| 1. |
- Bielecki, Johan, 1982, et al.
(författare)
-
Electrospray sample injection for single-particle imaging with x-ray lasers
- 2019
-
Ingår i: Science advances. - : American Association for the Advancement of Science (AAAS). - 2375-2548. ; 5:5
-
Tidskriftsartikel (refereegranskat)abstract
- The possibility of imaging single proteins constitutes an exciting challenge for x-ray lasers. Despite encouraging results on large particles, imaging small particles has proven to be difficult for two reasons: not quite high enough pulse intensity from currently available x-ray lasers and, as we demonstrate here, contamination of the aerosolized molecules by nonvolatile contaminants in the solution. The amount of contamination on the sample depends on the initial droplet size during aerosolization. Here, we show that, with our electrospray injector, we can decrease the size of aerosol droplets and demonstrate virtually contaminant-free sample delivery of organelles, small virions, and proteins. The results presented here, together with the increased performance of next-generation x-ray lasers, constitute an important stepping stone toward the ultimate goal of protein structure determination from imaging at room temperature and high temporal resolution.
|
|
| 2. |
- Carlsson, Gunilla H., et al.
(författare)
-
The elusive ligand complexes of the DWARF14 strigolactone receptor
- 2018
-
Ingår i: Journal of Experimental Botany. - : Oxford University Press (OUP). - 0022-0957 .- 1460-2431. ; 69:9, s. 2345-2354
-
Tidskriftsartikel (refereegranskat)abstract
- Strigolactones, a group of terpenoid lactones, control many aspects of plant growth and development, but the active forms of these plant hormones and their mode of action at the molecular level are still unknown. The strigolactone protein receptor is unusual because it has been shown to cleave the hormone and supposedly forms a covalent bond with the cleaved hormone fragment. This interaction is suggested to induce a conformational change in the receptor that primes it for subsequent interaction with partners in the signalling pathway. Substantial efforts have been invested into describing the interaction of synthetic strigolactone analogues with the receptor, resulting in a number of crystal structures. This investigation combines a re-evaluation of models in the Protein Data Bank with a search for new conditions that may permit the capture of a receptor-ligand complex. While weak difference density is frequently observed in the binding cavity, possibly due to a low-occupancy compound, the models often contain features not supported by the X-ray data. Thus, at this stage, we do not believe that any detailed deductions about the nature, conformation, or binding mode of the ligand can be made with any confidence.
|
|
| 3. |
- Daurer, Benedikt J., et al.
(författare)
-
Experimental strategies for imaging bioparticles with femtosecond hard X-ray pulses
- 2017
-
Ingår i: IUCrJ. - : INT UNION CRYSTALLOGRAPHY. - 2052-2525. ; 4, s. 251-262
-
Tidskriftsartikel (refereegranskat)abstract
- This study explores the capabilities of the Coherent X-ray Imaging Instrument at the Linac Coherent Light Source to image small biological samples. The weak signal from small samples puts a significant demand on the experiment. Aerosolized Omono River virus particles of similar to 40 nm in diameter were injected into the submicrometre X-ray focus at a reduced pressure. Diffraction patterns were recorded on two area detectors. The statistical nature of the measurements from many individual particles provided information about the intensity profile of the X-ray beam, phase variations in the wavefront and the size distribution of the injected particles. The results point to a wider than expected size distribution (from similar to 35 to similar to 300 nm in diameter). This is likely to be owing to nonvolatile contaminants from larger droplets during aerosolization and droplet evaporation. The results suggest that the concentration of nonvolatile contaminants and the ratio between the volumes of the initial droplet and the sample particles is critical in such studies. The maximum beam intensity in the focus was found to be 1.9 * 10(12) photons per mu m(2) per pulse. The full-width of the focus at half-maximum was estimated to be 500 nm (assuming 20% beamline transmission), and this width is larger than expected. Under these conditions, the diffraction signal from a sample-sized particle remained above the average background to a resolution of 4.25 nm. The results suggest that reducing the size of the initial droplets during aerosolization is necessary to bring small particles into the scope of detailed structural studies with X-ray lasers.
|
|
| 4. |
- Eisenhut, Marion, et al.
(författare)
-
The plant-like C2 glycolate cycle and the bacterial-like glycerate pathway cooperate in phosphoglycolate metabolism in cyanobacteria.
- 2006
-
Ingår i: Plant Physiology. - : Oxford University Press (OUP). - 0032-0889 .- 1532-2548. ; 142:1
-
Tidskriftsartikel (refereegranskat)abstract
- The occurrence of a photorespiratory 2-phosphoglycolate metabolism in cyanobacteria is not clear. In the genome of the cyanobacterium Synechocystis sp. strain PCC 6803, we have identified open reading frames encoding enzymes homologous to those forming the plant-like C2 cycle and the bacterial-type glycerate pathway. To study the route and importance of 2-phosphoglycolate metabolism, the identified genes were systematically inactivated by mutagenesis. With a few exceptions, most of these genes could be inactivated without leading to a high-CO(2)-requiring phenotype. Biochemical characterization of recombinant proteins verified that Synechocystis harbors an active serine hydroxymethyltransferase, and, contrary to higher plants, expresses a glycolate dehydrogenase instead of an oxidase to convert glycolate to glyoxylate. The mutation of this enzymatic step, located prior to the branching of phosphoglycolate metabolism into the plant-like C2 cycle and the bacterial-like glycerate pathway, resulted in glycolate accumulation and a growth depression already at high CO(2). Similar growth inhibitions were found for a single mutant in the plant-type C2 cycle and more pronounced for a double mutant affected in both the C2 cycle and the glycerate pathway after cultivation at low CO(2). These results suggested that cyanobacteria metabolize phosphoglycolate by the cooperative action of the C2 cycle and the glycerate pathway. When exposed to low CO(2), glycine decarboxylase knockout mutants accumulated far more glycine and lysine than wild-type cells or mutants with inactivated glycerate pathway. This finding and the growth data imply a dominant, although not exclusive, role of the C2 route in cyanobacterial phosphoglycolate metabolism.
|
|
| 5. |
- Ekeberg, Tomas, 1983-, et al.
(författare)
-
Observation of a single protein by ultrafast X-ray diffraction
- 2024
-
Ingår i: Light. - : Springer Nature. - 2095-5545 .- 2047-7538. ; 13:1
-
Tidskriftsartikel (refereegranskat)abstract
- The idea of using ultrashort X-ray pulses to obtain images of single proteins frozen in time has fascinated and inspired many. It was one of the arguments for building X-ray free-electron lasers. According to theory, the extremely intense pulses provide sufficient signal to dispense with using crystals as an amplifier, and the ultrashort pulse duration permits capturing the diffraction data before the sample inevitably explodes. This was first demonstrated on biological samples a decade ago on the giant mimivirus. Since then, a large collaboration has been pushing the limit of the smallest sample that can be imaged. The ability to capture snapshots on the timescale of atomic vibrations, while keeping the sample at room temperature, may allow probing the entire conformational phase space of macromolecules. Here we show the first observation of an X-ray diffraction pattern from a single protein, that of Escherichia coli GroEL which at 14 nm in diameter is the smallest biological sample ever imaged by X-rays, and demonstrate that the concept of diffraction before destruction extends to single proteins. From the pattern, it is possible to determine the approximate orientation of the protein. Our experiment demonstrates the feasibility of ultrafast imaging of single proteins, opening the way to single-molecule time-resolved studies on the femtosecond timescale.
|
|
| 6. |
- Gorkhover, Tais, et al.
(författare)
-
Femtosecond X-ray Fourier holography imaging of free-flying nanoparticles
- 2018
-
Ingår i: Nature Photonics. - : Springer Science and Business Media LLC. - 1749-4885 .- 1749-4893. ; 12:3, s. 150-153
-
Tidskriftsartikel (refereegranskat)abstract
- Ultrafast X-ray imaging on individual fragile specimens such as aerosols 1 , metastable particles 2 , superfluid quantum systems 3 and live biospecimens 4 provides high-resolution information that is inaccessible with conventional imaging techniques. Coherent X-ray diffractive imaging, however, suffers from intrinsic loss of phase, and therefore structure recovery is often complicated and not always uniquely defined 4,5 . Here, we introduce the method of in-flight holography, where we use nanoclusters as reference X-ray scatterers to encode relative phase information into diffraction patterns of a virus. The resulting hologram contains an unambiguous three-dimensional map of a virus and two nanoclusters with the highest lateral resolution so far achieved via single shot X-ray holography. Our approach unlocks the benefits of holography for ultrafast X-ray imaging of nanoscale, non-periodic systems and paves the way to direct observation of complex electron dynamics down to the attosecond timescale.
|
|
| 7. |
- Hagemann, Martin, et al.
(författare)
-
Detection of a phage genome carrying a zonula occludens like toxin gene (zot) in clinical isolates of Stenotrophomonas maltophilia.
- 2006
-
Ingår i: Archives of Microbiology. - : Springer Science and Business Media LLC. - 0302-8933 .- 1432-072X. ; 185:6
-
Tidskriftsartikel (refereegranskat)abstract
- During a study of the genetic diversity of Stenotrophomonas strains, we found an autonomous replicating DNA molecule in chromosomal DNA preparations of the clinical Stenotrophomonas maltophilia strain c5. The entire sequence of 6,907 bp of the isolated DNA molecule was determined, which was called phiSMA9. Seven ORFs, which code for proteins with considerable similarity to proteins in databases, were identified in the DNA sequence. The largest ORF shows high sequence similarities to the pI protein of the filamentous phage phiLf, which was later shown to be identical to toxin Zot of Vibrio cholerae. Beside the Zot-like protein, six other proteins with similarities to known phage proteins such as a phage replication protein RstA and phage absorption or coat protein are encoded on phiSMA9, which indicate that this circular DNA molecule represents the replicative form of a linear phage genome. A PCR-based screening showed that only five from the totally investigated 47 Stenotrophomonas strains of clinical and environmental origin harbor these genes. Altogether, we describe the first genome of a phage for the nosocomial pathogen Stenotrophomonas, which contains a Zot toxin like gene and might be regarded as the first Stenotrophomonas virulence factor.
|
|
| 8. |
- Hagemann, Martin, et al.
(författare)
-
The plant-associated bacterium Stenotrophomonas rhizophila expresses a new enzyme for the synthesis of the compatible solute glucosylglycerol.
- 2008
-
Ingår i: Journal of Bacteriology. - 0021-9193 .- 1098-5530. ; 190:17
-
Tidskriftsartikel (refereegranskat)abstract
- The rhizobacterium Stenotrophomonas rhizophila accumulates the compatible solutes glucosylglycerol (GG) and trehalose under salt stress conditions. The complete gene for the GG synthesis enzyme was cloned and sequenced. This enzyme from S. rhizophila represented a novel fusion protein composed of a putative C-terminal GG-phosphate synthase domain and an N-terminal putative GG-phosphate phosphatase domain, which was named GgpPS. A similar gene was cloned from Pseudomonas sp. strain OA146. The ggpPS gene was induced after a salt shock in S. rhizophila cells. After the salt-loaded cells reached stationary phase, the ggpPS mRNA content returned to the low level characteristic of the control cells, and GG was released into the medium. The complete ggpPS gene and a truncated version devoid of the phosphatase part were obtained as recombinant proteins. Enzyme activity tests revealed the expected abilities of the full-length protein to synthesize GG and the truncated GgpPS to synthesize GG-phosphate. However, dephosphorylation of GG-phosphate was detected only with the complete GgpPS protein. These enzyme activities were confirmed by complementation experiments using defined GG-defective mutants of the cyanobacterium Synechocystis sp. strain PCC 6803. Genes coding for proteins very similar to the newly identified fusion protein GgpPS for GG synthesis in S. rhizophila were found in genome sequences of related bacteria, where these genes are often linked to a gene coding for a transporter of the Mfs superfamily.
|
|
| 9. |
- Hantke, Max F., et al.
(författare)
-
A data set from flash X-ray imaging of carboxysomes
- 2016
-
Ingår i: Scientific Data. - : Springer Science and Business Media LLC. - 2052-4463. ; 3
-
Tidskriftsartikel (refereegranskat)abstract
- Ultra-intense femtosecond X-ray pulses from X-ray lasers permit structural studies on single particles and biomolecules without crystals. We present a large data set on inherently heterogeneous, polyhedral carboxysome particles. Carboxysomes are cell organelles that vary in size and facilitate up to 40% of Earth’s carbon fixation by cyanobacteria and certain proteobacteria. Variation in size hinders crystallization. Carboxysomes appear icosahedral in the electron microscope. A protein shell encapsulates a large number of Rubisco molecules in paracrystalline arrays inside the organelle. We used carboxysomes with a mean diameter of 115±26 nm from Halothiobacillus neapolitanus. A new aerosol sample-injector allowed us to record 70,000 low-noise diffraction patterns in 12 min. Every diffraction pattern is a unique structure measurement and high-throughput imaging allows sampling the space of structural variability. The different structures can be separated and phased directly from the diffraction data and open a way for accurate, high-throughput studies on structures and structural heterogeneity in biology and elsewhere.
|
|
| 10. |
- Hantke, Max F., et al.
(författare)
-
High-throughput imaging of heterogeneous cell organelles with an X-ray laser
- 2014
-
Ingår i: Nature Photonics. - : Springer Science and Business Media LLC. - 1749-4885 .- 1749-4893. ; 8:12, s. 943-949
-
Tidskriftsartikel (refereegranskat)abstract
- We overcome two of the most daunting challenges in single-particle diffractive imaging: collecting many high-quality diffraction patterns on a small amount of sample and separating components from mixed samples. We demonstrate this on carboxysomes, which are polyhedral cell organelles that vary in size and facilitate up to 40% of Earth's carbon fixation. A new aerosol sample-injector allowed us to record 70,000 low-noise diffraction patterns in 12 min with the Linac Coherent Light Source running at 120 Hz. We separate different structures directly from the diffraction data and show that the size distribution is preserved during sample delivery. We automate phase retrieval and avoid reconstruction artefacts caused by missing modes. We attain the highest-resolution reconstructions on the smallest single biological objects imaged with an X-ray laser to date. These advances lay the foundations for accurate, high-throughput structure determination by flash-diffractive imaging and offer a means to study structure and structural heterogeneity in biology and elsewhere.
|
|
