phosphate through a Heyns rearrangement in the active site of transaldolase B variants

Variants of the C—C bond-forming enzymes transaldolase B and D-fructose-6-phosphate aldolase A are irreversibly inhibited by D-tagatose 6-phosphate. A novel structure of a covalently bound ring sugar at the active-site lysine residue of the transaldolase B F178Y variant is revealed.

Direct-methods structure determination of a trypanosome RNA-editing substrate fragment with translational pseudosymmetry

The crystal structure of a 32-base-pair RNA double helix (675 non-H atoms) from a trypanosome RNA-editing substrate was determined with 1.05 Å resolution X-ray diffraction data starting from random phases using the direct-methods computer program SIR2014. Success was achieved in the presence of two levels of translational pseudosymmetry caused by three helical repeats.

Structure of a quinolone-stabilized cleavage complex of topoisomerase IV from Klebsiella pneumoniae and comparison with a related Streptococcus pneumoniae complex

Crystal structures of the cleavage complexes of topoisomerase IV from Gram-negative (K. pneumoniae) and Gram-positive (S. pneumoniae) bacterial pathogens stabilized by the clinically important antibacterial drug levofloxacin are presented, analysed and compared. For K. pneumoniae, this is the first high-resolution cleavage complex structure to be reported.

UHM–ULM interactions in the RBM39–U2AF65 splicing-factor complex

The first crystal structure of the RBM39-UHM–U2AF65-ULM complex has been determined at 2.2 Å resolution. Comparison to other UHM–ULM complexes reveals both common and unique interactions.

Grappling with anisotropic data, pseudo-merohedral twinning and pseudo-translational noncrystallographic symmetry: a case study involving pyruvate kinase

A clear narrative of complex structure determination, including crystallographic problems of pseudo-merohedral twinning, pseudo-translational symmetry and data anisotropy, is provided. This manuscript provides a description of how these issues encountered during the structural determination of E. coli type I pyruvate kinase to 2.28 Å resolution were overcome.

Microcrystal delivery by pulsed liquid droplet for serial femtosecond crystallography

A liquid-droplet injector delivers microcrystals of lysozyme in a pulse-by-pulse manner for conducting serial femtosecond crystallography with an X-ray free-electron laser. The structure was determined at a resolution of 2.3 Å with low sample consumption.

New conformations of linear polyubiquitin chains from crystallographic and solution-scattering studies expand the conformational space of polyubiquitin

A new crystal structure of linear Ub₂ reveals a more compact conformation than the previously known conformations. Solution conformations of linear Ub₂, Ub₃ and Ub₄ were probed by small-angle X-ray scattering, revealing that linear Ub₃ predominantly adopts compact conformations while linear Ub₂ and Ub₄ adopt an extended conformation. The results provide a full view of the conformational diversity of linear polyubiquitins.

T-to-R switch of muscle fructose-1,6-bis­phosphatase involves fundamental changes of secondary and quaternary structure

When crystallized in the absence of the allosteric inhibitor AMP, human muscle fructose-1,6-bisphosphatase has a totally unexpected quaternary structure of its active R form, with the two dimers of the homotetrameric molecule in a perpendicular orientation, in stark contrast to the coplanar arrangement of the closely related liver isozyme. The T-to-R switch of the muscle enzyme also involves a highly unusual α→β refolding of the N-terminus.

Phase improvement via the Phantom Derivative technique: ancils that are related to the target structure

Ancil structures correlated with the target structures are used to improve phases via the Phantom Derivative approach.

Diffraction-geometry refinement in the DIALS framework

A comprehensive description of the methods used within the DIALS framework for diffraction-geometry refinement using predicted reflection centroids is given. Examples of the advanced features of the software are provided.

A pipeline for structure determination of in vivo-grown crystals using in cellulo diffraction

A streamlined pipeline for structure determination is described that applies in cellulo X-ray diffraction analysis to crystal-containing cells isolated by flow cytometry. Application of this pipeline to in vivo-grown crystals of the recombinant CPV1 polyhedrin shows that in cellulo analysis is more efficient, is compatible with experimental phasing and does not compromise the quality of the final model.

XModeScore: a novel method for accurate protonation/tautomer-state determination using quantum-mechanically driven macromolecular X-ray crystallographic refinement

XModeScore determines the correct protomeric/tautomeric state or mode of active-site residues along with any bound ligand(s) using quantum-mechanics-based X-ray refinement followed by post-refinement scoring based on a combination of energetic strain (or ligand strain) and rigorous difference electron-density analysis.

Biophysics of DNA