aCHESYM: standardized placement of macromolecular models in the unit cell

Unique choice of the unit cell and the asymmetric unit are well defined and described in the International Tables for Crystallography vol. A. Unfortunately, the placement of molecules within the unit cell is not standardized. Since structure solution programs often use random numbers in their algorithms, the selected set of atomic coordinates may be different even with successive runs of the same program. Although formally correct, an arbitrary choice of molecular placement within the unit cell is confusing and may lead to interpretation errors [1]. With the use of the anti-Cheshire unit cell introduced by Dauter [2], for all space groups without inversion symmetry, it is possible to transform the molecular model such that its center of gravity falls within the anti-Cheshire asymmetric unit cell. It means that for macromolecular crystal structures it should be possible to standardize the placement of the molecules within the unit cell. In consequence, it should be easier for crystallographers and non-crystallographers to compare similar or related crystal structures. An implementation of the anti-Cheshire concept has been programmed in Python as a web service, aCHESYM. The aCHESYM program takes a PDB file as input and transforms the macromolecular model into the desired anti-Cheshire region. The program can also handle structure factor CIF files if the transformation used requires reindexing of the reflection data. The unit cell, coordinates and displacement parameters of all atoms after transformation are saved in a new PDB file. All the calculated transformations are reversible, so there is no danger of data loss. Moreover, the program helps the user to find the most compact assembly of the molecules (chains) in the structure when there are several chains in the asymmetric unit.

Modulated protein crystal structure with 28 molecules in the asymmetric unit

In modulated crystals short-range translational order is lost and the atomic structure cannot be defined by the contents of a single small unit cell. The wave of disorder is described by a modulation function, which restores long-range periodicity. If the modulation period divided by the unit cell translation is a rational number, then the modulation is commensurate, and can be described in an expanded unit cell. Otherwise it is incommensurate. The diffraction pattern of a modulated structure contains strong main reflections from the basic unit cell, surrounded by weaker satellites from the modulation wave. Modulated structures are rare in protein crystallography. Stress factors induce in plants the expression of Pathogenesis-Related (PR) proteins, divided into 17 classes. PR proteins of class 10 (PR-10) are well studied structurally but their biological function is unclear with an implication in phytohormone binding. PR-10/hormone complexes are studied using fluorescent probes such as ANS (8-anilino-1-naphthalene sulfonate). We crystallized Hyp-1, a PR-10 protein from St John's wort, in complex with ANS. Solution of the apparent P4(1)22 crystal structure was impossible by standard molecular replacement because of evident tetartohedral twinning and a bizarre modulation of reflection intensities with l periodicity of 7. The structure was solved using Phaser and data expanded to P1 symmetry. Ultimately, the structure turned out to have C2 symmetry with 28 independent protein molecules, arranged in dimers around a non-crystallographic (NCS) screw along c with a pitch of ~1/7. The seven-fold repetition along c is indicative of a commensurate modulated structure: the NCS copies are similar but not identical. For instance, the consecutive Hyp-1 molecules bind a varying number (0-3) of the ligand molecules. The structure has been successfully refined to R=22.2% using conventional methods, i.e. with unit cell expanded to encompass the entire commensurate modulation period.

Structural flexibility revealed by ultra-high resolution data

Not many macromolecular crystals diffract X-rays to ultra-high resolution, defined usually as higher than 0.8 Å, and in the Protein Data Bank there are currently 43 such submissions. These structures range in size from antibiotics of about a hundred atoms to proteins with more than 3,000 independent atoms in the asymmetric unit of the crystal cell. The unprecedented data resolution reveals a great wealth of structural details, which cannot be visualized by analyses at lower resolution. The accuracy of the refined stereochemical and geometrical parameters is then comparable with values typical for small-molecular crystallography and exceeds the accuracy of the library of the standard restraint target values, routinely used in refinement of proteins and nucleotides. Somewhat unexpectedly, the very high resolution diffraction does not necessarily relates to extreme stability of the crystallized molecules, so that the obtained electron density maps reveal significant parts of the atomic models existing in multiple conformations, slightly differing from each other. For example, about 1/3 of the protein chain in the 0.65 Å structure of lysozyme [1] and majority of phosphate groups in the 0.75 Å structure of Z-DNA dodecamer [2] could be modeled in double conformations.

Scoliosis of backbone in Z-DNA nucleotides

According to Wikipedia "scoliosis is a medical condition in which a person's spine is curved from side to side. Although it is a complex three-dimensional deformity, on an X-ray, viewed from the rear, the spine of an individual with scoliosis can resemble an `S' or a `?', rather than a straight line". This definition fits very well with the syndrome observed in many structures of Z-DNA nucleotides. In contrast to the A- and B-forms of DNA, where the sugar-phosphate backbone smoothly follows the right-handed helical line, in Z-DNA the backbone winds as a left-handed zig-zag pattern, repeating every two base pairs. On the basis of the first structures of Z-DNA it was observed that the phosphate groups can be rotated towards the outside of the helix in the ZI type or towards the inside in the ZII type of Z-DNA. A large number of crystal structures of Z-DNA oligomers are currently available in the Protein Data Bank, where both backbone types are observed, often co-existing as alternative, partially occupied conformations. At first it was postulated that the backbone conformation adopted by Z-DNA depends on the presence of metal cations and polyamines in the crystal structures, but later it was realized that the ZI and ZII conformations are not specific to the sequence or the interactions with metal ions [1]. A comparison of larger number of high-resolution Z-DNA structures reveals that the structures can adopt a range of backbone torsion angles between the two values representing the canonical ZI and ZII types [2].

Orange Fluorescent Proteins: Filling the Spectral Gap

Fluorescent proteins (FPs) have become valuable tools for molecular biology, biochemistry, medicine, and cancer research. Starting from parent green fluorescent protein (GFP), most challenging task of the FPs studies was the development of FPs with longer excitation/emission wavelength. This pursuit was motivated by advantages of so-called red-shifted FPs, namely, lower background of cellular autofluorescence in microscopy, lower light scattering and reduced tissue absorbance of longer wavelengths for in vivo imaging. In addition to FPs with regular spectral properties, there are proteins of other types available, including FPs with a large Stokes shift and photoconvertible FPs. These special kinds of FPs have become useful in super-resolution microscopy, imaging of enzyme activities, protein-protein interactions, photolabeling, and in vivo imaging. According to their emission wavelength, red-shifted FPs could be divided in the following groups: 520-540 nm yellow FPs (YFPs), 540-570 nm orange FPs (OFPs), 570-620 nm red FPs (RFPs), and > 620 nm far-RFPs. Red shift of the excitation/emission bands of these FPs is predominantly achieved by extension of the conjugated system of the chromophore and its protonation/deprotonation. The variety of spectral properties of FPs (excitation and emission wavelength, quantum yield, brightness, photo- and pH- stability, photoconversion, large Stokes shift, etc) results from the different chromophore structures and its interactions with surrounding amino acid residues. In this work we focus on structural studies and molecular mechanisms of FPs with orange emission.