The MORPHEUS protein crystallization screen

MORPHEUS is an initial protein crystallization screen with a unique organization which integrates components and ligands selected after analysing all crystal structure data deposited with the Protein Data Bank and local data gathered at the MRC Laboratory of Molecular Biology, Cambridge, England (MRC-LMB). Three challenging proteins from the MRC-LMB have already been crystallized exclusively using MORPHEUS

C-H...N hydrogen bonding in an overlayer of s-triazine physisorbed on a graphite surface

The structure of a crystalline monolayer of 1,3,5-triazine has been characterised using X-ray diffraction. The monolayer is found to exhibit a hexagonal unit cell with a lattice parameter of 6.161(5) Å, indicating the formation of C–H…N hydrogen bonds. DFT simulations have been performed exhibiting close agreement with the experimental structure. By comparing the strength of the intermolecular interactions both with and in the absence of Van der Waals corrections, it is possible to estimate an interaction strength for the weak C–H…N hydrogen bonds.We acknowledge financial support for JAD from an Engineering and Physical Sciences Research Council (EPSRC) DTA studentship award from the Department of Chemistry, University of Cambridge

2D constraint modifies packing behaviour: a halobenzene monolayer with X3 halogen-bonding motif.

Using a combination of X-ray diffraction and simulation techniques, we are able to identify a crystalline monolayer of 1,3,5-triiodotrifluorobenzene formed on graphite. The monolayer is found to exhibit an incommensurate hexagonal unit cell with a lattice parameter of 9.28(7) Å, exhibiting a trigonal arrangement of iodine atoms not found in the bulk structure. DFT simulations have been performed exhibiting close agreement with the experimental structure. Importantly these simulations can be used to compare the strength of the intermolecular interactions both with and without Van der Waals corrections. Thus it is possible to estimate that halogen bonding consists of approximately half the total interaction energy. This demonstrates that despite the presence of strong directional non-covalent bonding, dispersion interactions account for a very significant proportion of the total energy

The p110 delta structure: mechanisms for selectivity and potency of new PI(3)K inhibitors.

Deregulation of the phosphoinositide-3-OH kinase (PI(3)K) pathway has been implicated in numerous pathologies including cancer, diabetes, thrombosis, rheumatoid arthritis and asthma. Recently, small-molecule and ATP-competitive PI(3)K inhibitors with a wide range of selectivities have entered clinical development. In order to understand the mechanisms underlying the isoform selectivity of these inhibitors, we developed a new expression strategy that enabled us to determine to our knowledge the first crystal structure of the catalytic subunit of the class IA PI(3)K p110 delta. Structures of this enzyme in complex with a broad panel of isoform- and pan-selective class I PI(3)K inhibitors reveal that selectivity toward p110 delta can be achieved by exploiting its conformational flexibility and the sequence diversity of active site residues that do not contact ATP. We have used these observations to rationalize and synthesize highly selective inhibitors for p110 delta with greatly improved potencies

Pi sampling: a methodical and flexible approach to initial macromolecular crystallization screening

Pi sampling, derived from the incomplete factorial approach, is an effort to maximize the diversity of macromolecular crystallization conditions and to facilitate the preparation of 96-condition initial screens

Structural analysis of an eIF3 subcomplex reveals conserved interactions required for a stable and proper translation pre-initiation complex assembly

Translation initiation factor eIF3 acts as the key orchestrator of the canonical initiation pathway in eukaryotes, yet its structure is greatly unexplored. We report the 2.2 Å resolution crystal structure of the complex between the yeast seven-bladed β-propeller eIF3i/TIF34 and a C-terminal α-helix of eIF3b/PRT1, which reveals universally conserved interactions. Mutating these interactions displays severe growth defects and eliminates association of eIF3i/TIF34 and strikingly also eIF3g/TIF35 with eIF3 and 40S subunits in vivo. Unexpectedly, 40S-association of the remaining eIF3 subcomplex and eIF5 is likewise destabilized resulting in formation of aberrant pre-initiation complexes (PICs) containing eIF2 and eIF1, which critically compromises scanning arrest on mRNA at its AUG start codon suggesting that the contacts between mRNA and ribosomal decoding site are impaired. Remarkably, overexpression of eIF3g/TIF35 suppresses the leaky scanning and growth defects most probably by preventing these aberrant PICs to form. Leaky scanning is also partially suppressed by eIF1, one of the key regulators of AUG recognition, and its mutant sui1G107R but the mechanism differs. We conclude that the C-terminus of eIF3b/PRT1 orchestrates co-operative recruitment of eIF3i/TIF34 and eIF3g/TIF35 to the 40S subunit for a stable and proper assembly of 48S pre-initiation complexes necessary for stringent AUG recognition on mRNAs

Nouvelles conditions de criblage pour la cristallisation de macromolécules : applications à l'étude structurale des protéines par diffraction aux rayons X

La cristallographie aux rayons X permet la détermination des structures tridimensionnelles de macromolécules biologiques ainsi que de leurs complexes à haute résolution. Cependant, la cristallisation des protéines est un phénomène totalement aléatoire et peu de cristaux sont généralement produits, de plus leur qualité et résistance sont souvent insuffisantes. Ces travaux visent à présenter les différentes étapes pour résoudre des structures de protéines ainsi que deux développements innovants pour formuler des solutions de criblage pour la cristallisation (appelés Pi et MORPHEUS).X-ray crystallography enables the structure determination of biological macromolecules, their complexes and assemblies to high-resolution. Nevertheless, protein crystallisation is a stochastic process and the yield of crystals is typically very poor. In addition, crystal properties are often deceiving. Herein, we introduce the basic principles of protein structure determination process and we will discuss two innovative developments of screen formulation (called Pi and MORPHEUS)

The FUSION protein crystallization screen.

The success and speed of atomic structure determination of biological macromolecules by X-ray crystallography depends critically on the availability of diffraction-quality crystals. However, the process of screening crystallization conditions often consumes large amounts of sample and time. An innovative protein crystallization screen formulation called FUSION has been developed to help with the production of useful crystals. The concept behind the formulation of FUSION was to combine the most efficient components from the three MORPHEUS screens into a single screen using a systematic approach. The resulting formulation integrates 96 unique combinations of crystallization additives. Most of these additives are small molecules and ions frequently found in crystal structures of the Protein Data Bank (PDB), where they bind proteins and complexes. The efficiency of FUSION is demonstrated by obtaining high yields of diffraction-quality crystals for seven different test proteins. In the process, two crystal forms not currently in the PDB for the proteins α-amylase and avidin were discovered

Halogen Bonding in Bicomponent Monolayers: Self-Assembly of a Homologous Series of Iodinated Perfluoroalkanes with Bipyridine

A homologous series of halogen bonding monolayers based on terminally iodinated perfluoroalkanes and 4,4′-bipyridine have been observed on a graphitic surface and noninvasively probed using powder X-ray diffraction. An excellent agreement is observed between the X-ray structures and density functional theory calculations with dispersion force corrections. Theoretical analysis of the binding energies of the structures indicate that these halogen bonds are strong (25 kJ mol–1), indicating that the layers are highly stable. The monolayer structures are found to be distinct from any plane of the corresponding bulk structures, with limited evidence of partitioning of hydrocarbon and perfluoro tectons. The interchain interactions are found to be slightly stronger than those in related aromatic systems, with important implications for 2D crystal engineering

Halogen Bonding in Bicomponent Monolayers: Self-Assembly of a Homologous Series of Iodinated Perfluoroalkanes with Bipyridine.

A homologous series of halogen bonding monolayers based on terminally iodinated perfluoroalkanes and 4,4'-bipyridine have been observed on a graphitic surface and noninvasively probed using powder X-ray diffraction. An excellent agreement is observed between the X-ray structures and density functional theory calculations with dispersion force corrections. Theoretical analysis of the binding energies of the structures indicate that these halogen bonds are strong (25 kJ mol-1), indicating that the layers are highly stable. The monolayer structures are found to be distinct from any plane of the corresponding bulk structures, with limited evidence of partitioning of hydrocarbon and perfluoro tectons. The interchain interactions are found to be slightly stronger than those in related aromatic systems, with important implications for 2D crystal engineering