scholarly journals The future is bright-- structural biology at FELs

2014 ◽  
Vol 70 (a1) ◽  
pp. C35-C35
Author(s):  
Ilme Schlichting
Keyword(s):  
Data Collection ◽  
Radiation Damage ◽  
De Novo ◽  
Temperature Dependent ◽  
Short Pulses ◽  
High Resolution Data ◽  
X Ray ◽  
Radiation Sources ◽  
Damage Data ◽  
Theoretical Considerations

Protein crystallography using synchrotron radiation sources has had tremendous impact on biology, having yielded the structures of thousands of proteins and given detailed insight into their working mechanisms. However, the technique is limited by the requirement for macroscopic crystals, which can be difficult to obtain, as well as by the often severe radiation damage caused in diffraction experiments, in particular when using tiny crystals. To slow radiation damage, data collection is typically performed at cryogenic temperatures. With the advent of X-ray free-electron lasers (FELs) this situation appears remedied. Theoretical considerations had predicted that with sufficiently short pulses useful diffraction data can be collected before the onset of significant radiation damage that ultimately results in Coulomb explosion of the sample. This has been shown recently at the first hard X-ray FEL, the LCLS at Stanford. High resolution data collected of a stream of microcrystals of the model system lysozyme agree well with conventional data collected of a large macroscopic crystal [1] With the demonstration that de-novo phasing is feasible [2], serial femtosecond crystallography has been established as a useful tool for the analysis of tiny crystals [3] and thus the large group of proteins that resist yielding macroscopic crystals such as membrane proteins. In addition to ensure the required fast exchange of the microcrystals upon exposure, liquid jet delivery has the advantage of allowing data collection at room temperature. As demonstrated recently, this is important since structural dynamics and thus the observed conformation is often temperature dependent. Recent results will be described.

scholarly journals New opportunities in structural biology with XFEL: from sources to structures

2014 ◽  
Vol 70 (a1) ◽  
pp. C19-C19
Author(s):  
Soichi Wakatsuki
Keyword(s):  
Data Collection ◽  
Radiation Damage ◽  
Structural Biology ◽  
Single Particle ◽  
De Novo ◽  
Reconstruction Algorithm ◽  
Simultaneous Recording ◽  
Atomic Resolution ◽  
X Ray ◽  
Biology Research

X-ray free electron lasers (XFEL) have shown the promise of providing new opportunities in structural biology research with their extremely high peak brilliance and short pulses. It is reaching the stage where biologically important questions can be tackled using XFEL based on the "diffract-before-destroy" concept. The first part of this presentation will focus on macromolecular crystallography using XFEL with results obtained at LCLS so far and future scope. R&D efforts being pursued at SLAC/LCLS include new beam modes, (two-color beam for de novo phasing, wider bandwidth for SAXS/WAXS and spectroscopy), beam multiplexing, a dedicated new station for in-air data collection, next generation detectors, data analysis incorporating pulse-by-pulse spectrometer measurements and post refinement. These projects are being pursued in collaboration with many groups locally and globally with a goal to provide integrated facilities for cutting edge structural biology research. For example, two-color self-seeded XFEL mode is being developed for simultaneous recording of diffraction data at two energies in order to optimize the dispersive difference between the two wavelengths for phasing. Another area of collaborative effort is a development of dedicated station for in-air data collection with a variety of sample delivery schemes. The second part will discuss a possible roadmap towards atomic resolution single particle imaging using XFEL. Here, key questions are ·Can XFEL single particle 3D structural analysis at atomic resolution be done? ·What is the pulse characteristics required? ·Can we overcome the radiation damage at soft X-ray regime? ·What is the highest resolution attainable in comparison with cryoEM? A workshop at LCLS is being organized to discuss these questions in 4 areas: radiation damage, image reconstruction algorithm, beam modes and instrumentation, sample delivery and heterogeneity. The outcome of the workshop and follow-up discussions will be presented.

scholarly journals Serial femtosecond crystallography: the first five years

IUCrJ ◽  
2015 ◽  
Vol 2 (2) ◽  
pp. 246-255 ◽  
Author(s):  
Ilme Schlichting
Keyword(s):  
Radiation Damage ◽  
Diffraction Data ◽  
De Novo ◽  
Time Resolved ◽  
Radiation Sources ◽  
Damage Data ◽  
Serial Femtosecond Crystallography ◽  
Insight Into ◽  
Tremendous Impact

Protein crystallography using synchrotron radiation sources has had a tremendous impact on biology, having yielded the structures of thousands of proteins and given detailed insight into their mechanisms. However, the technique is limited by the requirement for macroscopic crystals, which can be difficult to obtain, as well as by the often severe radiation damage caused in diffraction experiments, in particular when using tiny crystals. To slow radiation damage, data collection is typically performed at cryogenic temperatures. With the advent of free-electron lasers (FELs) capable of delivering extremely intense femtosecond X-ray pulses, this situation appears to be remedied, allowing the structure determination of undamaged macromolecules using either macroscopic or microscopic crystals. The latter are exposed to the FEL beam in random orientations and their diffraction data are collected at cryogenic or room temperature in a serial fashion, since each crystal is destroyed upon a single exposure. The new approaches required for crystal growth and delivery, and for diffraction data analysis, includingde novophasing, are reviewed. The opportunities and challenges of SFX are described, including applications such as time-resolved measurements and the analysis of radiation damage-prone systems.

Intensity measurements

2002 ◽  
Author(s):  
David Blow
Keyword(s):  
Data Collection ◽  
Radiation Damage ◽  
Resolution Limit ◽  
Strategic Decisions ◽  
Experimental Conditions ◽  
X Ray ◽  
Intensity Measurements ◽  
Data Collection Technique ◽  
Molecular Structure Investigation ◽  
Disordered Crystal

Once a suitable crystal has been obtained, a molecular structure investigation requires measurement of the intensities of as many Bragg reflections as possible. In this chapter, some of the options that must be decided by the experimenter will be considered, and some of the criteria used to assess the accuracy and completeness of the data will be presented. The experimenter has to make a number of strategic decisions in collecting the crystal intensity data. These include: • What X-ray source should be used? • What X-ray detector should be used? • Under what conditions should the crystal be maintained? • How long should each crystal be exposed? • What data collection technique will be used? • What resolution limit should be applied? The choice of source and detector will depend largely on what is available, but the major decision is whether to use facilities in the home laboratory or whether to use a synchrotron at a central facility. The energy released by absorption of X-rays in a crystal inevitably damages it. The process of radiation damage increases crystal disorder and reduces the intensity of scattering. The experimenter will ultimately have to abandon data collection from the damaged and disordered crystal. Under ideal experimental conditions, all the useful diffraction data can be obtained from a crystal long before radiation damage takes its toll, and radiation damage does not create a practical problem. At the other end of the scale, it may be necessary to combine the measurements from many crystals in order to obtain a complete set of diffracted intensities. There is no definite criterion to decide when a crystal is so badly damaged that it must be discarded. But if the measurements are going to be of highest quality, any observable change is bad news. The most serious effects occur in the part of the diffraction pattern at the highest observed resolution, where the observed intensities of the Bragg reflections will be altered most rapidly. The first observable effect of radiation damage is usually a reduction of high angle intensities due to increased disorder.

scholarly journals Structural knowledge or X-ray damage? A case study on xylose isomerase illustrating both

2019 ◽  
Vol 26 (4) ◽  
pp. 931-944 ◽  
Author(s):  
Helena Taberman ◽  
Charles S. Bury ◽  
Mark J. van der Woerd ◽  
Edward H. Snell ◽  
Elspeth F. Garman
Keyword(s):  
Reaction Mechanism ◽  
Radiation Damage ◽  
Xylose Isomerase ◽  
Data Sets ◽  
High Resolution Data ◽  
X Ray ◽  
Metal Cofactor ◽  
Acid Environment ◽  
Radiation Induced ◽  
Catalytic Metal

Xylose isomerase (XI) is an industrially important metalloprotein studied for decades. Its reaction mechanism has been postulated to involve movement of the catalytic metal cofactor to several different conformations. Here, a dose-dependent approach was used to investigate the radiation damage effects on XI and their potential influence on the reaction mechanism interpreted from the X-ray derived structures. Radiation damage is still one of the major challenges for X-ray diffraction experiments and causes both global and site-specific damage. In this study, consecutive high-resolution data sets from a single XI crystal from the same wedge were collected at 100 K and the progression of radiation damage was tracked over increasing dose (0.13–3.88 MGy). The catalytic metal and its surrounding amino acid environment experience a build-up of free radicals, and the results show radiation-damage-induced structural perturbations ranging from an absolute metal positional shift to specific residue motions in the active site. The apparent metal movement is an artefact of global damage and the resulting unit-cell expansion, but residue motion appears to be driven by the dose. Understanding and identifying radiation-induced damage is an important factor in accurately interpreting the biological conclusions being drawn.

RADDOSE-3D: time- and space-resolved modelling of dose in macromolecular crystallography

2013 ◽  
Vol 46 (4) ◽  
pp. 1225-1230 ◽  
Author(s):  
Oliver B. Zeldin ◽  
Markus Gerstel ◽  
Elspeth F. Garman
Keyword(s):  
Data Collection ◽  
Radiation Damage ◽  
Diffraction Experiment ◽  
Macromolecular Crystallography ◽  
X Ray Diffraction ◽  
Time Resolved ◽  
X Ray ◽  
Online Methods ◽  
Crystal Volume ◽  
Number Of Iterations

RADDOSE-3D allows the macroscopic modelling of an X-ray diffraction experiment for the purpose of better predicting radiation-damage progression. The distribution of dose within the crystal volume is calculated for a number of iterations in small angular steps across one or more data collection wedges, providing a time-resolved picture of the dose state of the crystal. The code is highly modular so that future contributions from the community can be easily integrated into it, in particular to incorporate online methods for determining the shape of macromolecular crystals and better protocols for imaging real experimental X-ray beam profiles.

scholarly journals Research at the NSF STC for Biology with X-ray lasers

2014 ◽  
Vol 70 (a1) ◽  
pp. C296-C296
Author(s):  
John Spence
Keyword(s):  
Radiation Damage ◽  
Bragg Diffraction ◽  
Cubic Phase ◽  
Short Pulses ◽  
X Ray ◽  
Fast Solution ◽  
Linac Coherent Light Source ◽  
New Ideas ◽  
Diffraction Patterns ◽  
Dynamic Structures

"The NSF BioXFEL Science and Technology Center (STC) is a new consortium of six research campuses devoted to the application of x-ray free-electron lasers (XFELs) to structural biology. Over the last four years a variety of approaches have been made to the observation of protein structure and dynamics for various classes of proteins. The Linac Coherent Light source at SLAC, the first hard-Xray EXFEL, provides intense coherent hard X-ray pulses at 120 Hz which vaporize protein when focussed to a sub-micron beam. Atomic-resolution Bragg diffraction patterns are nevertheless obtained using 50 fs pulses prior to the onset of significant damage, in this ""diffract-then-destroy"" mode, which outruns radiation damage. This use of short pulses instead of freezing samples to reduce radiation damage therefore opens the way to the study of protein dynamics at room temperature in a native environment. I'll review the work of several groups using a range of approaches to different types of sample, including the following: 1. Differences between the frozen sychrotron structure of GPCR proteins and the RT XFEL structure [1]. 2. Pump-probe dynamic structures in Photosynthesis [2]. 3. XFEL study of 2D protein crystals [3]. 4. Prospects for improved resolution in XFEL imaging from single particles such as viruses, where patterns can be obtained from a single virus. 5. New ideas - the Lipid Cubic Phase injector (which allows protein nanocrystals to be studied also at sychrotrons) [4], prospects for fast Laue diffraction using coherent attosecond X-ray lasers, ab-initio phasing [5], the use of angular correlation functions for analysis of fast solution scattering, and two-color opportunites for serial femtosecond crystallography (SFX). See [6] for a recent review of the field. 1. W.Liu et al Science 342, 1521 (2013) 2. A.Aquila et al Optics Express 20, 2706 (2012) 3. M.Frank et al IUCrJ (2014) In press. 4. U.Weierstall et al Nature Comms. (2014) In press. 5. J. Spence et al Optics Express 19, 2866 (2011). 6. J. Spence et al Rep. Prog. Phys. 75, 102601 (2012)."

scholarly journals Advancing Native-SAD Phasing at Synchrotron with 13 Real-life Case Studies

2014 ◽  
Vol 70 (a1) ◽  
pp. C601-C601
Author(s):  
Meitian Wang
Keyword(s):  
Data Collection ◽  
Single Crystal ◽  
Measurement Errors ◽  
Model Building ◽  
De Novo ◽  
Real Life ◽  
Data Sets ◽  
X Ray ◽  
Recent Developments ◽  
Sad Phasing

The key step in elucidating de novo 3D X-ray structures relies on the incorporation of heavy elements into proteins or crystals. Selenomethionine incorporation or heavy metal derivatization are however not always possible and require additional efforts. Exploiting anomalous signals from intrinsically present elements like S, P, and Ca2+ from proteins and nucleic acids, as well as Cl-, SO42-, and PO42- from crystallization solutions, is therefore an appealing alternative. Such a method has been shown to be valid by collecting data from several crystals and combining them(1). Recent developments at macromolecular crystallography beamlines are however pushing the limits of what could be obtained out of a single crystal. Here we introduce a novel data collection routine for native-SAD phasing, which distributes tolerable X-ray life-doses to very high multiplicity X-ray diffraction data sets measured at 6 keV energy and at different crystal orientations on a single crystal. This allows the extraction of weak anomalous signals reliably by reducing both systematic and random measurement errors. The data collection method has been applied successfully to thirteen real-life examples including membrane proteins, a protein/DNA complex, and a large protein complex. In addition to de novo structure determination, we advocate such a data collection protocol for molecular replacement solvable structures where unbiased phase information is crucial in objective map interpretation and model building, especially for medium and low-resolution cases.

scholarly journals Synchrotron microcrystal native-SAD phasing at a low energy

IUCrJ ◽  
2019 ◽  
Vol 6 (4) ◽  
pp. 532-542 ◽  
Author(s):  
Gongrui Guo ◽  
Ping Zhu ◽  
Martin R. Fuchs ◽  
Wuxian Shi ◽  
Babak Andi ◽  
...  
Keyword(s):  
Data Analysis ◽  
Data Collection ◽  
Diffraction Data ◽  
De Novo ◽  
Low Energy ◽  
Anomalous Scattering ◽  
Free Electron Lasers ◽  
X Ray ◽  
Structural Evaluation ◽  
Sad Phasing

De novo structural evaluation of native biomolecules from single-wavelength anomalous diffraction (SAD) is a challenge because of the weakness of the anomalous scattering. The anomalous scattering from relevant native elements – primarily sulfur in proteins and phosphorus in nucleic acids – increases as the X-ray energy decreases toward their K-edge transitions. Thus, measurements at a lowered X-ray energy are promising for making native SAD routine and robust. For microcrystals with sizes less than 10 µm, native-SAD phasing at synchrotron microdiffraction beamlines is even more challenging because of difficulties in sample manipulation, diffraction data collection and data analysis. Native-SAD analysis from microcrystals by using X-ray free-electron lasers has been demonstrated but has required use of thousands of thousands of microcrystals to achieve the necessary accuracy. Here it is shown that by exploitation of anomalous microdiffraction signals obtained at 5 keV, by the use of polyimide wellmounts, and by an iterative crystal and frame-rejection method, microcrystal native-SAD phasing is possible from as few as about 1 200 crystals. Our results show the utility of low-energy native-SAD phasing with microcrystals at synchrotron microdiffraction beamlines.

scholarly journals X-ray and UV radiation-damage-induced phasing using synchrotron serial crystallography

2018 ◽  
Vol 74 (4) ◽  
pp. 366-378 ◽  
Author(s):  
Nicolas Foos ◽  
Carolin Seuring ◽  
Robin Schubert ◽  
Anja Burkhardt ◽  
Olof Svensson ◽  
...  
Keyword(s):  
Success Rate ◽  
Radiation Damage ◽  
Uv Radiation ◽  
De Novo ◽  
Data Sets ◽  
Individual Data ◽  
High Quality ◽  
X Ray ◽  
Serial Crystallography

Specific radiation damage can be used to determine phasesde novofrom macromolecular crystals. This method is known as radiation-damage-induced phasing (RIP). One limitation of the method is that the dose of individual data sets must be minimized, which in turn leads to data sets with low multiplicity. A solution to this problem is to use data from multiple crystals. However, the resulting signal can be degraded by a lack of isomorphism between crystals. Here, it is shown that serial synchrotron crystallography in combination with selective merging of data sets can be used to determine high-quality phases for insulin and thaumatin, and that the increased multiplicity can greatly enhance the success rate of the experiment.

scholarly journals Computer-controlled liquid-nitrogen drizzling device for removing frost from cryopreserved crystals

2020 ◽  
Vol 76 (12) ◽  
pp. 616-622
Author(s):  
Yuki Nakamura ◽  
Seiki Baba ◽  
Nobuhiro Mizuno ◽  
Takaki Irie ◽  
Go Ueno ◽  
...  
Keyword(s):  
Data Collection ◽  
Radiation Damage ◽  
Liquid Nitrogen ◽  
Room Temperature ◽  
Temperature Data ◽  
Macromolecular Crystallography ◽  
X Ray ◽  
Low Visibility ◽  
Computer Controlled

Cryocrystallography is a technique that is used more often than room-temperature data collection in macromolecular crystallography. One of its advantages is the significant reduction in radiation damage, which is especially useful in synchrotron experiments. Another advantage is that cryopreservation provides simple storage of crystals and easy transportation to a synchrotron. However, this technique sometimes results in the undesirable adhesion of frost to mounted crystals. The frost produces noisy diffraction images and reduces the optical visibility of crystals, which is crucial for aligning the crystal position with the incident X-ray position. To resolve these issues, a computer-controlled device has been developed that drizzles liquid nitrogen over a crystal to remove frost. It was confirmed that the device works properly, reduces noise from ice rings in diffraction images and enables the centering of crystals with low visibility owing to frost adhesion.

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