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1.
Nature ; 626(8000): 905-911, 2024 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-38355794

RESUMO

High-intensity femtosecond pulses from an X-ray free-electron laser enable pump-probe experiments for the investigation of electronic and nuclear changes during light-induced reactions. On timescales 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 transfer1,2. However, all ultrafast TR-SFX studies to date have employed such high pump laser energies that nominally several photons were absorbed per chromophore3-17. As multiphoton absorption may force the protein response into non-physiological pathways, it is of great concern18,19 whether this experimental approach20 allows valid conclusions to be drawn vis-à-vis biologically relevant single-photon-induced reactions18,19. Here we describe ultrafast pump-probe SFX experiments on the 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 dynamics21) are seen to depend strongly on pump laser energy, in line with quantum chemical analysis. Our results confirm both the feasibility and necessity of performing ultrafast TR-SFX pump-probe experiments in the linear photoexcitation regime. We consider this to be a starting point for reassessing both the design and the interpretation of ultrafast TR-SFX pump-probe experiments20 such that mechanistically relevant insight emerges.


Assuntos
Artefatos , Lasers , Mioglobina , Cristalografia/instrumentação , Cristalografia/métodos , Elétrons , Mioglobina/química , Mioglobina/metabolismo , Mioglobina/efeitos da radiação , Fótons , Conformação Proteica/efeitos da radiação , Teoria Quântica , Raios X
2.
Nat Methods ; 17(7): 681-684, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32451477

RESUMO

Time-resolved crystallography with X-ray free-electron lasers enables structural characterization of light-induced reactions on ultrafast timescales. To be biologically and chemically relevant, such studies must be carried out in an appropriate photoexcitation regime to avoid multiphoton artifacts, a common issue in recent studies. We describe numerical and experimental approaches to determine how many photons are needed for single-photon excitation in microcrystals, taking into account losses by scattering.


Assuntos
Cristalografia por Raios X/métodos , Fótons , Radiação Eletromagnética , Lasers , Luz , Espalhamento de Radiação
3.
J Synchrotron Radiat ; 27(Pt 5): 1415-1429, 2020 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-32876619

RESUMO

BioMAX is the first macromolecular crystallography beamline at the MAX IV Laboratory 3 GeV storage ring, which is the first operational multi-bend achromat storage ring. Due to the low-emittance storage ring, BioMAX has a parallel, high-intensity X-ray beam, even when focused down to 20 µm × 5 µm using the bendable focusing mirrors. The beam is tunable in the energy range 5-25 keV using the in-vacuum undulator and the horizontally deflecting double-crystal monochromator. BioMAX is equipped with an MD3 diffractometer, an ISARA high-capacity sample changer and an EIGER 16M hybrid pixel detector. Data collection at BioMAX is controlled using the newly developed MXCuBE3 graphical user interface, and sample tracking is handled by ISPyB. The computing infrastructure includes data storage and processing both at MAX IV and the Lund University supercomputing center LUNARC. With state-of-the-art instrumentation, a high degree of automation, a user-friendly control system interface and remote operation, BioMAX provides an excellent facility for most macromolecular crystallography experiments. Serial crystallography using either a high-viscosity extruder injector or the MD3 as a fixed-target scanner is already implemented. The serial crystallography activities at MAX IV Laboratory will be further developed at the microfocus beamline MicroMAX, when it comes into operation in 2022. MicroMAX will have a 1 µm × 1 µm beam focus and a flux up to 1015 photons s-1 with main applications in serial crystallography, room-temperature structure determinations and time-resolved experiments.

4.
Nature ; 505(7482): 244-7, 2014 Jan 09.
Artigo em Inglês | MEDLINE | ID: mdl-24270807

RESUMO

The determination of protein crystal structures is hampered by the need for macroscopic crystals. X-ray free-electron lasers (FELs) provide extremely intense pulses of femtosecond duration, which allow data collection from nanometre- to micrometre-sized crystals in a 'diffraction-before-destruction' approach. So far, all protein structure determinations carried out using FELs have been based on previous knowledge of related, known structures. Here we show that X-ray FEL data can be used for de novo protein structure determination, that is, without previous knowledge about the structure. Using the emerging technique of serial femtosecond crystallography, we performed single-wavelength anomalous scattering measurements on microcrystals of the well-established model system lysozyme, in complex with a lanthanide compound. Using Monte-Carlo integration, we obtained high-quality diffraction intensities from which experimental phases could be determined, resulting in an experimental electron density map good enough for automated building of the protein structure. This demonstrates the feasibility of determining novel protein structures using FELs. We anticipate that serial femtosecond crystallography will become an important tool for the structure determination of proteins that are difficult to crystallize, such as membrane proteins.


Assuntos
Cristalografia/métodos , Elétrons , Lasers , Proteínas/química , Difração de Raios X/métodos , Raios X , Animais , Galinhas , Cristalização , Feminino , Gadolínio , Proteínas de Membrana/química , Modelos Moleculares , Método de Monte Carlo , Muramidase/química , Conformação Proteica , Fatores de Tempo
5.
Proc Natl Acad Sci U S A ; 114(9): 2247-2252, 2017 02 28.
Artigo em Inglês | MEDLINE | ID: mdl-28202732

RESUMO

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.


Assuntos
Cristalografia/métodos , Elétrons , Granulovirus/ultraestrutura , Peptídeos e Proteínas de Sinalização Intercelular/química , Lasers , Cristalografia/instrumentação , Granulovirus/química , Modelos Moleculares , Progranulinas , Estrutura Secundária de Proteína , Síncrotrons
6.
Opt Express ; 26(6): 7190-7203, 2018 Mar 19.
Artigo em Inglês | MEDLINE | ID: mdl-29609405

RESUMO

To conduct X-ray Free-Electron Laser (XFEL) measurements at megahertz (MHz) repetition rates, sample solution must be delivered in a micron-sized liquid free-jet moving at up to 100 m/s. This exceeds by over a factor of two the jet speeds measurable with current high-speed camera techniques. Accordingly we have developed and describe herein an alternative jet velocimetry based on dual-pulse nanosecond laser illumination. Three separate implementations are described, including a small laser-diode system that is inexpensive and highly portable. We have also developed and describe analysis techniques to automatically and rapidly extract jet speed from dual-pulse images.

7.
Nat Methods ; 11(9): 923-6, 2014 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-25108686

RESUMO

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.


Assuntos
Transferência de Energia/efeitos da radiação , Lasers , Ficobiliproteínas/efeitos da radiação , Ficobiliproteínas/ultraestrutura , Espalhamento a Baixo Ângulo , Difração de Raios X/métodos , Ficobiliproteínas/química , Conformação Proteica/efeitos da radiação , Doses de Radiação
8.
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
9.
Nature ; 470(7332): 78-81, 2011 Feb 03.
Artigo em Inglês | MEDLINE | ID: mdl-21293374

RESUMO

X-ray lasers offer new capabilities in understanding the structure of biological systems, complex materials and matter under extreme conditions. Very short and extremely bright, coherent X-ray pulses can be used to outrun key damage processes and obtain a single diffraction pattern from a large macromolecule, a virus or a cell before the sample explodes and turns into plasma. The continuous diffraction pattern of non-crystalline objects permits oversampling and direct phase retrieval. Here we show that high-quality diffraction data can be obtained with a single X-ray pulse from a non-crystalline biological sample, a single mimivirus particle, which was injected into the pulsed beam of a hard-X-ray free-electron laser, the Linac Coherent Light Source. Calculations indicate that the energy deposited into the virus by the pulse heated the particle to over 100,000 K after the pulse had left the sample. The reconstructed exit wavefront (image) yielded 32-nm full-period resolution in a single exposure and showed no measurable damage. The reconstruction indicates inhomogeneous arrangement of dense material inside the virion. We expect that significantly higher resolutions will be achieved in such experiments with shorter and brighter photon pulses focused to a smaller area. The resolution in such experiments can be further extended for samples available in multiple identical copies.


Assuntos
Mimiviridae/química , Difração de Raios X/instrumentação , Difração de Raios X/métodos , Elétrons , Temperatura Alta , Lasers , Fótons , Fatores de Tempo , Raios X
10.
Proc Natl Acad Sci U S A ; 111(35): 12769-74, 2014 Sep 02.
Artigo em Inglês | MEDLINE | ID: mdl-25136092

RESUMO

It has long been known that toxins produced by Bacillus thuringiensis (Bt) are stored in the bacterial cells in crystalline form. Here we describe the structure determination of the Cry3A toxin found naturally crystallized within Bt cells. When whole Bt cells were streamed into an X-ray free-electron laser beam we found that scattering from other cell components did not obscure diffraction from the crystals. The resolution limits of the best diffraction images collected from cells were the same as from isolated crystals. The integrity of the cells at the moment of diffraction is unclear; however, given the short time (∼ 5 µs) between exiting the injector to intersecting with the X-ray beam, our result is a 2.9-Å-resolution structure of a crystalline protein as it exists in a living cell. The study suggests that authentic in vivo diffraction studies can produce atomic-level structural information.


Assuntos
Bacillus thuringiensis/química , Proteínas de Bactérias/química , Cristalografia por Raios X/métodos , Endotoxinas/química , Proteínas Hemolisinas/química , Esporos Bacterianos/química , Bacillus thuringiensis/ultraestrutura , Toxinas de Bacillus thuringiensis , Cristalização , Cristalografia por Raios X/instrumentação , Lasers , Esporos Bacterianos/ultraestrutura , Síncrotrons , Difração de Raios X
11.
Acta Crystallogr D Biol Crystallogr ; 71(Pt 2): 387-97, 2015 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-25664750

RESUMO

Recent advances in synchrotron sources, beamline optics and detectors are driving a renaissance in room-temperature data collection. The underlying impetus is the recognition that conformational differences are observed in functionally important regions of structures determined using crystals kept at ambient as opposed to cryogenic temperature during data collection. In addition, room-temperature measurements enable time-resolved studies and eliminate the need to find suitable cryoprotectants. Since radiation damage limits the high-resolution data that can be obtained from a single crystal, especially at room temperature, data are typically collected in a serial fashion using a number of crystals to spread the total dose over the entire ensemble. Several approaches have been developed over the years to efficiently exchange crystals for room-temperature data collection. These include in situ collection in trays, chips and capillary mounts. Here, the use of a slowly flowing microscopic stream for crystal delivery is demonstrated, resulting in extremely high-throughput delivery of crystals into the X-ray beam. This free-stream technology, which was originally developed for serial femtosecond crystallography at X-ray free-electron lasers, is here adapted to serial crystallography at synchrotrons. By embedding the crystals in a high-viscosity carrier stream, high-resolution room-temperature studies can be conducted at atmospheric pressure using the unattenuated X-ray beam, thus permitting the analysis of small or weakly scattering crystals. The high-viscosity extrusion injector is described, as is its use to collect high-resolution serial data from native and heavy-atom-derivatized lysozyme crystals at the Swiss Light Source using less than half a milligram of protein crystals. The room-temperature serial data allow de novo structure determination. The crystal size used in this proof-of-principle experiment was dictated by the available flux density. However, upcoming developments in beamline optics, detectors and synchrotron sources will enable the use of true microcrystals. This high-throughput, high-dose-rate methodology provides a new route to investigating the structure and dynamics of macromolecules at ambient temperature.


Assuntos
Cristalografia por Raios X/instrumentação , Síncrotrons/instrumentação , Animais , Galinhas , Cristalografia por Raios X/economia , Cristalografia por Raios X/métodos , Desenho de Equipamento , Modelos Moleculares , Muramidase/química , Temperatura , Viscosidade
12.
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
13.
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
14.
J Synchrotron Radiat ; 22(3): 634-43, 2015 May.
Artigo em Inglês | MEDLINE | ID: mdl-25931079

RESUMO

X-ray free-electron laser sources such as the Linac Coherent Light Source offer very exciting possibilities for unique research. However, beam time at such facilities is very limited and in high demand. This has led to significant efforts towards beam multiplexing of various forms. One such effort involves re-using the so-called spent beam that passes through the hole in an area detector after a weak interaction with a primary sample. This beam can be refocused into a secondary interaction region and used for a second, independent experiment operating in series. The beam profile of this refocused beam was characterized for a particular experimental geometry at the Coherent X-ray Imaging instrument at LCLS. A demonstration of this multiplexing capability was performed with two simultaneous serial femtosecond crystallography experiments, both yielding interpretable data of sufficient quality to produce electron density maps.

15.
J Synchrotron Radiat ; 22(3): 644-52, 2015 May.
Artigo em Inglês | MEDLINE | ID: mdl-25931080

RESUMO

Serial femtosecond crystallography (SFX) is an emerging method for data collection at free-electron lasers (FELs) in which single diffraction snapshots are taken from a large number of crystals. The partial intensities collected in this way are then combined in a scheme called Monte Carlo integration, which provides the full diffraction intensities. However, apart from having to perform this merging, the Monte Carlo integration must also average out all variations in crystal quality, crystal size, X-ray beam properties and other factors, necessitating data collection from thousands of crystals. Because the pulses provided by FELs running in the typical self-amplified spontaneous emission (SASE) mode of operation have very irregular, spiky spectra that vary strongly from pulse to pulse, it has been suggested that this is an important source of variation contributing to inaccuracies in the intensities, and that, by using monochromatic pulses produced through a process called self-seeding, fewer images might be needed for Monte Carlo integration to converge, resulting in more accurate data. This paper reports the results of two experiments performed at the Linac Coherent Light Source in which data collected in both SASE and self-seeded mode were compared. Importantly, no improvement attributable to the use of self-seeding was detected. In addition, other possible sources of variation that affect SFX data quality were investigated, such as crystal-to-crystal variations reflected in the unit-cell parameters; however, these factors were found to have no influence on data quality either. Possibly, there is another source of variation as yet undetected that affects SFX data quality much more than any of the factors investigated here.

16.
J Synchrotron Radiat ; 22(2): 225-38, 2015 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-25723924

RESUMO

Proteins that contain metal cofactors are expected to be highly radiation sensitive since the degree of X-ray absorption correlates with the presence of high-atomic-number elements and X-ray energy. To explore the effects of local damage in serial femtosecond crystallography (SFX), Clostridium ferredoxin was used as a model system. The protein contains two [4Fe-4S] clusters that serve as sensitive probes for radiation-induced electronic and structural changes. High-dose room-temperature SFX datasets were collected at the Linac Coherent Light Source of ferredoxin microcrystals. Difference electron density maps calculated from high-dose SFX and synchrotron data show peaks at the iron positions of the clusters, indicative of decrease of atomic scattering factors due to ionization. The electron density of the two [4Fe-4S] clusters differs in the FEL data, but not in the synchrotron data. Since the clusters differ in their detailed architecture, this observation is suggestive of an influence of the molecular bonding and geometry on the atomic displacement dynamics following initial photoionization. The experiments are complemented by plasma code calculations.


Assuntos
Ferredoxinas/efeitos da radiação , Metaloproteínas/efeitos da radiação , Síncrotrons , Clostridium/efeitos da radiação , Cristalografia por Raios X/métodos , Relação Dose-Resposta à Radiação , Humanos , Modelos Moleculares , Lesões por Radiação , Sensibilidade e Especificidade
17.
Artigo em Inglês | MEDLINE | ID: mdl-23989164

RESUMO

High-resolution ribosome structures determined by X-ray crystallography have provided important insights into the mechanism of translation. Such studies have thus far relied on large ribosome crystals kept at cryogenic temperatures to reduce radiation damage. Here, the application of serial femtosecond X-ray crystallography (SFX) using an X-ray free-electron laser (XFEL) to obtain diffraction data from ribosome microcrystals in liquid suspension at ambient temperature is described. 30S ribosomal subunit microcrystals diffracted to beyond 6 Šresolution, demonstrating the feasibility of using SFX for ribosome structural studies. The ability to collect diffraction data at near-physiological temperatures promises to provide fundamental insights into the structural dynamics of the ribosome and its functional complexes.


Assuntos
Elétrons , Subunidades Ribossômicas Menores de Bactérias/ultraestrutura , Thermus thermophilus/química , Cristalização , Cristalografia por Raios X , Lasers , Subunidades Ribossômicas Menores de Bactérias/química , Temperatura , Difração de Raios X
18.
J Appl Crystallogr ; 56(Pt 3): 903-907, 2023 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-37284264

RESUMO

A 'catcher' based on a revolving cylindrical collector is described. The simple and inexpensive device reduces free-jet instabilities inherent to high-viscosity extrusion injection, facilitating delivery of microcrystals for serial diffraction X-ray crystallography.

19.
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.

20.
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
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