Journal of Physics B: Atomic, Molecular and Optical Physics, 2010
In a flash diffraction experiment, a short and extremely intense X-ray pulse illuminates the samp... more In a flash diffraction experiment, a short and extremely intense X-ray pulse illuminates the sample to obtain a diffraction pattern before the onset of significant radiation damage. The over-sampled diffraction pattern permits phase retrieval through iterative phasing methods. Flash diffractive imaging was first demonstrated on an inorganic test object (Chapman et al. Nature Physics 2, 839-843, 2006). We report here experiments on biological systems where individual cells were imaged, using single, 10-15 femtoseconds soft X-ray pulses at 13.5 nm wavelength from the FLASH free-electron laser in Hamburg. Simulations show the pulse heated the sample to about 160,000 K but not before an interpretable diffraction pattern could be obtained. The reconstructed projection images return the structures of the intact cells. The simulations suggest the average displacement of ions and atoms in the hottest surface layers remained below 3 Å during the pulse. Introduction Confidential: not for distribution.
Single-particle experiments using X-ray Free Electron Lasers produce more than 10 5 snapshots per... more 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.
Advances in X-ray Free-Electron Lasers: Radiation Schemes, X-ray Optics, and Instrumentation, 2011
... and Karol Nass“ and Dusko Odie“ and Emanuele Pedersoli“ and Christian Reich“ and Daniel Rolle... more ... and Karol Nass“ and Dusko Odie“ and Emanuele Pedersoli“ and Christian Reich“ and Daniel Rollesci“ and Benedikt Rudek“'“ and Artem Rudenko“'“ and Carlo Schmidt“'“ and Joachim Schulz“ and M. Marvin Seibert“ and Robert L. Shoeman“ and Raymond G. Sierra“ and Heike ...
Theory predicts that with an ultrashort and extremely bright coherent X-ray pulse, a single diffr... more Theory predicts that with an ultrashort and extremely bright coherent X-ray pulse, a single diffraction pattern may be recorded from a large macromolecule, a virus, or a cell before the sample explodes and turns into a plasma. Here we report the first experimental demonstration of this principle using the FLASH soft X-ray free-electron laser. An intense 25 fs, 4 × 10 13 W/cm 2 pulse, containing 10 12 photons at 32 nm wavelength, produced a coherent diffraction pattern from a nano-structured non-periodic object, before destroying it at 60,000 • K. A novel X-ray camera assured single photon detection sensitivity by filtering out parasitic scattering and plasma radiation. The reconstructed image, obtained directly from the coherent pattern by phase retrieval through oversampling , shows no measurable damage, and extends to diffraction-limited resolution. A three-dimensional data set may be assembled from such images when copies of a reproducible sample are exposed to the beam one by one [10].
X-ray free-electron lasers have enabled new approaches to the structural determination of protein... more 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 analysis(1). 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 information(1-4). 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 technology(5) should enable structural determination from submicrometre protein crystals with atomic resolution.
Journal of Physics B: Atomic, Molecular and Optical Physics, 2010
In a flash diffraction experiment, a short and extremely intense X-ray pulse illuminates the samp... more In a flash diffraction experiment, a short and extremely intense X-ray pulse illuminates the sample to obtain a diffraction pattern before the onset of significant radiation damage. The over-sampled diffraction pattern permits phase retrieval through iterative phasing methods. Flash diffractive imaging was first demonstrated on an inorganic test object (Chapman et al. Nature Physics 2, 839-843, 2006). We report here experiments on biological systems where individual cells were imaged, using single, 10-15 femtoseconds soft X-ray pulses at 13.5 nm wavelength from the FLASH free-electron laser in Hamburg. Simulations show the pulse heated the sample to about 160,000 K but not before an interpretable diffraction pattern could be obtained. The reconstructed projection images return the structures of the intact cells. The simulations suggest the average displacement of ions and atoms in the hottest surface layers remained below 3 Å during the pulse. Introduction Confidential: not for distribution.
X-ray crystallography provides the vast majority of macromolecular structures, but the success of... more 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...
X-ray lasers offer new capabilities in understanding the structure of biological systems, complex... more 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 si...
Protein crystallization in cells has been observed several times in nature. However, owing to the... more 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.
X-ray free electron laser (X-FEL)-based serial femtosecond crystallography is an emerging method ... more 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.
The ultrafast pulses from X-ray free-electron lasers will enable imaging of non-periodic objects ... more The ultrafast pulses from X-ray free-electron lasers will enable imaging of non-periodic objects at near-atomic resolution [1, Neutze]. These objects could include single molecules, protein complexes, or virus particles. The specimen would be completely destroyed by the pulse in a Coulomb explosion, but that destruction will only happen after the pulse. The scattering from the sample will give structural information about the undamaged object. There are many technical challenges that must be addressed before ...
Structural studies of biological macromolecules are severely limited by radiation damage. Traditi... more Structural studies of biological macromolecules are severely limited by radiation damage. Traditional crystallography curbs the effects of damage by spreading damage over many copies of the molecule of interest. X-ray lasers, such as the recently built LINAC Coherent Light Source (LCLS) [1], offer an additional opportunity for limiting damage by out-running damage processes with ultrashort and very intense X-ray pulses. Such pulses may allow the imaging of single molecules, clusters or nanoparticles, but coherent flash imaging will also open up new avenues for structural studies on nano-and microcrystalline substances. This paper addresses the theoretical potentials and limitations of nanocrystallography with extremely intense coherent Xray pulses. We use urea nanocrystals as a model for generic biological substances and simulate primary and secondary ionization dynamics in the crystalline sample. Our results establish conditions for ultrafast nanocrystallography diffraction experiments as a function of fluence and pulse duration. Any sample exposed to an intense X-ray pulse will be ionized and extensive ionization destroys the sample. The time scale on which this process occurs is critical for obtaining an interpretable diffraction pattern that conveys an atomic structure of the sample. In principle, the X-ray pulse must be short enough for the entire pulse to pass through the sample before major disarrangement of atomic and electronic configurations takes place. The ionizations due to direct photoabsorption and subsequent secondary processes affect the ability to get useful structural information from the diffraction pattern in three ways: (i) Ionization decreases the elastic X-ray scattering power of atoms and induces considerable changes in diffracted intensities due to ionization stochasticity. (ii) Removal of electrons from atoms leaves behind positively charged ions that repel each other due *
We demonstrate the use of an X-ray free electron laser synchronized with an optical pump laser to... more 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 s 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.
Journal of Physics B: Atomic, Molecular and Optical Physics, 2010
In a flash diffraction experiment, a short and extremely intense X-ray pulse illuminates the samp... more In a flash diffraction experiment, a short and extremely intense X-ray pulse illuminates the sample to obtain a diffraction pattern before the onset of significant radiation damage. The over-sampled diffraction pattern permits phase retrieval through iterative phasing methods. Flash diffractive imaging was first demonstrated on an inorganic test object (Chapman et al. Nature Physics 2, 839-843, 2006). We report here experiments on biological systems where individual cells were imaged, using single, 10-15 femtoseconds soft X-ray pulses at 13.5 nm wavelength from the FLASH free-electron laser in Hamburg. Simulations show the pulse heated the sample to about 160,000 K but not before an interpretable diffraction pattern could be obtained. The reconstructed projection images return the structures of the intact cells. The simulations suggest the average displacement of ions and atoms in the hottest surface layers remained below 3 Å during the pulse. Introduction Confidential: not for distribution.
Single-particle experiments using X-ray Free Electron Lasers produce more than 10 5 snapshots per... more 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.
Advances in X-ray Free-Electron Lasers: Radiation Schemes, X-ray Optics, and Instrumentation, 2011
... and Karol Nass“ and Dusko Odie“ and Emanuele Pedersoli“ and Christian Reich“ and Daniel Rolle... more ... and Karol Nass“ and Dusko Odie“ and Emanuele Pedersoli“ and Christian Reich“ and Daniel Rollesci“ and Benedikt Rudek“'“ and Artem Rudenko“'“ and Carlo Schmidt“'“ and Joachim Schulz“ and M. Marvin Seibert“ and Robert L. Shoeman“ and Raymond G. Sierra“ and Heike ...
Theory predicts that with an ultrashort and extremely bright coherent X-ray pulse, a single diffr... more Theory predicts that with an ultrashort and extremely bright coherent X-ray pulse, a single diffraction pattern may be recorded from a large macromolecule, a virus, or a cell before the sample explodes and turns into a plasma. Here we report the first experimental demonstration of this principle using the FLASH soft X-ray free-electron laser. An intense 25 fs, 4 × 10 13 W/cm 2 pulse, containing 10 12 photons at 32 nm wavelength, produced a coherent diffraction pattern from a nano-structured non-periodic object, before destroying it at 60,000 • K. A novel X-ray camera assured single photon detection sensitivity by filtering out parasitic scattering and plasma radiation. The reconstructed image, obtained directly from the coherent pattern by phase retrieval through oversampling , shows no measurable damage, and extends to diffraction-limited resolution. A three-dimensional data set may be assembled from such images when copies of a reproducible sample are exposed to the beam one by one [10].
X-ray free-electron lasers have enabled new approaches to the structural determination of protein... more 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 analysis(1). 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 information(1-4). 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 technology(5) should enable structural determination from submicrometre protein crystals with atomic resolution.
Journal of Physics B: Atomic, Molecular and Optical Physics, 2010
In a flash diffraction experiment, a short and extremely intense X-ray pulse illuminates the samp... more In a flash diffraction experiment, a short and extremely intense X-ray pulse illuminates the sample to obtain a diffraction pattern before the onset of significant radiation damage. The over-sampled diffraction pattern permits phase retrieval through iterative phasing methods. Flash diffractive imaging was first demonstrated on an inorganic test object (Chapman et al. Nature Physics 2, 839-843, 2006). We report here experiments on biological systems where individual cells were imaged, using single, 10-15 femtoseconds soft X-ray pulses at 13.5 nm wavelength from the FLASH free-electron laser in Hamburg. Simulations show the pulse heated the sample to about 160,000 K but not before an interpretable diffraction pattern could be obtained. The reconstructed projection images return the structures of the intact cells. The simulations suggest the average displacement of ions and atoms in the hottest surface layers remained below 3 Å during the pulse. Introduction Confidential: not for distribution.
X-ray crystallography provides the vast majority of macromolecular structures, but the success of... more 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...
X-ray lasers offer new capabilities in understanding the structure of biological systems, complex... more 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 si...
Protein crystallization in cells has been observed several times in nature. However, owing to the... more 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.
X-ray free electron laser (X-FEL)-based serial femtosecond crystallography is an emerging method ... more 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.
The ultrafast pulses from X-ray free-electron lasers will enable imaging of non-periodic objects ... more The ultrafast pulses from X-ray free-electron lasers will enable imaging of non-periodic objects at near-atomic resolution [1, Neutze]. These objects could include single molecules, protein complexes, or virus particles. The specimen would be completely destroyed by the pulse in a Coulomb explosion, but that destruction will only happen after the pulse. The scattering from the sample will give structural information about the undamaged object. There are many technical challenges that must be addressed before ...
Structural studies of biological macromolecules are severely limited by radiation damage. Traditi... more Structural studies of biological macromolecules are severely limited by radiation damage. Traditional crystallography curbs the effects of damage by spreading damage over many copies of the molecule of interest. X-ray lasers, such as the recently built LINAC Coherent Light Source (LCLS) [1], offer an additional opportunity for limiting damage by out-running damage processes with ultrashort and very intense X-ray pulses. Such pulses may allow the imaging of single molecules, clusters or nanoparticles, but coherent flash imaging will also open up new avenues for structural studies on nano-and microcrystalline substances. This paper addresses the theoretical potentials and limitations of nanocrystallography with extremely intense coherent Xray pulses. We use urea nanocrystals as a model for generic biological substances and simulate primary and secondary ionization dynamics in the crystalline sample. Our results establish conditions for ultrafast nanocrystallography diffraction experiments as a function of fluence and pulse duration. Any sample exposed to an intense X-ray pulse will be ionized and extensive ionization destroys the sample. The time scale on which this process occurs is critical for obtaining an interpretable diffraction pattern that conveys an atomic structure of the sample. In principle, the X-ray pulse must be short enough for the entire pulse to pass through the sample before major disarrangement of atomic and electronic configurations takes place. The ionizations due to direct photoabsorption and subsequent secondary processes affect the ability to get useful structural information from the diffraction pattern in three ways: (i) Ionization decreases the elastic X-ray scattering power of atoms and induces considerable changes in diffracted intensities due to ionization stochasticity. (ii) Removal of electrons from atoms leaves behind positively charged ions that repel each other due *
We demonstrate the use of an X-ray free electron laser synchronized with an optical pump laser to... more 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 s 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.
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