Difficult macromolecular structures determined using X-ray diffraction techniques

Macromolecular crystallography has been, for the last few decades, the main source of structural information of biological macromolecular systems and it is one of the most powerful techniques for the analysis of enzyme mechanisms and macromolecular interactions at the atomic level. In addition, it is also an extremely powerful tool for drug design. Recent technological and methodological developments in macromolecular X-ray crystallography have allowed solving structures that until recently were considered difficult or even impossible, such as structures at atomic or subatomic resolution or large macromolecular complexes and assemblies at low resolution. These developments have also helped to solve the 3D-structure of macromolecules from twin crystals. Recently, this technique complemented with cryo-electron microscopy and neutron crystallography has provided the structure of large macromolecular machines with great precision allowing understanding of the mechanisms of their function.     

X-ray diffraction analysis of dehydrated myelin

Improved X-ray diffraction data from dry nerve myelin are presented. In addition to the spacings of approx. 150 Å, 60 Å, 44 Å and 34.6 Å, which have been previously reported, we identify a 14 Å series. The data suggests that the hydrocarbon chains in the single bilayer ($\text{≈ 60}\text{A}\text{?}$) is ordered, whereas in the double bilayer ($\text{≈ 150}\text{A}\text{?}$) and in the fluid phase ($\text{≈ 44}\text{A}\text{?}$) it is disordered. It is shown that cholesterol ($\text{≈34.6}\text{A}\text{?}$) exists as a bilayer, and the 14 Å series is probably another cholesterol phase.     

An X-ray diffraction study of model membrane raft structures

Protein sorting and assembly in membrane biogenesis and function involves the creation of ordered domains of lipids known as membrane rafts. The rafts are comprised of all the major classes of lipids, including glycerophospholipids, sphingolipids and sterol. Cholesterol is known to interact with sphingomyelin to form a liquid-ordered bilayer phase. Domains formed by sphingomyelin and cholesterol, however, represent relatively small proportions of the lipids found in membrane rafts and the properties of other raft lipids are not well characterized. We examined the structure of lipid bilayers comprised of aqueous dispersions of ternary mixtures of phosphatidylcholines and sphingomyelins from tissue extracts and cholesterol using synchrotron X-ray powder diffraction methods. Analysis of the Bragg reflections using peak-fitting methods enables the distinction of three coexisting bilayer structures: (a) a quasicrystalline structure comprised of equimolar proportions of phosphatidylcholine and sphingomyelin, (b) a liquid-ordered bilayer of phospholipid and cholesterol, and (c) fluid phospholipid bilayers. The structures have been assigned on the basis of lamellar repeat spacings, relative scattering intensities and bilayer thickness of binary and ternary lipid mixtures of varying composition subjected to thermal scans between 20 and 50 °C. The results suggest that the order created by the quasicrystalline phase may provide an appropriate scaffold for the organization and assembly of raft proteins on both sides of the membrane. Co-existing liquid-ordered structures comprised of phospholipid and cholesterol provides an additional membrane environment for assembly of different raft proteins.     

Time-resolved analysis of macromolecular structures during reactions by stopped-flow electrooptics.

A stopped-flow field-jump instrument and its use for the analysis of macromolecular structure changes during reactions is described. The operation of the new instrument is simple and reliable, owing to a new type of cell construction with electrodes directly integrated in a quartz cuvette: major advantages are the relatively low demand on sample quantities and a high time resolution. The stopped flow is characterized by a dead time of approximately 0.5 ms. Electric field pulses with field strengths up to 20 kV/cm and rise times in the nanosecond range are applied at adjustable times after stop of the flow. The time resolution of the optical detection is up to the nanosecond time range. The instrument may be used for the combination of stopped flow with temperature-jump and field-jump experiments. A particularly useful new application is the analysis of macromolecular reactions by electrooptical measurements, because electrooptical data provide information about structures. This is demonstrated for the intercalation of ethidium into double-helical DNA. The transients, measured at 313 nm, where the signal is exclusively due to ethidium bound to the DNA, demonstrate a relatively high negative dichroism at 0.5 ms after mixing. The absolute value of this negative dichroism increases in the millisecond time range and approaches the equilibrium value within about a second. The dichroism decay time constants demonstrate a clear increase of the effective DNA length due to ethidium binding, already 0.5 ms after mixing; a further increase to the equilibrium value is found in the millisecond time range. The analysis of these data demonstrate the existence of up to three relaxation processes, depending on the conditions of the experiments. The dichroism amplitudes, together with the decay time constants, indicate that all the reaction states found in the present investigation are complexes with insertion of ethidium residues between basepairs. Moreover, the data clearly show the degree of intercalation in the intermediate states, which is very useful information for the quantitative assignment of the mechanism.     

MOLMOL: A program for display and analysis of macromolecular structures

MOLMOL is a molecular graphics program for display, analysis, and manipulation of three-dimensional structures of biological macromolecules, with special emphasis on nuclear magnetic resonance (NMR) solution structures of proteins and nucleic acids. MOLMOL has a graphical user interface with menus, dialog boxes, and on-line help. The display possibilities include conventional presentation, as well as novel schematic drawings, with the option of combining different presentations in one view of a molecule. Covalent molecular structures can be modified by addition or removal of individual atoms and bonds, and three-dimensional structures can be manipulated by interactive rotation about individual bonds. Special efforts were made to allow for appropriate display and analysis of the sets of typically 20–40 conformers that are conventionally used to represent the result of an NMR structure determination, using functions for superimposing sets of conformers, calculation of root mean square distance (RMSD) values, identification of hydrogen bonds, checking and displaying violations of NMR constraints, and identification and listing of short distances between pairs of hydrogen atoms.     

X-ray diffraction analysis of internal wool lipids

Polarised optical microscopy (POM) and X-ray diffraction techniques were applied to intercellular lipids extracted from wool to study their structural arrangement in order to determine their role in the diffusion properties of wool fibre. Intercellular wool lipids (IWL) arranged as concentrated liposomes were shown to be a good intercellular lipid model, allowing their study by X-ray diffraction techniques. The results confirm that intercellular lipids of wool fibre are organised in a lamellar structure of 5.0–8.0 nm width, termed β-layer, which had been assumed to be lipids arranged as a bilayer. Structurally, internal wool lipids are distributed at least in two domains at low temperatures: an ordered phase made up of ceramides and free fatty acids (FFA) alone, arranged in crystal orthorhombic states separately, and a liquid crystal state when mixed together. At 40 °C there is a reversible phase transition produced by the melt of the crystal orthorhombic states, whereas the liquid crystal state remains until 65 °C.     

New developments in X-ray optics for macromolecular crystallography using laboratory X-ray sources.

A variety of different devices has been described recently for conditioning the X-ray beam incident on the sample for structural studies on proteins and other macromolecular crystals.     

Crystallization and preliminary X-ray diffraction analysis of recombinant pentalenene synthase.

Recombinant pentalenene synthase, a 42.5-kDa sesquiterpene cyclase originally isolated from Streptomyces UC5319 and cloned in Escherichia coli, has been crystallized in space group P6(3) with unit cell dimensions a = b = 183.5 A and c = 56.5 A. Hexagonal prismatic crystals, approximately 0.2 x 0.2 x 0.3 mm, diffract to approximately 2.9 A resolution using monochromatic synchrotron radiation. From the universal (and achiral) building block, farnesyl pyrophosphate, pentalenene synthase catalyzes the formation of four stereocenters in the construction of the three fused five-membered rings of pentalenene; this novel sesquiterpene is a precursor to the pentalenolactone family of antibiotics.     

Molecular symmetry of fructose-1,6-diphosphatase by X-ray diffraction analysis.

Large single crystals of rabbit liver fructose-1,6-diphosphatase suitable for a high resolution structure analysis have been grown from polyethylene glycol. The space group of these crystals is I222 with a = 75 A, b = 81 A, and c = 132 A and there are 2 tetrameric molecules in the unit cell. These crystals have one protein subunit as the crystallographic asymmetric unit and establish point group symmetry 222 as the molecular symmetry.     

A new crystal form of tropomyosin. Preliminary X-ray diffraction analysis

A new crystalline form of tropomyosin has been produced that diffracts to about 4 A resolution. The crystals are grown at room temperature by slowly lowering the concentration of spermine. This polyamine apparently neutralizes the acidic amino acid side-chains of tropomyosin and allows close side-by-side packing of molecules. The space group is C2, with unit cell dimensions a = 259.7 A, b = 55.3 A, c = 135.6 A, and beta = 97.2 degrees. The tropomyosin molecules appear to be bonded head-to-tail to form straight filaments that run along the crystallographic (332) direction in an arrangement closely related to thin crystalline sheets previously described.     

Methods of preparing well-orientated sols of f-actin containing filaments suitable for X-ray diffraction

In this paper we describe methods of preparing orientated f-actin and reconstituting thin filaments that are suitable for X-ray diffraction that allow us to analyse the structure of f-actin to at least 15 A resolution (1 A = 0.1 nm). We described problems that occur during the process of orientation and ways of solving them.     

Methods development for diffraction and spectroscopy studies of metalloenzymes at X-ray free-electron lasers

X-ray free-electron lasers (XFELs) open up new possibilities for X-ray crystallographic and spectroscopic studies of radiation-sensitive biological samples under close to physiological conditions. To facilitate these new X-ray sources, tailored experimental methods and data-processing protocols have to be developed. The highly radiation-sensitive photosystem II (PSII) protein complex is a prime target for XFEL experiments aiming to study the mechanism of light-induced water oxidation taking place at a Mn cluster in this complex. We developed a set of tools for the study of PSII at XFELs, including a new liquid jet based on electrofocusing, an energy dispersive von Hamos X-ray emission spectrometer for the hard X-ray range and a high-throughput soft X-ray spectrometer based on a reflection zone plate. While our immediate focus is on PSII, the methods we describe here are applicable to a wide range of metalloenzymes. These experimental developments were complemented by a new software suite, cctbx.xfel. This software suite allows for near-real-time monitoring of the experimental parameters and detector signals and the detailed analysis of the diffraction and spectroscopy data collected by us at the Linac Coherent Light Source, taking into account the specific characteristics of data measured at an XFEL.     

Study on crystal structures of enzyme-hydrolyzed cellulosic materials by X-ray diffraction

Different cellulosic materials were treated with different extraceller microbial enzymes. Changes in structure and properties of the cellulose caused by enzymatic treatment depend on the composition, the type of enzyme and the type of cellulosic materials. Both endoglucanase and crude cellulase have pronounced effects on the structure of cellulose. The variation of crystal structure was found to attack preferentially the (0 0 2) crystal planes for cellulose I during enzymatic hydrolysis.     

Preliminary X-ray diffraction analysis of human chorionic gonadotropin

Hexagonal bipyramidal crystals of deglycosylated human chorionic gonadotropin have been grown using the method of vapor diffusion against ammonium sulfate. These crystals grow to nearly 0.4 mm along each axis, diffract to better than 3.5-A resolution and are relatively stable to irradiation. The crystals belong to the hexagonal space group P6(1)22 or enantiomer, and have unit cell parameters a = b = 88.7 A and c = 177.3 A.     

Preliminary X-ray diffraction analysis of HhaII endonuclease-DNA cocrystals

HhaII restriction endonuclease purified from an overproducing recombinant E. coli clone has been cocrystallized with a heptanucleotide duplex, d-GGAGTCC:GGACTCC. The cocrystals are monoclonic and belong to the space group C2. The unit cell dimensions are a = 199.0 +/- 1.0 A, b = 100.0 +/- 0.5 A, c = 80.3 +/- 0.4 A, and beta = 101.0 +/- 1.0 degrees. There appear to be two dimers per asymmetric unit and the crystals diffract to 4-A resolution.