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X-ray lasers in biology - techniques

Event

Starts:

October
162013

09:00

Ends:

October
172013

17:00

Location

Kavli Royal Society Centre, Chicheley Hall, Newport Pagnell, Buckinghamshire, MK16 9JJ

Overview

Satellite meeting organised by Professor John Spence and Professor Henry Chapman

X-ray image of a human handing holding an egg containing a map of the molecule, lysozyme, of which egg-white consists. Dr A Barty.

Event details

This meeting brings together leaders in the development of new techniques for the study of molecular structure and interactions in biology using the recently invented hard X-ray laser. Topics will include time-resolved protein nanocrystallography, femtosecond wide-angle X-ray diffraction, sample delivery devices, data analysis and diffraction theory, and detector systems.

Biographies of the organisers and speakers are available below. Recorded audio of the presentations will be available on this page after the event.

Attending this event

Participants are also encouraged to attend the related scientific discussion meeting X-ray lasers in biology which immediately precedes this event.

Enquiries: Contact the events team

Event organisers

Select an organiser for more information

Schedule of talks

Session 1

3 talks Show detail Hide detail

Chair of Session 1

Professor Ian Robinson, University College London, UK

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Structure, conformations and dynamics from random snapshots

Dr Abbas Ourmazd, University of Wisconsin-Milwaukee, USA

Abstract

Cryo-EM now routinely produces low-dose snapshots of biological objects. The advent of the X-ray Free Electron Laser has made it possible to record diffraction snapshots with intense short pulses before the onset of radiation damage. In both cases, each snapshot stems from an unknown orientation of a weakly scattering object at a poorly defined time-point along its dynamic trajectory. This presentation describes a new generation of algorithms with the potential to recover the structure, conformations, and time evolution of single particles from ultralow signal XFEL and cryo-EM snapshots.

Co-authors:

P Schwander, R Fung, A Hosseinizadeh, and A Dashti

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Observation and modelling of long-range electronic correlations in femtosecond nanocrystallography

Dr Harry Quiney, University of Melbourne, Australia

Abstract

We have recently observed an abrupt change in the diffraction pattern of crystalline C60 that is initiated by intense femtosecond X-ray illumination using the LCLS XFEL source. This change in the scattering characteristics is attributable to a reduction in the symmetry of the electronic structure over a femtosecond timescale that is too brief in duration to allow  any significant nuclear rearrangement.  We have been able to fit the diffraction data to a crystallographic model by allowing the electronic structures of the C60 molecules to polarize under the influence of an internal electric field that is characteristic of long-range correlations within the crystal. In order to determine whether this modification to the electronic structure can be regarded as static or in a dynamic equilibrium between symmetry-equivalent configurations, an electrodynamical model has been constructed to simulate the temporal evolution of the coherent electronic structure from a random distribution of electronically excited molecular states. We find that an initial state consisting of a periodic array of C60 molecule ions in randomly-allocated electronic states evolves on a femtosecond timescale into a coherently polarized structure under the influence of long-range internal electronic correlations. The dominant electronic structure of the crystal during the interaction with the femtosecond XFEL pulse corresponds to the observed experimental diffraction pattern and to the empirical crystallographic model that has been used to fit the data.  We will also report on recent observations of radiation-induced modification of the electronic structure of  β-hematin in nanocrystallographic experiments using XFEL sources.

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Session 2

3 talks Show detail Hide detail

Chair of Session 2

Dr Michael J Bogan, Stanford PULSE Institute, SLAC National Accelerator Laboratory, USA

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Will single particle structural imaging be possible with X-ray free-electron lasers

Dr Duane Loh, SLAC National Accelerator Laboratory, USA

Abstract

Reconstruction algorithms have demonstrably recovered the three-dimensional structure of biologically relevant objects from noisy and incomplete two-dimensional diffraction patterns from single copies of the object, both in simulations and experiments. These algorithms, however, do not consider random sample injection and are frustrated by the resultant computational demands by an overwhelming stream of largely spurious patterns. This is a pressing computational challenge in three-dimensional imaging. 

In this talk, I will share intuition and strategies to differentiate, from many spurious patterns, a subset of weak diffraction patterns actually due to the object of interest. I will also discuss other expected hurdles on the path towards three-dimensional single-particle structural imaging with X-ray free-electron lasers.

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When crystals meet single particles

Dr Anton Barty, Center for Free-Electron Laser Science, Germany

Abstract

The unprecedented peak power provided by X-ray free electron lasers enables the study of sub micron sized protein crystals using the principle of diffraction before destruction.  Coherent illumination across the entire crystal, which in some cases may consist of only a few unit cells on each side, opens up the possibility of studying nanocrystal diffraction using techniques originally developed for single particle analysis.  We will describe developments in mapping the three-dimensional diffraction space produced by nanocrystals and its application to structural analysis using data measured at LCLS.

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Session 3

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Chair of Session 3

Dr Robert Stroud, UCSF, USA

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Processing of FEL crystallographic data

Dr Thomas White, Center for Free-Electron Laser Science, Germany

Abstract

The availability of suitable processing software for data acquired using the method of serial femtosecond crystallography (SFX) is an important contribution to the development of structural biology using free-electron lasers.  Since early 2012, the open-source software suite CrystFEL has been freely available for this purpose and under continuous development as new understanding emerges about the characteristics of SFX data compared to data from more conventional crystallographic data acquisition techniques.

In this talk, I will describe some of the main issues surrounding data processing for  data acquired using SFX.  Special challenges arise which are unique to this data acquisition methodology, and I will discuss some recent developments in overcoming them.

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Methods for phasing coherently illuminated nanocrystals

Dr Rick Kirian, Center for Free-Electron Laser Science, Germany

Abstract

Diffraction patterns from femtosecond protein nanocrystallography experiments performed at x-ray free-electron lasers contain measurable intensity in regions that lie between Bragg peaks.  The extra information contained in inter-Bragg intensity measurements presents a unique opportunity to determine diffraction phases ab initio, and hence to retrieve the electron density of the molecules that compose the crystal.  In order to realize this goal, we are developing iterative phase retrieval algorithms and methods for processing the continuous diffraction from size-varying nanocrystals.  In this talk I will present an overview of these ongoing efforts.

Co-authors:

Richard Bean, Oleksandr Yevanof, Kenneth Beyerlein, Thomas White, Anton Barty, Henry Chapman

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Session 4

2 talks Show detail Hide detail

Chair of Session 4

Dr Frank von Delft, University of Oxford, UK

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Structure from an ensemble of multiple randomly oriented particles

Dr Dilano Saldin, University of Wisconsin-Milwaukee, USA

Abstract

The published quality of images of single molecules or viruses reconstructed from experimental XFEL data seems to lag behind those and from simulations. We investigate the cause. For example the requirement that a single particle in an aqueous environment be hit by the XFEL beam may be quite difficult to achieve. If the particle concentration is made so low to allow this, the scattering signal from the particle may be drowned by the scattering by the solvent. On the other hand, increasing the particle concentration may mean that more than a single particle is hit. We describe an approach to analyzing an ensemble of XFEL measurements which may allow the reconstruction of single particle structure from diffraction patterns from multiple randomly oriented particles. We illustrate the theory with the reconstruction of icosahedral and helical viruses. Another circumstance which admits a solution is when a structure sought is a small deviation from a known structure as in time-resolved experiments. We show that it is possible to recover difference electron densities from randomly oriented single proteins.

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Session 5

3 talks Show detail Hide detail

Chair of Session 5

Professor David Stuart FRS FMedSci, Diamond Light Source & Wellcome Trust Centre for Human Genetics, UK

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New methods for analysing nanocrystal and single-particle diffraction

Dr Andrew Martin, ARC Centre of Excellence for Coherent X-ray Science, School of Physics, University of Melbourne, Australia

Abstract

In many ways, the extent of our theoretical knowledge about coherent diffraction determines the scope of current experiments and directs our efforts toward future goals, like studying single biological molecules. A collection of theoretical results will be presented for non-periodic and nanocrystalline samples with the goal of measuring basic parameters, like size or orientation, or to aid the ongoing development coherent imaging algorithms and structure determination methods. Some of the issues discussed are missing data due to the detector geometry, the impact of radiation damage and the variable surface configurations of nanocrystals.

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Detectors for X-ray lasers

Dr Jan Kern, Lawrence Berkeley National Laboratory, USA

Abstract

Our ability to harness the advances in microelectronics over the last decade(s) for X-ray detection has resulted in significant improvements in the state-of-the-art. Biology with Free Electron Lasers (FELs) present daunting detector challenges: all of the photons arrive at the same time, and individual high peak power pulses must be read out shot-by-shot.  Direct X-ray detection in silicon pixel detectors – monolithic or hybrid – are the standard for FELs today.  This talk will compare different detector implementations, and discuss both fundamental and practical limitations (and what might be done to overcome them) inherent in today’s detectors.  The goal is elicit discussions on detector needs (and what tradeoffs might be required) rather than to suggest a specific solution.

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Session 6

3 talks Show detail Hide detail

Chair of Session 6

Dr Keith Hodgson, Stanford University, USA

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Nanofocusing of X-ray free electron laser for coherent X-ray science

Professor Kazuto Yamauchi, Osaka University, Japan

Abstract

X-ray free-electron lasers produce intense femtosecond pulses, which have applications in exploring new frontiers of coherent X-ray diffraction imaging. In particular, in biomedical sample imaging, extremely short pulses can be used to visualize nanostructures without radiation damage, which is of critical importance but has never been achieved using synchrotron ring facilities. The unique characteristics of X-ray free-electron laser radiation can be enhanced significantly using focusing optics. Here we present reflective optics comprising elliptically figured total-reflection mirrors with nanometer accuracy for preserving a coherent wavefront, and successfully focus a 10 keV X-ray free-electron laser on areas of 1 mm and less than 50 nm. The peak power density reached a value of 1020 W/cm2. This achievement can be applied to realize the further condensation of an X-ray free-electron laser by employing a highly accurate multilayer mirror. Our focusing optics are expected to play a crucial role in the advance of microscopic research towards achieving ultimate resolution, as well as in the development of nonlinear optical sciences under extreme conditions. The current status and future challenges of the X-ray free electron laser optics development will be outlined with discussion of the mirror design, fabrication, and wavefront measurement methods.

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Analysis of X-ray diffuse scattering in X-FEL experiments – opportunities, challenges and strategies

Dr Kristoffer Haldrup, DTU-Physics, Denmark

Abstract

This presentation details some of the challenges and opportunities encountered when utilizing ultra-bright pulses of X-rays for X-ray Diffuse Scattering experiments on solution-phase chemistry on femtosecond time scales. In particular, the use of Singular Value Decomposition as a tool for detecting and removing highly variable (pulse-to-pulse) background contributions is discussed. These efforts have been motivated by the recent arrival of X-ray Free Electron Lasers, where the unique beam characteristics of these facilities have opened up new possibilities for research at the ultrashort time scales, including structural investigations of photo-chemical reactions in liquids.

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Session 7

3 talks Show detail Hide detail

Chair of Session 7

Professor Bruce Doak, Max-Planck-Institut fur Medizinische Forschung, Germany and Arizona State University, USA

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Liquid jet injectors for X-ray lasers

Dr Uwe Weierstall, Arizona State University, USA

Abstract

The emerging method of serial femtosecond crystallography (SFX), which uses ultrashort X-ray pulses from an X-ray free electron laser to outrun radiation damage, has been shown recently to achieve atomic resolution from protein microcrystals. An essential requirement for SFX is a sample delivery method, which can keep up with the repetition rate of the XFEL and provides fresh sample for every X-ray pulse. A liquid jet injector has been developed at ASU for this purpose and allows data collection from hydrated biomolecules and microcrystals at room temperature. In order to use protein microcrystals grown in Lipidic cubic phase (LCP) for SFX experiments, a new approach was needed to generate a stream of the gel-like LCP with tens of micrometer diameter. Therefore a new LCP injector has been developed, which allows the collection of data from a contiguous stream of nanocrystals embedded in LCP. An overview of the current injection devices and recent results will be presented.

Coauthors:

Daniel James, Dingjie Wang, John C H Spence, R B Doak, Petra Fromme, Arizona State University, USA
Martin Caffrey, Trinity College Dublin, Republic of Ireland
Vadim Cherezov, The Scripps Research Institute, USA

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SFX vs PX: comparing crystallographic data from FELs and synchrotrons

Dr Thomas Barends, Max-Planck-Institut fur Medizinische Forschung, Germany

Abstract

Free-electron lasers (FELs) are pushing back the limits of possibility in protein crystallography. The high-intensity, femtosecond duration pulses afforded by FELs allow data collection from micrometer-sized crystals while outrunning radiation damage. Moreover, FELs may be used for pump-probe experiments with unprecedented time resolution.

However, the intricacies of FEL data collection pose specific challenges: as every FEL pulse destroys the sample, data are mostly collected from a stream of microcrystals and averaged to remove the variations in crystal size and quality as well as shot-to-shot variations in beam parameters. This technique, dubbed serial femtosecond crystallography (SFX) requires specific data processing methods.

Alternatively, data may be collected from a single crystal using an attenuated beam. This, too, requires special processing methods because of the limited sampling of reciprocal space possible in the short duration of the FEL pulse.

We will compare FEL data collected using both methods with comparable data collected using classical methods at synchrotrons, to explore the determinants of data quality in terms of beam and crystal properties.

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Session 8

3 talks Show detail Hide detail

Chair of Session 8

Dr Liz Carpenter, University of Oxford, UK

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Serial femtosecond crystallography of membrane proteins using the lipidic cubic mesophase

Professor Martin Caffrey, Trinity College Dublin, Ireland

Abstract

The lipid-based bicontinuous cubic mesophase is a nanoporous membrane mimetic with wide ranging applications in areas that include medicine, personal care products, foods, and the basic sciences. An application of particular note concerns it use as a medium in which to grow crystals of membrane proteins for structure determination by X-ray crystallography (1). At least two variations of the mesophase exist. One is the highly viscous and sticky cubic phase which has well developed long-range order. The other, referred to as the sponge phase, is considerably more fluid and lacks long-range order. The sponge phase has recently been shown to be a convenient vehicle for delivering microcrystals of membrane proteins for serial femtosecond crystallography (SFX) at the Linac Coherent Light Source, SLAC National Accelerator Laboratory (2). Unfortunately, the sponge phase approach calls for large amounts of protein which are not always available in the case of membrane proteins. The cubic phase offers the advantage of requiring significantly less protein for SFX but comes with its own challenges. In this talk, I will describe the physico-chemical bases for these challenges, solutions to them and prospects for future uses of lipidic mesophases in the SFX arena.

Caffrey, M., Li, D., Dukkupati, A. 2012. Membrane protein structure determination using crystallography and lipidic mesophases: Recent advances and successes. Biochemistry 51: 6266

Johansson, L. C., et al. 2012. Lipidic phase membrane protein serial femtosecond crystallography. Nat. Meth. 9:293

Co-authors:

Dianfan Li, Trinity College Dublin, Ireland

Vadim Cherezov, The Scripps Research Institute, USA

Uwe Weierstall, John C H Spence, Petra Fromme, Arizona State University, USA

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XFEL 2D protein crystallography on fixed targets

Dr Bill Pedrini, Paul Scherrer Institute, Switzerland

Abstract

We report on the first X-FEL single shot experiments of bacteriorhodopsin 2D crystals, recently performed at LCLS.

The pattern from single crystals are clearly recognized in the diffraction images, and the Bragg peak intensities can be reliably extracted down to resolutions of 6-7 A.

These results are promising, but indicate that substantial progress is necessary to achieve atomic resolution.

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X-ray lasers in biology - techniques Kavli Royal Society Centre, Chicheley Hall Newport Pagnell Buckinghamshire MK16 9JJ