| 11. |
- Andersson, Magnus, et al.
(författare)
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Structural Dynamics of Light-Driven Proton Pumps
- 2009
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Ingår i: Structure. - : Elsevier BV. - 0969-2126 .- 1878-4186. ; 17:9, s. 1265-1275
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Tidskriftsartikel (refereegranskat)abstract
- Bacteriorhodopsin and proteorhodopsin are simple heptahelical proton pumps containing a retinal chromophore covalently bound to helix G via a protonated Schiff base. Following the absorption of a photon, all-trans retinal is isomerized to a 13-cis conformation, initiating a sequence of conformational changes driving vectorial proton transport. In this study we apply time-resolved wide-angle X-ray scattering to visualize in real time the helical motions associated with proton pumping by bacteriorhodopsin and proteorhodopsin. Our results establish that three conformational states are required to describe their photocycles. Significant motions of the cytoplasmic half of helix F and the extracellular half of helix C are observed prior to the primary proton transfer event, which increase in amplitude following proton transfer. These results both simplify the structural description to emerge from intermediate trapping studies of bacteriorhodopsin and reveal shared dynamical principles for proton pumping.
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| 12. |
- Andersson, Rebecka, 1988, et al.
(författare)
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Serial femtosecond crystallography structure of cytochrome c oxidase at room temperature.
- 2017
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Ingår i: Scientific reports. - : Springer Science and Business Media LLC. - 2045-2322. ; 7:1
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Tidskriftsartikel (refereegranskat)abstract
- Cytochrome c oxidase catalyses the reduction of molecular oxygen to water while the energy released in this process is used to pump protons across a biological membrane. Although an extremely well-studied biological system, the molecular mechanism of proton pumping by cytochrome c oxidase is still not understood. Here we report a method to produce large quantities of highly diffracting microcrystals of ba 3-type cytochrome c oxidase from Thermus thermophilus suitable for serial femtosecond crystallography. The room-temperature structure of cytochrome c oxidase is solved to 2.3Å resolution from data collected at an X-ray Free Electron Laser. We find overall agreement with earlier X-ray structures solved from diffraction data collected at cryogenic temperature. Previous structures solved from synchrotron radiation data, however, have shown conflicting results regarding the identity of the active-site ligand. Our room-temperature structure, which is free from the effects of radiation damage, reveals that a single-oxygen species in the form of a water molecule or hydroxide ion is bound in the active site. Structural differences between the ba 3-type and aa 3-type cytochrome c oxidases around the proton-loading site are also described.
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| 13. |
- Andersson, Rebecka, 1988, et al.
(författare)
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Well-based crystallization of lipidic cubic phase microcrystals for serial X-ray crystallography experiments.
- 2019
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Ingår i: Acta crystallographica. Section D, Structural biology. - 2059-7983. ; 75:Pt 10, s. 937-946
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Tidskriftsartikel (refereegranskat)abstract
- Serial crystallography is having an increasing impact on structural biology. This emerging technique opens up new possibilities for studying protein structures at room temperature and investigating structural dynamics using time-resolved X-ray diffraction. A limitation of the method is the intrinsic need for large quantities of well ordered micrometre-sized crystals. Here, a method is presented to screen for conditions that produce microcrystals of membrane proteins in the lipidic cubic phase using a well-based crystallization approach. A key advantage over earlier approaches is that the progress of crystal formation can be easily monitored without interrupting the crystallization process. In addition, the protocol can be scaled up to efficiently produce large quantities of crystals for serial crystallography experiments. Using the well-based crystallization methodology, novel conditions for the growth of showers of microcrystals of three different membrane proteins have been developed. Diffraction data are also presented from the first user serial crystallography experiment performed at MAX IV Laboratory.
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| 14. |
- Aponte-Santamaria, C., et al.
(författare)
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Temperature dependence of protein-water interactions in a gated yeast aquaporin
- 2017
-
Ingår i: Scientific Reports. - : Springer Science and Business Media LLC. - 2045-2322. ; 7
-
Tidskriftsartikel (refereegranskat)abstract
- Regulation of aquaporins is a key process of living organisms to counteract sudden osmotic changes. Aqy1, which is a water transporting aquaporin of the yeast Pichia pastoris, is suggested to be gated by chemo-mechanical stimuli as a protective regulatory-response against rapid freezing. Here, we tested the influence of temperature by determining the X-ray structure of Aqy1 at room temperature (RT) at 1.3 angstrom resolution, and by exploring the structural dynamics of Aqy1 during freezing through molecular dynamics simulations. At ambient temperature and in a lipid bilayer, Aqy1 adopts a closed conformation that is globally better described by the RT than by the low-temperature (LT) crystal structure. Locally, for the blocking-residue Tyr31 and the water molecules inside the pore, both LT and RT data sets are consistent with the positions observed in the simulations at room-temperature. Moreover, as the temperature was lowered, Tyr31 adopted a conformation that more effectively blocked the channel, and its motion was accompanied by a temperature-driven rearrangement of the water molecules inside the channel. We therefore speculate that temperature drives Aqy1 from a loosely-to a tightly-blocked state. This analysis provides high-resolution structural evidence of the influence of temperature on membrane-transport channels.
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| 15. |
- Arnlund, David, et al.
(författare)
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Visualizing a protein quake with time-resolved X-ray scattering at a free-electron laser
- 2014
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Ingår i: Nature Methods. - : Springer Science and Business Media LLC. - 1548-7091 .- 1548-7105. ; 11:9, s. 923-926
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Tidskriftsartikel (refereegranskat)abstract
- We describe a method to measure ultrafast protein structural changes using time-resolved wide-angle X-ray scattering at an X-ray free-electron laser. We demonstrated this approach using multiphoton excitation of the Blastochloris viridis photosynthetic reaction center, observing an ultrafast global conformational change that arises within picoseconds and precedes the propagation of heat through the protein. This provides direct structural evidence for a 'protein quake': the hypothesis that proteins rapidly dissipate energy through quake-like structural motions.
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| 16. |
- Backmark, Anna, 1979, et al.
(författare)
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Affinity tags can reduce merohedral twinning of membrane protein crystals
- 2008
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Ingår i: Acta Crystallographica. Section D: Biological Crystallography. - 1399-0047 .- 0907-4449. ; D64, s. 1183-1186
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Tidskriftsartikel (refereegranskat)abstract
- This work presents a comparison of the crystal packing of three eukaryotic membrane proteins: human aquaporin 1, human aquaporin 5 and a spinach plasma membrane aquaporin. All were purified from expression constructs both with and without affinity tags. With the exception of tagged aquaporin 1, all constructs yielded crystals. Two significant effects of the affinity tags were observed: crystals containing a tag typically diffracted to lower resolution than those from constructs encoding the protein sequence alone and constructs without a tag frequently produced crystals that suffered from merohedral twinning. Twinning is a challenging crystallographic problem that can seriously hinder solution of the structure. Thus, for integral membrane proteins, the addition of an affinity tag may help to disrupt the approximate symmetry of the protein and thereby reduce or avoid merohedral twinning.
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| 17. |
- Barty, A., et al.
(författare)
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Self-terminating diffraction gates femtosecond X-ray nanocrystallography measurements
- 2012
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Ingår i: Nature Photonics. - 1749-4885 .- 1749-4893. ; 6:1, s. 35-40
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Tidskriftsartikel (refereegranskat)abstract
- X-ray free-electron lasers have enabled new approaches to the structural determination of protein crystals that are too small or radiation-sensitive for conventional analysis1. For sufficiently short pulses, diffraction is collected before significant changes occur to the sample, and it has been predicted that pulses as short as 10 fs may be required to acquire atomic-resolution structural information1, 2, 3, 4. Here, we describe a mechanism unique to ultrafast, ultra-intense X-ray experiments that allows structural information to be collected from crystalline samples using high radiation doses without the requirement for the pulse to terminate before the onset of sample damage. Instead, the diffracted X-rays are gated by a rapid loss of crystalline periodicity, producing apparent pulse lengths significantly shorter than the duration of the incident pulse. The shortest apparent pulse lengths occur at the highest resolution, and our measurements indicate that current X-ray free-electron laser technology5 should enable structural determination from submicrometre protein crystals with atomic resolution.
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| 18. |
- Bill, Roslyn M., et al.
(författare)
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Overcoming barriers to membrane protein structure determination.
- 2011
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Ingår i: Nature biotechnology. - : Springer Science and Business Media LLC. - 1546-1696 .- 1087-0156. ; 29:4, s. 335-40
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Tidskriftsartikel (refereegranskat)abstract
- After decades of slow progress, the pace of research on membrane protein structures is beginning to quicken thanks to various improvements in technology, including protein engineering and microfocus X-ray diffraction. Here we review these developments and, where possible, highlight generic new approaches to solving membrane protein structures based on recent technological advances. Rational approaches to overcoming the bottlenecks in the field are urgently required as membrane proteins, which typically comprise ~30% of the proteomes of organisms, are dramatically under-represented in the structural database of the Protein Data Bank.
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| 19. |
- Boutet, S., et al.
(författare)
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High-Resolution Protein Structure Determination by Serial Femtosecond Crystallography
- 2012
-
Ingår i: Science. - : American Association for the Advancement of Science (AAAS). - 0036-8075 .- 1095-9203. ; 337:6092, s. 362-364
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Tidskriftsartikel (refereegranskat)abstract
- Structure determination of proteins and other macromolecules has historically required the growth of high-quality crystals sufficiently large to diffract x-rays efficiently while withstanding radiation damage. We applied serial femtosecond crystallography (SFX) using an x-ray free-electron laser (XFEL) to obtain high-resolution structural information from microcrystals (less than 1 micrometer by 1 micrometer by 3 micrometers) of the well-characterized model protein lysozyme. The agreement with synchrotron data demonstrates the immediate relevance of SFX for analyzing the structure of the large group of difficult-to-crystallize molecules.
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| 20. |
- Brändén, Gisela, 1975, et al.
(författare)
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Advances and challenges in time-resolved macromolecular crystallography.
- 2021
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Ingår i: Science. - : American Association for the Advancement of Science (AAAS). - 1095-9203 .- 0036-8075. ; 373:6558
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Forskningsöversikt (refereegranskat)abstract
- Conformational changes within biological macromolecules control a vast array of chemical reactions in living cells. Time-resolved crystallography can reveal time-dependent structural changes that occur within protein crystals, yielding chemical insights in unparalleled detail. Serial crystallography approaches developed at x-ray free-electron lasers are now routinely used for time-resolved diffraction studies of macromolecules. These techniques are increasingly being applied at synchrotron radiation sources and to a growing diversity of macromolecules. Here, we review recent progress in the field, including visualizing ultrafast structural changes that guide the initial trajectories of light-driven reactions as well as capturing biologically important conformational changes on slower time scales, for which bacteriorhodopsin and photosystem II are presented as illustrative case studies.
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