Dorothee Liebschner

An improved experimental databank of transferable multipolar atom models – ELMAM2. Construction details and applications

ELMAM2 is a generalized and improved library of experimentally derived multipolar atom types. The previously published ELMAM database is restricted mostly to protein atoms. The current database is extended to common functional groups encountered in organic molecules and is based on optimized local axes systems taking into account the local pseudosymmetry of the molecular fragment. In this approach, the symmetry-restricted multipoles have zero populations, while others take generally significant values. The various applications of the database are described. The deformation electron densities, electrostatic potentials and interaction energies calculated for several tripeptides and aromatic molecules are calculated using ELMAM2 electron-density parameters and compared with the former ELMAM database and density functional theory calculations.

Elucidation of the phosphate binding mode of DING proteins revealed by subangstrom X-ray crystallography.

PfluDING is a bacterial protein isolated from Pseudomonas fluorescens that belongs to the DING protein family, which is ubiquitous in eukaryotes and extends to prokaryotes. DING proteins and PfluDING have very similar topologies to phosphate Solute Binding Proteins (SBPs). The three-dimensional structure of PfluDING was obtained at subangstrom resolution (0.88 and 0.98 A) at two different pHs (4.5 and 8.5), allowing us to discuss the hydrogen bond network that sequesters the phosphate ion in the binding site. From this high resolution data, we experimentally elucidated the molecular basis of phosphate binding in phosphate SBPs. The phosphate ion is tightly bound to the protein via 12 hydrogen bonds between phosphate oxygen atoms and OH and NH groups of the protein. The proton on one oxygen atom of the phosphate dianion forms a 2.5 A low barrier hydrogen bond with an aspartate, with the energy released by forming this strong bond ensuring the specificity for the dianion even at pH 4.5. In particular, contrary to previous theories on phosphate SBPs, accurate electrostatic potential calculations show that the binding cleft is positively charged. PfluDING structures reveal that only dibasic phosphate binds to the protein at both acidic and basic phosphate, suggesting that the protein binding site environment stabilizes the HPO(4)(2-) form of phosphate.

Topological analysis of hydrogen bonds and weak interactions in protein helices via transferred experimental charge density parameters.

Helices represent the most abundant secondary structure motif in proteins and are often involved in various functional roles. They are stabilized by a network of hydrogen bonds between main chain carbonyl and amide groups. Several surveys scrutinized these hydrogen bonds, mostly based on the geometry of the interaction. Alternatively, the topological analysis of the electron density provides a powerful technique to investigate hydrogen bonds. For the first time, transferred experimental charge density parameters (ELMAM database) were used to carry out a topological analysis of the electron density in protein helices. New parameters for the description of the hydrogen bond geometry are proposed. Bonding contacts between the amide N and carbonyl O atoms (N···O) of helices, poorly addressed in the literature so far, were characterized from topological, geometrical, and local energetic analyses. Particularly, a geometrical criterion allowing for the discrimination between hydrogen bonds and N···O contacts is proposed.

Hydrogen atoms in protein structures: high-resolution X-ray diffraction structure of the DFPase

BackgroundHydrogen atoms represent about half of the total number of atoms in proteins and are often involved in substrate recognition and catalysis. Unfortunately, X-ray protein crystallography at usual resolution fails to access directly their positioning, mainly because light atoms display weak contributions to diffraction. However, sub-Ångstrom diffraction data, careful modeling and a proper refinement strategy can allow the positioning of a significant part of hydrogen atoms.ResultsA comprehensive study on the X-ray structure of the diisopropyl-fluorophosphatase (DFPase) was performed, and the hydrogen atoms were modeled, including those of solvent molecules. This model was compared to the available neutron structure of DFPase, and differences in the protein and the active site solvation were noticed.ConclusionsA further examination of the DFPase X-ray structure provides substantial evidence about the presence of an activated water molecule that may constitute an interesting piece of information as regard to the enzymatic hydrolysis mechanism.

On the influence of crystal size and wavelength on native SAD phasing.

Native SAD is an emerging phasing technique that uses the anomalous signal of native heavy atoms to obtain crystallographic phases. The method does not require specific sample preparation to add anomalous scatterers, as the light atoms contained in the native sample are used as marker atoms. The most abundant anomalous scatterer used for native SAD, which is present in almost all proteins, is sulfur. However, the absorption edge of sulfur is at low energy (2.472 keV = 5.016 Å), which makes it challenging to carry out native SAD phasing experiments as most synchrotron beamlines are optimized for shorter wavelength ranges where the anomalous signal of sulfur is weak; for longer wavelengths, which produce larger anomalous differences, the absorption of X-rays by the sample, solvent, loop and surrounding medium (e.g. air) increases tremendously. Therefore, a compromise has to be found between measuring strong anomalous signal and minimizing absorption. It was thus hypothesized that shorter wavelengths should be used for large crystals and longer wavelengths for small crystals, but no thorough experimental analyses have been reported to date. To study the influence of crystal size and wavelength, native SAD experiments were carried out at different wavelengths (1.9 and 2.7 Å with a helium cone; 3.0 and 3.3 Å with a helium chamber) using lysozyme and ferredoxin reductase crystals of various sizes. For the tested crystals, the results suggest that larger sample sizes do not have a detrimental effect on native SAD data and that long wavelengths give a clear advantage with small samples compared with short wavelengths. The resolution dependency of substructure determination was analyzed and showed that high-symmetry crystals with small unit cells require higher resolution for the successful placement of heavy atoms.

Structural insights and ab initio sequencing within the DING proteins family

DING proteins constitute a recently discovered protein family that is ubiquitous in eukaryotes. The structural insights and the physiological involvements of these intriguing proteins are hereby deciphered.

Frontier Applications of Experimental Charge Density and Electrostatics to Bio-macromolecules

Experimental charge density methodologies have been extended to macromolecular structures and biocrystallography. Ultra-high resolution diffraction data can now be collected at third-generation synchrotron sources for well-ordered protein crystals. The molecular structure can then be refined using multipolar expansion of the atomic electron density, which is a more sophisticated model than the independent atom model (neutral spherical atoms). Several databases describing average electron densities in terms of multipolar atoms were built by different research groups. These library charge density parameters have been transferred, in the literature, to several small molecules and a few biomacromolecules. The construction of the molecular electron densities through database transfer yields a better crystallographic refinement, notably when the X-ray diffraction data are measured at atomic resolution (0.6–1 A). The use of an aspherical atom model for the electron density in atomic resolution protein structures allows for the accurate description of their electrostatic properties. The applications to several protein structures including syntenin PDZ2 domain, influenza neuraminidase, human aldose reductase (at 0.66 A resolution) and a DING phosphate binding protein have been reported.

Crystallographic analysis of murine p24γ2 Golgi dynamics domain

The p24 family proteins form homo‐ and hetero‐oligomeric complexes for efficient transport of cargo proteins from the endoplasmic reticulum to the Golgi apparatus. It consists of four subfamilies (p24α, p24β, p24γ, and p24δ). p24γ2 plays crucial roles in the selective transport of glycosylphosphatidylinositol‐anchored proteins. Here, we determined the crystal structure of mouse p24γ2 Golgi dynamics (GOLD) domain at 2.8 Å resolution by the single anomalous diffraction method using intrinsic sulfur atoms. In spite of low sequence identity among p24 family proteins, p24γ2 GOLD domain assumes a β‐sandwich fold, similar to that of p24β1 or p24δ1. An additional short α‐helix is observed at the C‐terminus of the p24γ2 GOLD domain. Intriguingly, p24γ2 GOLD domains crystallize as dimers, and dimer formation seems assisted by the short α‐helix. Dimerization modes of GOLD domains are compared among p24 family proteins. Proteins 2017; 85:764–770.

Video tutorials for the Phenix software suite

The World Wide Web is abundant of video tutorials covering many different topics. For example, a search for “tutorial” yields 178 million results on the video sharing website Youtube. The success of the tutorials is without doubt aided by easy-to-use software for creating the videos and the broad coverage of high-speed internet connections enabling convenient upload and access. This also reflects the desire of the audience to have an audio-visual support for procedures, which could be given in written format instead. In 2017, the Phenix Tutorials Youtube channel [1] was launched. Phenix [2] is a program for the automated determination of molecular structures using X-ray crystallography and other methods. The extensive online manual [3] covers about 180 separate html pages (more than 600 letter size printed pages), including tutorials, FAQs and descriptions of many Phenix tools. To expand the documentation resources, video tutorials for commonly used tools were created and made accessible on YouTube. The videos give a short introduction about a Phenix tool, summarize the required input files and parameters, explain how to run the program and – when appropriate – discuss the results. It is planned to keep adding tutorials addressing new tools or commonly asked questions from users. Here, we present our experience with the Phenix video tutorials, such as feedback from the community, viewing statistics and how to create them.

Case studies of sulfur SAD phasing with LigM from Sphingobium sp. SYK-6

Sphingobium sp. SYK-6 grows on a lignin-related biphenyl compound as the sole carbon and energy source and was initially isolated from a pond waste liquor from a kraft pulp mill. In SYK-6, 5-CH3-H4folate is synthesized from aromatic compounds such as a vanillate by a O-demethylase, LigM. The 5-CH3-H4folate is then converted to 5,10-CH2-H4folate, which is utilized for syntheses of DNA, repair DNA, and methylate DNA as well as to act as a cofactor in certain biological reactions, by another enzyme, MetF. In other bacterial speceis, 5,10-CH2-H4folate is directly synthesized by Tand H-proteins that are enzymes in Glycine Cleavage System. It is considered that SYK-6 has evolved to acquire this unique pathway for the 5,10-CH2-H4folate production, in order to survive in extreme environmental condition. To elucidate the molecular mechanisim of this pathway, we have carried out the structural analysis of LigM. LigM was purified by using IMPACT system provided from NEB, which use intein and affinity chitin-binding tag. After crystallization screening, a reservoir solution of 0.2 M Mg acetate, 0.1 M Acetate buffer pH 4.6, and 20 %(w/v) PEG8000 gave a needle crystals with approximate dimensions of 0.3×0.1×0.01 mm3. A diffraction data set was collected with 1.1 Å wavelength at BL1-A in the Photon Factory. However, phasing trials via molecular replacement (using a model with 19% sequence identity) failed. Because LigM is a 53 kDa protein and contains fourteen sulfur atoms, LigM is an interesting candidate for SAD phasing with sulfur (S-SAD). Diffraction data sets of LigM crystals were collected with 1.9 and 2.7 Å wavelengths, reaching a maximum resolution of 2.3Å. Preliminary results are promising for solving the phase problem via S-SAD. This study is also of methodological interest as the phasing capability of two different wavelengths can be compared. A thorough analysis of the diffraction data is in progress.