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1.
J Synchrotron Radiat ; 25(Pt 5): 1529-1540, 2018 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-30179194

RESUMO

The non-monochromatic beamline BL1 at the FLASH free-electron laser facility at DESY was upgraded with new transport and focusing optics, and a new permanent end-station, CAMP, was installed. This multi-purpose instrument is optimized for electron- and ion-spectroscopy, imaging and pump-probe experiments at free-electron lasers. It can be equipped with various electron- and ion-spectrometers, along with large-area single-photon-counting pnCCD X-ray detectors, thus enabling a wide range of experiments from atomic, molecular, and cluster physics to material and energy science, chemistry and biology. Here, an overview of the layout, the beam transport and focusing capabilities, and the experimental possibilities of this new end-station are presented, as well as results from its commissioning.

2.
Nature ; 470(7332): 73-7, 2011 Feb 03.
Artigo em Inglês | MEDLINE | ID: mdl-21293373

RESUMO

X-ray crystallography provides the vast majority of macromolecular structures, but the success of the method relies on growing crystals of sufficient size. In conventional measurements, the necessary increase in X-ray dose to record data from crystals that are too small leads to extensive damage before a diffraction signal can be recorded. It is particularly challenging to obtain large, well-diffracting crystals of membrane proteins, for which fewer than 300 unique structures have been determined despite their importance in all living cells. Here we present a method for structure determination where single-crystal X-ray diffraction 'snapshots' are collected from a fully hydrated stream of nanocrystals using femtosecond pulses from a hard-X-ray free-electron laser, the Linac Coherent Light Source. We prove this concept with nanocrystals of photosystem I, one of the largest membrane protein complexes. More than 3,000,000 diffraction patterns were collected in this study, and a three-dimensional data set was assembled from individual photosystem I nanocrystals (∼200 nm to 2 µm in size). We mitigate the problem of radiation damage in crystallography by using pulses briefer than the timescale of most damage processes. This offers a new approach to structure determination of macromolecules that do not yield crystals of sufficient size for studies using conventional radiation sources or are particularly sensitive to radiation damage.


Assuntos
Cristalografia por Raios X/métodos , Nanopartículas/química , Nanotecnologia/métodos , Complexo de Proteína do Fotossistema I/química , Cristalografia por Raios X/instrumentação , Lasers , Modelos Moleculares , Nanotecnologia/instrumentação , Conformação Proteica , Fatores de Tempo , Raios X
3.
Nat Methods ; 9(3): 263-5, 2012 Jan 29.
Artigo em Inglês | MEDLINE | ID: mdl-22286383

RESUMO

X-ray free electron laser (X-FEL)-based serial femtosecond crystallography is an emerging method with potential to rapidly advance the challenging field of membrane protein structural biology. Here we recorded interpretable diffraction data from micrometer-sized lipidic sponge phase crystals of the Blastochloris viridis photosynthetic reaction center delivered into an X-FEL beam using a sponge phase micro-jet.


Assuntos
Cristalografia por Raios X/métodos , Bicamadas Lipídicas/química , Proteínas de Membrana/química , Proteínas de Membrana/ultraestrutura , Ligação Proteica , Conformação Proteica/efeitos da radiação , Raios X
4.
Nat Methods ; 9(3): 259-62, 2012 Jan 29.
Artigo em Inglês | MEDLINE | ID: mdl-22286384

RESUMO

Protein crystallization in cells has been observed several times in nature. However, owing to their small size these crystals have not yet been used for X-ray crystallographic analysis. We prepared nano-sized in vivo-grown crystals of Trypanosoma brucei enzymes and applied the emerging method of free-electron laser-based serial femtosecond crystallography to record interpretable diffraction data. This combined approach will open new opportunities in structural systems biology.


Assuntos
Cristalografia por Raios X/métodos , Cristalografia/métodos , Proteínas/química , Proteínas/ultraestrutura , Ligação Proteica/efeitos da radiação , Conformação Proteica/efeitos da radiação , Proteínas/efeitos da radiação , Solubilidade/efeitos da radiação , Raios X
5.
Acta Crystallogr D Biol Crystallogr ; 69(Pt 5): 838-42, 2013 May.
Artigo em Inglês | MEDLINE | ID: mdl-23633593

RESUMO

X-ray free-electron lasers (FELs) enable crystallographic data collection using extremely bright femtosecond pulses from microscopic crystals beyond the limitations of conventional radiation damage. This diffraction-before-destruction approach requires a new crystal for each FEL shot and, since the crystals cannot be rotated during the X-ray pulse, data collection requires averaging over many different crystals and a Monte Carlo integration of the diffraction intensities, making the accurate determination of structure factors challenging. To investigate whether sufficient accuracy can be attained for the measurement of anomalous signal, a large data set was collected from lysozyme microcrystals at the newly established `multi-purpose spectroscopy/imaging instrument' of the SPring-8 Ångstrom Compact Free-Electron Laser (SACLA) at RIKEN Harima. Anomalous difference density maps calculated from these data demonstrate that serial femtosecond crystallography using a free-electron laser is sufficiently accurate to measure even the very weak anomalous signal of naturally occurring S atoms in a protein at a photon energy of 7.3 keV.


Assuntos
Cristalografia por Raios X/métodos , Lasers , Conformação Proteica , Enxofre/química , Cristalografia por Raios X/instrumentação , Cisteína/química , Modelos Moleculares , Muramidase/química
6.
Opt Express ; 20(4): 4149-58, 2012 Feb 13.
Artigo em Inglês | MEDLINE | ID: mdl-22418172

RESUMO

We describe femtosecond X-ray diffraction data sets of viruses and nanoparticles collected at the Linac Coherent Light Source. The data establish the first large benchmark data sets for coherent diffraction methods freely available to the public, to bolster the development of algorithms that are essential for developing this novel approach as a useful imaging technique. Applications are 2D reconstructions, orientation classification and finally 3D imaging by assembling 2D patterns into a 3D diffraction volume.

7.
Opt Express ; 20(3): 2706-16, 2012 Jan 30.
Artigo em Inglês | MEDLINE | ID: mdl-22330507

RESUMO

We demonstrate the use of an X-ray free electron laser synchronized with an optical pump laser to obtain X-ray diffraction snapshots from the photoactivated states of large membrane protein complexes in the form of nanocrystals flowing in a liquid jet. Light-induced changes of Photosystem I-Ferredoxin co-crystals were observed at time delays of 5 to 10 µs after excitation. The result correlates with the microsecond kinetics of electron transfer from Photosystem I to ferredoxin. The undocking process that follows the electron transfer leads to large rearrangements in the crystals that will terminally lead to the disintegration of the crystals. We describe the experimental setup and obtain the first time-resolved femtosecond serial X-ray crystallography results from an irreversible photo-chemical reaction at the Linac Coherent Light Source. This technique opens the door to time-resolved structural studies of reaction dynamics in biological systems.


Assuntos
Cristalografia por Raios X/métodos , Ferredoxinas/ultraestrutura , Lasers , Nanoestruturas/ultraestrutura , Difração de Raios X/métodos , Elétrons , Conformação Proteica , Raios X
8.
Opt Express ; 19(17): 16542-9, 2011 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-21935018

RESUMO

Single-particle experiments using X-ray Free Electron Lasers produce more than 10(5) snapshots per hour, consisting of an admixture of blank shots (no particle intercepted), and exposures of one or more particles. Experimental data sets also often contain unintentional contamination with different species. We present an unsupervised method able to sort experimental snapshots without recourse to templates, specific noise models, or user-directed learning. The results show 90% agreement with manual classification.

9.
Nat Photonics ; 6: 35-40, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-24078834

RESUMO

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 information1-4. 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.

10.
Phys Rev B Condens Matter Mater Phys ; 84(21): 214111, 2011 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-24089594

RESUMO

X-ray free-electron lasers deliver intense femtosecond pulses that promise to yield high resolution diffraction data of nanocrystals before the destruction of the sample by radiation damage. Diffraction intensities of lysozyme nanocrystals collected at the Linac Coherent Light Source using 2 keV photons were used for structure determination by molecular replacement and analyzed for radiation damage as a function of pulse length and fluence. Signatures of radiation damage are observed for pulses as short as 70 fs. Parametric scaling used in conventional crystallography does not account for the observed effects.

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