196 research outputs found
Cryptotomography: reconstructing 3D Fourier intensities from randomly oriented single-shot diffraction patterns
We reconstructed the 3D Fourier intensity distribution of mono-disperse
prolate nano-particles using single-shot 2D coherent diffraction patterns
collected at DESY's FLASH facility when a bright, coherent, ultrafast X-ray
pulse intercepted individual particles of random, unmeasured orientations. This
first experimental demonstration of cryptotomography extended the
Expansion-Maximization-Compression (EMC) framework to accommodate unmeasured
fluctuations in photon fluence and loss of data due to saturation or background
scatter. This work is an important step towards realizing single-shot
diffraction imaging of single biomolecules.Comment: 4 pages, 4 figure
Amino-terminal Polypeptides of Vimentin Are Responsible for the Changes in Nuclear Architecture Associated with Human Immunodeficiency Virus Type 1 Protease Activity in Tissue Culture Cells
Electronic damage in S atoms in a native protein crystal induced by an intense X-ray free-electron laser pulse
Current hard X-ray free-electron laser (XFEL) sources can deliver doses to biological macromolecules well exceeding 1 GGy, in timescales of a few tens of femtoseconds. During the pulse, photoionization can reach the point of saturation in which certain atomic species in the sample lose most of their electrons. This electronic radiation damage causes the atomic scattering factors to change, affecting, in particular, the heavy atoms, due to their higher photoabsorption cross sections. Here, it is shown that experimental serial femtosecond crystallography data collected with an extremely bright XFEL source exhibit a reduction of the effective scattering power of the sulfur atoms in a native protein. Quantitative methods are developed to retrieve information on the effective ionization of the damaged atomic species from experimental data, and the implications of utilizing new phasing methods which can take advantage of this localized radiation damage are discussed
Viscous hydrophilic injection matrices for serial crystallography
Serial (femtosecond) crystallography at synchrotron and X-ray free-electron
laser (XFEL) sources distributes the absorbed radiation dose over all crystals
used for data collection and therefore allows measurement of radiation damage
prone systems, including the use of microcrystals for room-temperature
measurements. Serial crystallography relies on fast and efficient exchange of
crystals upon X-ray exposure, which can be achieved using a variety of
methods, including various injection techniques. The latter vary significantly
in their flow rates – gas dynamic virtual nozzle based injectors provide very
thin fast-flowing jets, whereas high-viscosity extrusion injectors produce
much thicker streams with flow rates two to three orders of magnitude lower.
High-viscosity extrusion results in much lower sample consumption, as its
sample delivery speed is commensurate both with typical XFEL repetition rates
and with data acquisition rates at synchrotron sources. An obvious viscous
injection medium is lipidic cubic phase (LCP) as it is used for in meso
membrane protein crystallization. However, LCP has limited compatibility with
many crystallization conditions. While a few other viscous media have been
described in the literature, there is an ongoing need to identify additional
injection media for crystal embedding. Critical attributes are reliable
injection properties and a broad chemical compatibility to accommodate samples
as heterogeneous and sensitive as protein crystals. Here, the use of two novel
hydroÂgels as viscous injection matrices is described, namely sodium
carbÂoxyÂmethyl cellulose and the thermo-reversible block polymer Pluronic
F-127. Both are compatible with various crystallization conditions and yield
acceptable X-ray background. The stability and velocity of the extruded stream
were also analysed and the dependence of the stream velocity on the flow rate
was measured. In contrast with previously characterized injection media, both
new matrices afford very stable adjustable streams suitable for time-resolved
measurements
Atomic structure of granulin determined from native nanocrystalline granulovirus using an X-ray free-electron laser
To understand how molecules function in biological systems, new methods are required to obtain atomic resolution structures from biological material under physiological conditions. Intense femtosecond-duration pulses from X-ray free-electron lasers (XFELs) can outrun most damage processes, vastly increasing the tolerable dose before the specimen is destroyed. This in turn allows structure determination from crystals much smaller and more radiation sensitive than previously considered possible, allowing data collection from room temperature structures and avoiding structural changes due to cooling. Regardless, high-resolution structures obtained from XFEL data mostly use crystals far larger than 1 μm3 in volume, whereas the X-ray beam is often attenuated to protect the detector from damage caused by intense Bragg spots. Here, we describe the 2 Å resolution structure of native nanocrystalline granulovirus occlusion bodies (OBs) that are less than 0.016 μm3 in volume using the full power of the Linac Coherent Light Source (LCLS) and a dose up to 1.3 GGy per crystal. The crystalline shell of granulovirus OBs consists, on average, of about 9,000 unit cells, representing the smallest protein crystals to yield a high-resolution structure by X-ray crystallography to date. The XFEL structure shows little to no evidence of radiation damage and is more complete than a model determined using synchrotron data from recombinantly produced, much larger, cryocooled granulovirus granulin microcrystals. Our measurements suggest that it should be possible, under ideal experimental conditions, to obtain data from protein crystals with only 100 unit cells in volume using currently available XFELs and suggest that single-molecule imaging of individual biomolecules could almost be within reach
Influence of pump laser fluence on ultrafast structural changes in myoglobin
High-intensity femtosecond pulses from an X-ray free-electron laser enable pump probe experiments for investigating electronic and nuclear changes during light-induced reactions. On time scales ranging from femtoseconds to milliseconds and for a variety of biological systems, time-resolved serial femtosecond crystallography (TR-SFX) has provided detailed structural data for light-induced isomerization, breakage or formation of chemical bonds and electron transfer. However, all ultra-fast TR-SFX studies to date have employed such high pump laser energies that several photons were nominally absorbed per chromophore. As multiphoton absorption may force the protein response into nonphysiological pathways, it is of great concern whether this experimental approach allows valid inferences to be drawn vis-a-vis biologically relevant single-photon-induced reactions. Here we describe ultrafast pump-probe SFX experiments on photodissociation of carboxymyoglobin, showing that different pump laser fluences yield markedly different results. In particular, the dynamics of structural changes and observed indicators of the mechanistically important coherent oscillations of the Fe-CO bond distance (predicted by recent quantum wavepacket dynamics) are seen to depend strongly on pump laser energy. Our results confirm both the feasibility and necessity of performing TR-SFX pump probe experiments in the linear photoexcitation regime. We consider this to be a starting point for reassessing design and interpretation of ultrafast TR-SFX pump probe experiments such that biologically relevant insight emerges
crystal and solution structures of the multidomain cochaperone DnaJ
Hsp70 chaperones assist in a large variety of protein-folding processes in the
cell. Crucial for these activities is the regulation of Hsp70 by Hsp40
cochaperones. DnaJ, the bacterial homologue of Hsp40, stimulates ATP
hydrolysis by DnaK (Hsp70) and thus mediates capture of substrate protein, but
is also known to possess chaperone activity of its own. The first structure of
a complete functional dimeric DnaJ was determined and the mobility of its
individual domains in solution was investigated. Crystal structures of the
complete molecular cochaperone DnaJ from Thermus thermophilus comprising the
J, GF and C-terminal domains and of the J and GF domains alone showed an
ordered GF domain interacting with the J domain. Structure-based EPR spin-
labelling studies as well as cross-linking results showed the existence of
multiple states of DnaJ in solution with different arrangements of the various
domains, which has implications for the function of DnaJ.1\. Auflag
Single-shot diffraction data from the Mimivirus particle using an X-ray free-electron laser
Citation: Ekeberg, T., Svenda, M., Seibert, M. M., Abergel, C., Maia, F. R. N. C., Seltzer, V., . . . Hajdu, J. (2016). Single-shot diffraction data from the Mimivirus particle using an X-ray free-electron laser. Scientific Data, 3. doi:10.1038/sdata.2016.60Free-electron lasers (FEL) hold the potential to revolutionize structural biology by producing X-ray pules short enough to outrun radiation damage, thus allowing imaging of biological samples without the limitation from radiation damage. Thus, a major part of the scientific case for the first FELs was three-dimensional (3D) reconstruction of non-crystalline biological objects. In a recent publication we demonstrated the first 3D reconstruction of a biological object from an X-ray FEL using this technique. The sample was the giant Mimivirus, which is one of the largest known viruses with a diameter of 450 nm. Here we present the dataset used for this successful reconstruction. Data-analysis methods for single-particle imaging at FELs are undergoing heavy development but data collection relies on very limited time available through a highly competitive proposal process. This dataset provides experimental data to the entire community and could boost algorithm development and provide a benchmark dataset for new algorithms
The Different Function of Single Phosphorylation Sites of Drosophila melanogaster Lamin Dm and Lamin C
Lamins' functions are regulated by phosphorylation at specific sites but our understanding of the role of such modifications is practically limited to the function of cdc 2 (cdk1) kinase sites in depolymerization of the nuclear lamina during mitosis. In our study we used Drosophila lamin Dm (B-type) to examine the function of particular phosphorylation sites using pseudophosphorylated mutants mimicking single phosphorylation at experimentally confirmed in vivo phosphosites (S25E, S45E, T435E, S595E). We also analyzed lamin C (A-type) and its mutant S37E representing the N-terminal cdc2 (mitotic) site as well as lamin Dm R64H mutant as a control, non-polymerizing lamin. In the polymerization assay we could observe different effects of N-terminal cdc2 site pseudophosphorylation on A- and B-type lamins: lamin Dm S45E mutant was insoluble, in contrast to lamin C S37E. Lamin Dm T435E (C-terminal cdc2 site) and R64H were soluble in vitro. We also confirmed that none of the single phosphorylation site modifications affected the chromatin binding of lamin Dm, in contrast to the lamin C N-terminal cdc2 site. In vivo, all lamin Dm mutants were incorporated efficiently into the nuclear lamina in transfected Drosophila S2 and HeLa cells, although significant amounts of S45E and T435E were also located in cytoplasm. When farnesylation incompetent mutants were expressed in HeLa cells, lamin Dm T435E was cytoplasmic and showed higher mobility in FRAP assay
Structural Organization of DNA in Chlorella Viruses
Chlorella viruses have icosahedral capsids with an internal membrane enclosing their large dsDNA genomes and associated proteins. Their genomes are packaged in the particles with a predicted DNA density of ca. 0.2 bp nm−3. Occasionally infection of an algal cell by an individual particle fails and the viral DNA is dynamically ejected from the capsid. This shows that the release of the DNA generates a force, which can aid in the transfer of the genome into the host in a successful infection. Imaging of ejected viral DNA indicates that it is intimately associated with proteins in a periodic fashion. The bulk of the protein particles detected by atomic force microscopy have a size of ∼60 kDa and two proteins (A278L and A282L) of about this size are among 6 basic putative DNA binding proteins found in a proteomic analysis of DNA binding proteins packaged in the virion. A combination of fluorescence images of ejected DNA and a bioinformatics analysis of the DNA reveal periodic patterns in the viral DNA. The periodic distribution of GC rich regions in the genome provides potential binding sites for basic proteins. This DNA/protein aggregation could be responsible for the periodic concentration of fluorescently labeled DNA observed in ejected viral DNA. Collectively the data indicate that the large chlorella viruses have a DNA packaging strategy that differs from bacteriophages; it involves proteins and share similarities to that of chromatin structure in eukaryotes
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