| 1. |
- Boutet, S., et al.
(författare)
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High-Resolution Protein Structure Determination by Serial Femtosecond Crystallography
- 2012
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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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| 2. |
- Woitowich, N. C., et al.
(författare)
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Structural basis for light control of cell development revealed by crystal structures of a myxobacterial phytochrome
- 2018
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Ingår i: Iucrj. - : International Union of Crystallography (IUCr). - 2052-2525. ; 5:Part 5, s. 619-634
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Tidskriftsartikel (refereegranskat)abstract
- Phytochromes are red-light photoreceptors that were first characterized in plants, with homologs in photosynthetic and non-photosynthetic bacteria known as bacteriophytochromes (BphPs). Upon absorption of light, BphPs interconvert between two states denoted Pr and Pfr with distinct absorption spectra in the red and far-red. They have recently been engineered as enzymatic photoswitches for fluorescent-marker applications in non-invasive tissue imaging of mammals. This article presents cryo- and room-temperature crystal structures of the unusual phytochrome from the non-photosynthetic myxobacterium Stigmatella aurantiaca (SaBphP1) and reveals its role in the fruitingbody formation of this photomorphogenic bacterium. SaBphP1 lacks a conserved histidine (His) in the chromophore-binding domain that stabilizes the Pr state in the classical BphPs. Instead it contains a threonine (Thr), a feature that is restricted to several myxobacterial phytochromes and is not evolutionarily understood. SaBphP1 structures of the chromophore binding domain (CBD) and the complete photosensory core module (PCM) in wild-type and Thr-to-His mutant forms reveal details of the molecular mechanism of the Pr/Pfr transition associated with the physiological response of this myxobacterium to red light. Specifically, key structural differences in the CBD and PCM between the wild-type and the Thr-to-His mutant involve essential chromophore contacts with proximal amino acids, and point to how the photosignal is transduced through the rest of the protein, impacting the essential enzymatic activity in the photomorphogenic response of this myxobacterium.
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| 3. |
- 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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| 4. |
- Sanchez, J. C., et al.
(författare)
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High-resolution crystal structures of amyxobacterial phytochrome at cryo and roomtemperatures
- 2019
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Ingår i: Structural Dynamics-Us. - : AIP Publishing. - 2329-7778. ; 6:5
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Tidskriftsartikel (refereegranskat)abstract
- Phytochromes (PHYs) are photoreceptor proteins first discovered in plants, where they control a variety of photomorphogenesis events. PHYs as photochromic proteins can reversibly switch between two distinct states: a red light (Pr) and a far-red light (Pfr) absorbing form. The discovery of Bacteriophytochromes (BphPs) in nonphotosynthetic bacteria has opened new frontiers in our understanding of the mechanisms by which these natural photoswitches can control single cell development, although the role of BphPs in vivo remains largely unknown. BphPs are dimeric proteins that consist of a photosensory core module (PCM) and an enzymatic domain, often a histidine kinase. The PCM is composed of three domains (PAS, GAF, and PHY). It holds a covalently bound open-chain tetrapyrrole (biliverdin, BV) chromophore. Upon absorption of light, the double bond between BV rings C and D isomerizes and reversibly switches the protein between Pr and Pfr states. We report crystal structures of the wild-type and mutant (His275Thr) forms of the canonical BphP from the nonphotosynthetic myxobacterium Stigmatella aurantiaca (SaBphP2) in the Pr state. Structures were determined at 1.65A degrees and 2.2A degrees (respectively), the highest resolution of any PCM construct to date. We also report the room temperature wild-type structure of the same protein determined at 2.1A degrees at the SPring-8 Angstrom Compact free electron LAser (SACLA), Japan. Our results not only highlight and confirm important amino acids near the chromophore that play a role in Pr-Pfr photoconversion but also describe the signal transduction into the PHY domain which moves across tens of angstroms after the light stimulus. (C) 2019 Author(s). All article content, except where otherwise noted, is licensed under a Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).
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| 5. |
- Johansson, Linda C, 1983, et al.
(författare)
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Structure of a photosynthetic reaction centre determined by serial femtosecond crystallography.
- 2013
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Ingår i: Nature communications. - : Springer Science and Business Media LLC. - 2041-1723. ; 4
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Tidskriftsartikel (refereegranskat)abstract
- Serial femtosecond crystallography is an X-ray free-electron-laser-based method with considerable potential to have an impact on challenging problems in structural biology. Here we present X-ray diffraction data recorded from microcrystals of the Blastochloris viridis photosynthetic reaction centre to 2.8Å resolution and determine its serial femtosecond crystallography structure to 3.5Å resolution. Although every microcrystal is exposed to a dose of 33MGy, no signs of X-ray-induced radiation damage are visible in this integral membrane protein structure.
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| 6. |
- Liu, Wei, et al.
(författare)
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Serial Femtosecond Crystallography of G Protein-Coupled Receptors
- 2013
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Ingår i: Science. - : American Association for the Advancement of Science (AAAS). - 0036-8075 .- 1095-9203. ; 342:6165, s. 1521-1524
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Tidskriftsartikel (refereegranskat)abstract
- X-ray crystallography of G protein-coupled receptors and other membrane proteins is hampered by difficulties associated with growing sufficiently large crystals that withstand radiation damage and yield high-resolution data at synchrotron sources. We used an x-ray free-electron laser (XFEL) with individual 50-femtosecond-duration x-ray pulses to minimize radiation damage and obtained a high-resolution room-temperature structure of a human serotonin receptor using sub-10-micrometer microcrystals grown in a membrane mimetic matrix known as lipidic cubic phase. Compared with the structure solved by using traditional microcrystallography from cryo-cooled crystals of about two orders of magnitude larger volume, the room-temperature XFEL structure displays a distinct distribution of thermal motions and conformations of residues that likely more accurately represent the receptor structure and dynamics in a cellular environment.
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| 7. |
- Redecke, Lars, et al.
(författare)
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Natively inhibited Trypanosoma brucei cathepsin B structure determined by using an X-ray laser.
- 2013
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Ingår i: Science (New York, N.Y.). - : American Association for the Advancement of Science (AAAS). - 1095-9203 .- 0036-8075. ; 339:6116, s. 227-30
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Tidskriftsartikel (refereegranskat)abstract
- The Trypanosoma brucei cysteine protease cathepsin B (TbCatB), which is involved in host protein degradation, is a promising target to develop new treatments against sleeping sickness, a fatal disease caused by this protozoan parasite. The structure of the mature, active form of TbCatB has so far not provided sufficient information for the design of a safe and specific drug against T. brucei. By combining two recent innovations, in vivo crystallization and serial femtosecond crystallography, we obtained the room-temperature 2.1 angstrom resolution structure of the fully glycosylated precursor complex of TbCatB. The structure reveals the mechanism of native TbCatB inhibition and demonstrates that new biomolecular information can be obtained by the "diffraction-before-destruction" approach of x-ray free-electron lasers from hundreds of thousands of individual microcrystals.
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| 8. |
- Weierstall, Uwe, et al.
(författare)
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Lipidic cubic phase injector facilitates membrane protein serial femtosecond crystallography
- 2014
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Ingår i: Nature Communications. - : Springer Science and Business Media LLC. - 2041-1723. ; 5, s. 3309-
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Tidskriftsartikel (refereegranskat)abstract
- Lipidic cubic phase (LCP) crystallization has proven successful for high-resolution structure determination of challenging membrane proteins. Here we present a technique for extruding gel-like LCP with embedded membrane protein microcrystals, providing a continuously renewed source of material for serial femtosecond crystallography. Data collected from sub-10-mu m-sized crystals produced with less than 0.5 mg of purified protein yield structural insights regarding cyclopamine binding to the Smoothened receptor.
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| 9. |
- Wiedorn, Max O., et al.
(författare)
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Megahertz serial crystallography
- 2018
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Ingår i: Nature Communications. - : Nature Publishing Group. - 2041-1723. ; 9
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Tidskriftsartikel (refereegranskat)abstract
- The new European X-ray Free-Electron Laser is the first X-ray free-electron laser capable of delivering X-ray pulses with a megahertz inter-pulse spacing, more than four orders of magnitude higher than previously possible. However, to date, it has been unclear whether it would indeed be possible to measure high-quality diffraction data at megahertz pulse repetition rates. Here, we show that high-quality structures can indeed be obtained using currently available operating conditions at the European XFEL. We present two complete data sets, one from the well-known model system lysozyme and the other from a so far unknown complex of a beta-lactamase from K. pneumoniae involved in antibiotic resistance. This result opens up megahertz serial femtosecond crystallography (SFX) as a tool for reliable structure determination, substrate screening and the efficient measurement of the evolution and dynamics of molecular structures using megahertz repetition rate pulses available at this new class of X-ray laser source.
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| 10. |
- Edlund, Petra, et al.
(författare)
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The room temperature crystal structure of a bacterial phytochrome determined by serial femtosecond crystallography
- 2016
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Ingår i: Scientific Reports. - : Springer Science and Business Media LLC. - 2045-2322. ; 6
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Tidskriftsartikel (refereegranskat)abstract
- Phytochromes are a family of photoreceptors that control light responses of plants, fungi and bacteria. A sequence of structural changes, which is not yet fully understood, leads to activation of an output domain. Time-resolved serial femtosecond crystallography (SFX) can potentially shine light on these conformational changes. Here we report the room temperature crystal structure of the chromophore-binding domains of the Deinococcus radiodurans phytochrome at 2.1 angstrom resolution. The structure was obtained by serial femtosecond X-ray crystallography from microcrystals at an X-ray free electron laser. We find overall good agreement compared to a crystal structure at 1.35 angstrom resolution derived from conventional crystallography at cryogenic temperatures, which we also report here. The thioether linkage between chromophore and protein is subject to positional ambiguity at the synchrotron, but is fully resolved with SFX. The study paves the way for time-resolved structural investigations of the phytochrome photocycle with time-resolved SFX.
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| 11. |
- Kupitz, Christopher, et al.
(författare)
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Serial time-resolved crystallography of photosystem II using a femtosecond X-ray laser
- 2014
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Ingår i: Nature. - : Springer Science and Business Media LLC. - 0028-0836 .- 1476-4687. ; 513:7517, s. 261-265
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Tidskriftsartikel (refereegranskat)abstract
- Photosynthesis, a process catalysed by plants, algae and cyanobacteria converts sunlight to energy thus sustaining all higher life on Earth. Two large membrane protein complexes, photosystem I and II (PSI and PSII), act in series to catalyse the light-driven reactions in photosynthesis. PSII catalyses the light-driven water splitting process, which maintains the Earth's oxygenic atmosphere. In this process, the oxygen-evolving complex (OEC) of PSII cycles through five states, S0 to S4, in which four electrons are sequentially extracted from the OEC in four light-driven charge-separation events. Here we describe time resolved experiments on PSII nano/microcrystals from Thermosynechococcus elongatus performed with the recently developed technique of serial femtosecond crystallography. Structures have been determined from PSII in the dark S1 state and after double laser excitation (putative S3 state) at 5 and 5.5 Å resolution, respectively. The results provide evidence that PSII undergoes significant conformational changes at the electron acceptor side and at the Mn4CaO5 core of the OEC. These include an elongation of the metal cluster, accompanied by changes in the protein environment, which could allow for binding of the second substrate water molecule between the more distant protruding Mn (referred to as the 'dangler' Mn) and the Mn3CaOx cubane in the S2 to S3 transition, as predicted by spectroscopic and computational studies. This work shows the great potential for time-resolved serial femtosecond crystallography for investigation of catalytic processes in biomolecules.
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| 12. |
- Lee, Ho-Hsien, et al.
(författare)
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Expression, purification and crystallization of CTB-MPR, a candidate mucosal vaccine component against HIV-1
- 2014
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Ingår i: IUCrJ. - 2052-2525. ; 1:5, s. 305-317
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Tidskriftsartikel (refereegranskat)abstract
- CTB-MPR is a fusion protein between the B subunit of cholera toxin (CTB) andthe membrane-proximal region of gp41 (MPR), the transmembrane envelopeprotein ofHuman immunodeficiency virus 1(HIV-1), and has previously beenshown to induce the production of anti-HIV-1 antibodies with antiviralfunctions. To further improve the design of this candidate vaccine, X-raycrystallography experiments were performed to obtain structural informationabout this fusion protein. Several variants of CTB-MPR were designed,constructed and recombinantly expressed inEscherichia coli. The first variantcontained a flexible GPGP linker between CTB and MPR, and yielded crystalsthat diffracted to a resolution of 2.3 A ̊, but only the CTB region was detectedin the electron-density map. A second variant, in which the CTB was directlyattached to MPR, was shown to destabilize pentamer formation. A thirdconstruct containing a polyalanine linker between CTB and MPR proved tostabilize the pentameric form of the protein during purification. The purificationprocedure was shown to produce a homogeneously pure and monodispersesample for crystallization. Initial crystallization experiments led to pseudo-crystals which were ordered in only two dimensions and were disordered inthe third dimension. Nanocrystals obtained using the same precipitant showedpromising X-ray diffraction to 5 A ̊resolution in femtosecond nanocrystallo-graphy experiments at the Linac Coherent Light Source at the SLAC NationalAccelerator Laboratory. The results demonstrate the utility of femtosecondX-ray crystallography to enable structural analysis based on nano/microcrystalsof a protein for which no macroscopic crystals ordered in three dimensions havebeen observed before.
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