Hardware and software solutions for MX data-collection strategies using the EMBL/ESRF miniaturized multi-axis goniometer head are presented.
Most macromolecular crystallography (MX) diffraction experiments at synchrotrons use a single-axis goniometer. This markedly contrasts with small-molecule crystallography, in which the majority of the diffraction data are collected using multi-axis goniometers. A novel miniaturized κ-goniometer head, the MK3, has been developed to allow macromolecular crystals to be aligned. It is available on the majority of the structural biology beamlines at the ESRF, as well as elsewhere. In addition, the Strategy for the Alignment of Crystals (STAC) software package has been developed to facilitate the use of the MK3 and other similar devices. Use of the MK3 and STAC is streamlined by their incorporation into online analysis tools such as EDNA. The current use of STAC and MK3 on the MX beamlines at the ESRF is discussed. It is shown that the alignment of macromolecular crystals can result in improved diffraction data quality compared with data obtained from randomly aligned crystals.
kappa goniometer; crystal alignment; data-collection strategies
Finding alternatives for insulin therapy and making advances in etiology of type 1 diabetes benefits from a full structural and functional insight into Islets of Langerhans. Electron microscopy (EM) can visualize Islet morphology at the highest possible resolution, however, conventional EM only provides biased snapshots and lacks context. We developed and employed large scale EM and compiled a resource of complete cross sections of rat Islets during immuno-destruction to provide unbiased structural insight of thousands of cells at macromolecular resolution. The resource includes six datasets, totalling 25.000 micrographs, annotated for cellular and ultrastructural changes during autoimmune diabetes. Granulocytes are attracted to the endocrine tissue, followed by extravasation of a pleiotrophy of leukocytes. Subcellullar changes in beta cells include endoplasmic reticulum stress, insulin degranulation and glycogen accumulation. Rare findings include erythrocyte extravasation and nuclear actin-like fibers. While we focus on a rat model of autoimmune diabetes, our approach is general applicable.
Using transmission electron microscopy, automated data collection, and image stitching, biological specimens as large as one square millimeter can be ultrastructurally mapped at nanometer resolution.
A key obstacle in uncovering the orchestration between molecular and cellular events is the vastly different length scales on which they occur. We describe here a methodology for ultrastructurally mapping regions of cells and tissue as large as 1 mm2 at nanometer resolution. Our approach employs standard transmission electron microscopy, rapid automated data collection, and stitching to create large virtual slides. It greatly facilitates correlative light-electron microscopy studies to relate structure and function and provides a genuine representation of ultrastructural events. The method is scalable as illustrated by slides up to 281 gigapixels in size. Here, we applied virtual nanoscopy in a correlative light-electron microscopy study to address the role of the endothelial glycocalyx in protein leakage over the glomerular filtration barrier, in an immunogold labeling study of internalization of oncolytic reovirus in human dendritic cells, in a cryo-electron microscopy study of intact vitrified mouse embryonic cells, and in an ultrastructural mapping of a complete zebrafish embryo slice.
Cryo-electron microscopy images of vitrified large macromolecular complexes can become blurred due to beam-induced specimen alterations. Exposure series are examined, and rigid and non-rigid image registration schemes are applied to reduce such blurring.
The typical dose used to record cryo-electron microscopy images from vitrified biological specimens is so high that radiation-induced structural alterations are bound to occur during data acquisition. Integration of all scattered electrons into one image can lead to significant blurring, particularly if the data are collected from an unsupported thin layer of ice suspended over the holes of a support film. Here, the dose has been fractioned and exposure series have been acquired in order to study beam-induced specimen movements under low dose conditions, prior to bubbling. Gold particles were added to the protein sample as fiducial markers. These were automatically localized and tracked throughout the exposure series and showed correlated motions within small patches, with larger amplitudes of motion vectors at the start of a series compared with the end of each series. A non-rigid scheme was used to register all images within each exposure series, using natural neighbor interpolation with the gold particles as anchor points. The procedure increases the contrast and resolution of the examined macromolecules.
cryo-electron microscopy; single particle; beam-induced movements; exposure series; radiation damage
The effects of dose and dose-rate were investigated for single-particle cryo-electron microscopy using stroboscopic data collection. A dose-rate effect was observed favoring lower flux densities.
Radiation damage is an important resolution limiting factor both in macromolecular X-ray crystallography and cryo-electron microscopy. Systematic studies in macromolecular X-ray crystallography greatly benefited from the use of dose, expressed as energy deposited per mass unit, which is derived from parameters including incident flux, beam energy, beam size, sample composition and sample size. In here, the use of dose is reintroduced for electron microscopy, accounting for the electron energy, incident flux and measured sample thickness and composition. Knowledge of the amount of energy deposited allowed us to compare doses with experimental limits in macromolecular X-ray crystallography, to obtain an upper estimate of radical concentrations that build up in the vitreous sample, and to translate heat-transfer simulations carried out for macromolecular X-ray crystallography to cryo-electron microscopy. Stroboscopic exposure series of 50–250 images were collected for different incident flux densities and integration times from Lumbricus terrestris extracellular hemoglobin. The images within each series were computationally aligned and analyzed with similarity metrics such as Fourier ring correlation, Fourier ring phase residual and figure of merit. Prior to gas bubble formation, the images become linearly brighter with dose, at a rate of approximately 0.1% per 10 MGy. The gradual decomposition of a vitrified hemoglobin sample could be visualized at a series of doses up to 5500 MGy, by which dose the sample was sublimed. Comparison of equal-dose series collected with different incident flux densities showed a dose-rate effect favoring lower flux densities. Heat simulations predict that sample heating will only become an issue for very large dose rates (50 e−Å−2 s−1 or higher) combined with poor thermal contact between the grid and cryo-holder. Secondary radiolytic effects are likely to play a role in dose-rate effects. Stroboscopic data collection combined with an improved understanding of the effects of dose and dose rate will aid single-particle cryo-electron microscopists to have better control of the outcome of their experiments.
single-particle cryo-electron microscopy; radiation damage; dose; dose-rate effect; macromolecular X-ray crystallography
A rapid, easy-to-perform translation calibration procedure has been developed for use with the EMBL/ESRF mini-κ goniometer head and for other inverse-kappa goniometers designed for macromolecular crystallography. Regular calibration ensures the precision of experiments that rely on many degrees of freedom in crystal reorientation.
Precise and convenient crystal reorientation is of experimental importance in macromolecular crystallography (MX). The development of multi-axis goniometers, such as the ESRF/EMBL mini-κ, necessitates the corresponding development of calibration procedures that can be used for the setup, maintenance and troubleshooting of such devices. While traditional multi-axis goniometers require all rotation axes to intersect the unique point of the sample position, recently developed miniaturized instruments for sample reorientation in MX are not as restricted. However, the samples must always be re-centred following a change in orientation. To overcome this inconvenience and allow the use of multi-axis goniometers without the fundamental restriction of having all axes intersecting in the same point, an automatic translation correction protocol has been developed for such instruments. It requires precise information about the direction and location of the rotation axes. To measure and supply this information, a general, easy-to-perform translation calibration (TC) procedure has also been developed. The TC procedure is routinely performed on most MX beamlines at the ESRF and some results are presented for reference.
kappa goniometer; macromolecular crystallography; reorientation; calibration
MxCuBE is a beamline control environment optimized for the needs of macromolecular crystallography. This paper describes the design of the software and the features that MxCuBE currently provides.
The design and features of a beamline control software system for macromolecular crystallography (MX) experiments developed at the European Synchrotron Radiation Facility (ESRF) are described. This system, MxCuBE, allows users to easily and simply interact with beamline hardware components and provides automated routines for common tasks in the operation of a synchrotron beamline dedicated to experiments in MX. Additional functionality is provided through intuitive interfaces that enable the assessment of the diffraction characteristics of samples, experiment planning, automatic data collection and the on-line collection and analysis of X-ray emission spectra. The software can be run in a tandem client-server mode that allows for remote control and relevant experimental parameters and results are automatically logged in a relational database, ISPyB. MxCuBE is modular, flexible and extensible and is currently deployed on eight macromolecular crystallography beamlines at the ESRF. Additionally, the software is installed at MAX-lab beamline I911-3 and at BESSY beamline BL14.1.
automation; macromolecular crystallography; synchrotron beamline control; graphical user interface
The improvement of the X-ray beam quality achieved on ID14-4 by the installation of new X-ray optical elements is described.
ID14-4 at the ESRF is the first tunable undulator-based macromolecular crystallography beamline that can celebrate a decade of user service. During this time ID14-4 has not only been instrumental in the determination of the structures of biologically important molecules but has also contributed significantly to the development of various instruments, novel data collection schemes and pioneering radiation damage studies on biological samples. Here, the evolution of ID14-4 over the last decade is presented, and some of the major improvements that were carried out in order to maintain its status as one of the most productive macromolecular crystallography beamlines are highlighted. The experimental hutch has been upgraded to accommodate a high-precision diffractometer, a sample changer and a large CCD detector. More recently, the optical hutch has been refurbished in order to improve the X-ray beam quality on ID14-4 and to incorporate the most modern and robust optical elements used at other ESRF beamlines. These new optical elements will be described and their effect on beam stability discussed. These studies may be useful in the design, construction and maintenance of future X-ray beamlines for macromolecular crystallography and indeed other applications, such as those planned for the ESRF upgrade.
macromolecular crystallography; X-ray beam quality; beamline diagnostics; beamline automation
A summary of the 2008 New algorithms in macromolecular crystallography and electron microscopy Max-Inf2/Lorentz Center workshop in Leiden is given.
The resolution gap between macromolecular crystallography and electron microscopy continues to decrease. Recent advances in specimen preparation, instrumentation and computational power have allowed accurate structure determination of larger macromolecular complexes by crystallography and/or by electron microscopy on cryovitrified samples. New possibilities in structural biology have opened up and new challenges are faced to further reduce the resolution gap. A workshop at the Lorentz Center, Leiden, The Netherlands, which took place in May 2008, was organized to push further the limits of both complementary techniques through improved computational methods.
macromolecular crystallography; electron microscopy; computational algorithms; Lorentz Center/Max-Inf2 Leiden workshop
A portable and readily aligned online microspectrophotometer that can be easily installed on macromolecular crystallography beamlines is described. It allows measurement of the spectral characteristics of macromolecular crystals prior, during, and after the X-ray diffraction experiment.
X-rays can produce a high concentration of radicals within cryo-cooled macromolecular crystals. Some radicals have large extinction coefficients in the visible (VIS) range of the electromagnetic spectrum, and can be observed optically and spectrally. An online microspectrophotometer with high temporal resolution has been constructed that is capable of measuring UV/VIS absorption spectra (200–1100 nm) during X-ray data collection. The typical X-ray-induced blue colour that is characteristic of a wide range of cryo-conditions has been identified as trapped solvated electrons. Disulphide-containing proteins are shown to form disulphide radicals at millimolar concentrations, with absorption maxima around 400 nm. The solvated electrons and the disulphide radicals seem to have a lifetime in the range of seconds up to minutes at 100 K. The temperature dependence of the kinetics of X-ray-induced radical formation is different for the solvated electrons compared with the disulphide radicals. The online microspectrophotometer provides a technique complementary to X-ray diffraction for analysing and characterizing intermediates and redox states of proteins and enzymes.
radiation damage; macromolecular crystallography; online microspectrophotometry
Controller (C) proteins regulate the timing of the expression of restriction and modification (R–M) genes through a combination of positive and negative feedback circuits. A single dimer bound to the operator switches on transcription of the C-gene and the endonuclease gene; at higher concentrations, a second dimer bound adjacently switches off these genes. Here we report the first structure of a C protein–DNA operator complex, consisting of two C protein dimers bound to the native 35 bp operator sequence of the R–M system Esp1396I. The structure reveals a role for both direct and indirect DNA sequence recognition. The structure of the DNA in the complex is highly distorted, with severe compression of the minor groove resulting in a 50° bend within each operator site, together with a large expansion of the major groove in the centre of the DNA sequence. Cooperative binding between dimers governs the concentration-dependent activation–repression switch and arises, in part, from the interaction of Glu25 and Arg35 side chains at the dimer–dimer interface. Competition between Arg35 and an equivalent residue of the σ70 subunit of RNA polymerase for the Glu25 site underpins the switch from activation to repression of the endonuclease gene.
Many pathogenic mitochondrial DNA mutations are heteroplasmic, with a mixture of mutated and wild-type mtDNA present within individual cells. The severity and extent of the clinical phenotype is largely due to the distribution of mutated molecules between cells in different tissues, but mechanisms underpinning segregation are not fully understood. To facilitate mtDNA segregation studies we developed assays that measure m.3243A>G point mutation loads directly in hundreds of individual cells to determine the mechanisms of segregation over time. In the first study of this size, we observed a number of discrete shifts in cellular heteroplasmy between periods of stable heteroplasmy. The observed patterns could not be parsimoniously explained by random mitotic drift of individual mtDNAs. Instead, a genetically metastable, heteroplasmic mtDNA segregation unit provides the likely explanation, where stable heteroplasmy is maintained through the faithful replication of segregating units with a fixed wild-type/m.3243A>G mutant ratio, and shifts occur through the temporary disruption and re-organization of the segregation units. While the nature of the physical equivalent of the segregation unit remains uncertain, the factors regulating its organization are of major importance for the pathogenesis of mtDNA diseases.