Protein Crystal Motions from Time-Resolved Diffracted X-ray Blinking

The distinctive features of the physics-based probes used in understanding the structure of matter focusing on biological sciences, but not exclusively, are described in the modern context. This is set in a wider scope of holistic biology and the scepticism about `reductionism', what is called the `molecular level', and how to respond constructively. These topics will be set alongside the principles of accuracy and precision, and their boundaries. The combination of probes and their application together is the usual way of realizing accuracy. The distinction between precision and accuracy can be blurred by the predictive force of a precise structure, thereby lending confidence in its potential accuracy. These descriptions will be applied to the comparison of cryo and room-temperature protein crystal structures as well as the solid state of a crystal and the same molecules studied by small-angle X-ray scattering in solution and by electron microscopy on a sample grid. Examples will include: time-resolved X-ray Laue crystallography of an enzyme Michaelis complex formed directly in a crystal equivalent to in vivo; a new iodoplatin for radiation therapy predicted from studies of platin crystal structures; and the field of colouration of carotenoids, as an effective assay of function, i.e. their colouration, when unbound and bound to a protein. The complementarity of probes, as well as their combinatory use, is then at the foundation of real (biologically relevant), probe-artefacts-free, structure–function studies. The foundations of our methodologies are being transformed by colossal improvements in technologies of X-ray and neutron sources and their beamline instruments, as well as improved electron microscopes and NMR spectrometers. The success of protein structure prediction from gene sequence recently reported by CASP14 also opens new doors to change and extend the foundations of the structural sciences.

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Serial Femtosecond Crystallography user's consortium apparatus at European XFEL

Acta Crystallographica Section A Foundations and Advances ◽

10.1107/s2053273314082515 ◽

2014 ◽

Vol 70 (a1) ◽

pp. C1748-C1748

Author(s):

Marc Messerschmidt ◽

Leonard Chavas ◽

Sunil Ananthaneni ◽

Hamidreza Dadgostar ◽

Heinz Graafsma ◽

...

Keyword(s):

Protein Crystal ◽

Pixel Detector ◽

Single Particles ◽

Time Resolved ◽

X Ray ◽

Overall Design ◽

Laser Facility ◽

Common Problems ◽

Automated Procedures ◽

Serial Femtosecond Crystallography

The Serial Femtosecond Crystallography (SFX) user's consortium apparatus is to be installed within the Single Particles, Clusters and Biomolecules (SPB) instrument of the European X-ray Free-Electron Laser facility (XFEL.EU) [1, 2]. The XFEL.EU will provide ultra-short, highly intense, coherent X-ray pulses at an unprecedented repetition rate. The experimental setup and methodological approaches of many scientific areas will be transformed, including structural biology that could potentially overcome common problems and bottlenecks encountered in crystallography, such as creating large crystals, dealing with radiation damage, or understanding sub-picosecond time-resolved phenomena. The key concept of the SFX method is based on the kinetic insertion of protein crystal samples in solution via a gas dynamic virtual nozzle jet and recording diffraction signals of individual, randomly oriented crystals passing through the XFEL beam, as first demonstrated by Chapman et al. [3]. The SFX-apparatus will refocus the beam spent by the SPB instrument into a second interaction region, in some cases enabling two parallel experiments. The planned photon energy range at the SPB instrument is from 3 to 16 keV. The Adaptive Gain Integrating Pixel Detector (AGIPD) is to be implemented in the SPB instrument, including a 4 Megapixel version for the SFX-apparatus. The AGIPD is designed to store over 350 data frames from successive pulses, and aims to collect more than 3,000 images per second. Together with the implementation of automated procedures for sample exchange and injection, high-throughput nanocrystallography experiments can be integrated at the SFX-apparatus. In this work, we review the overall design of the SFX-apparatus and discuss the main parameters and challenges

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X-ray diffraction in temporally and spatially resolved biomolecular science

Faraday Discussions ◽

10.1039/c4fd00166d ◽

2015 ◽

Vol 177 ◽

pp. 429-441 ◽

Cited By ~ 1

Author(s):

John R. Helliwell ◽

Alice Brink ◽

Surasak Kaenket ◽

Victoria Laurina Starkey ◽

Simon W. M. Tanley

Keyword(s):

Single Molecule ◽

Structural Changes ◽

Protein Crystallography ◽

European Synchrotron Radiation Facility ◽

Protein Crystal ◽

Structural Studies ◽

X Ray Diffraction ◽

Microscopy Imaging ◽

Time Resolved ◽

X Ray

Time-resolved Laue protein crystallography at the European Synchrotron Radiation Facility (ESRF) opened up the field of sub-nanosecond protein crystal structure analyses. There are a limited number of such time-resolved studies in the literature. Why is this? The X-ray laser now gives us femtosecond (fs) duration pulses, typically 10 fs up to ∼50 fs. Their use is attractive for the fastest time-resolved protein crystallography studies. It has been proposed that single molecules could even be studied with the advantage of being able to measure X-ray diffraction from a ‘crystal lattice free’ single molecule, with or without temporal resolved structural changes. This is altogether very challenging R&D. So as to assist this effort we have undertaken studies of metal clusters that bind to proteins, both ‘fresh’ and after repeated X-ray irradiation to assess their X-ray-photo-dynamics, namely Ta6Br12, K2PtI6 and K2PtBr6 bound to a test protein, hen egg white lysozyme. These metal complexes have the major advantage of being very recognisable shapes (pseudo spherical or octahedral) and thereby offer a start to (probably very difficult) single molecule electron density map interpretations, both static and dynamic. A further approach is to investigate the X-ray laser beam diffraction strength of a well scattering nano-cluster; an example from nature being the iron containing ferritin. Electron crystallography and single particle electron microscopy imaging offers alternatives to X-ray structural studies; our structural studies of crustacyanin, a 320 kDa protein carotenoid complex, can be extended either by electron based techniques or with the X-ray laser representing a fascinating range of options. General outlook remarks concerning X-ray, electron and neutron macromolecular crystallography as well as ‘NMR crystallography’ conclude the article.

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The emergence of the synchrotron Laue method for rapid data collection from protein crystals

Proceedings of the Royal Society of London. Series A: Mathematical and Physical Sciences ◽

10.1098/rspa.1993.0098 ◽

1993 ◽

Vol 442 (1914) ◽

pp. 177-192 ◽

Cited By ~ 5

Keyword(s):

Multiplicity Distribution ◽

Spectral Curve ◽

Three Dimensional ◽

Protein Crystal ◽

Photographic Film ◽

Macromolecular Crystallography ◽

Time Resolved ◽

X Ray ◽

Laue Geometry ◽

Laue Method

Synchrotron X-radiation (SR) is intense, polychromatic and collimated. It is widely exploited, in macromolecular crystallography, particularly using a monochromatized short wavelength beam. The spectral curve of SR, however, ideally lends itself to use of Laue geometry, i. e. the original diffraction experimental arrangement based on a stationary crystal and a polychromatic X-ray beam. Rapid exposure times and time-resolved crystallography studies, e. g. of enzymes, are now possible. Historical objections to the use of Laue diffraction data, particularly the multiplicity distribution, have been found not to be as limiting as once thought. The credentials of the Laue method have been established through a variety of Laue crystal structure analyses, involving photographic film as detector. Recently a three-dimensional arrangement of films, known as a toast-rack, has been used to alleviate problems with spatially overlapping spots. This paper provides a review of these results and then reports several developments. In particular, one of the first Laue analyses using an image plate as detector, namely of a cobalt substituted concanavalin A crystal, is discussed. Recent experimental developments, also at the Daresbury synchrotron, are then described. First, a large toast-rack has been used to record Laue data from a protein crystal. Secondly, a transmission X-ray mirror has been constructed from thin mylar (1.5 μm) and used to provide a λ max filter instead of using aluminium foils. Thirdly, since the Laue method suffers from poor sampling of the low resolution data, a new method (known as LOT) has been introduced.

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Segmented flow generator for serial crystallography at the European X-ray free electron laser

Nature Communications ◽

10.1038/s41467-020-18156-7 ◽

2020 ◽

Vol 11 (1) ◽

Author(s):

Austin Echelmeier ◽

Jorvani Cruz Villarreal ◽

Marc Messerschmidt ◽

Daihyun Kim ◽

Jesse D. Coe ◽

...

Keyword(s):

Free Electron ◽

Structural Features ◽

Protein Crystal ◽

Liquid Sample ◽

Time Resolved ◽

X Ray ◽

Flow Generator ◽

Serial Crystallography ◽

Serial Femtosecond Crystallography

Abstract Serial femtosecond crystallography (SFX) with X-ray free electron lasers (XFELs) allows structure determination of membrane proteins and time-resolved crystallography. Common liquid sample delivery continuously jets the protein crystal suspension into the path of the XFEL, wasting a vast amount of sample due to the pulsed nature of all current XFEL sources. The European XFEL (EuXFEL) delivers femtosecond (fs) X-ray pulses in trains spaced 100 ms apart whereas pulses within trains are currently separated by 889 ns. Therefore, continuous sample delivery via fast jets wastes >99% of sample. Here, we introduce a microfluidic device delivering crystal laden droplets segmented with an immiscible oil reducing sample waste and demonstrate droplet injection at the EuXFEL compatible with high pressure liquid delivery of an SFX experiment. While achieving ~60% reduction in sample waste, we determine the structure of the enzyme 3-deoxy-D-manno-octulosonate-8-phosphate synthase from microcrystals delivered in droplets revealing distinct structural features not previously reported.

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Co-flow injection for serial crystallography at X-ray free-electron lasers

Journal of Applied Crystallography ◽

10.1107/s1600576721011079 ◽

2022 ◽

Vol 55 (1) ◽

Author(s):

Diandra Doppler ◽

Mohammad T. Rabbani ◽

Romain Letrun ◽

Jorvani Cruz Villarreal ◽

Dai Hyun Kim ◽

...

Keyword(s):

Free Electron ◽

Protein Crystal ◽

Free Electron Lasers ◽

Data Set ◽

Time Resolved ◽

High Background ◽

X Ray ◽

Flow Generation ◽

Structural Details ◽

3D Printed

Serial femtosecond crystallography (SFX) is a powerful technique that exploits X-ray free-electron lasers to determine the structure of macromolecules at room temperature. Despite the impressive exposition of structural details with this novel crystallographic approach, the methods currently available to introduce crystals into the path of the X-ray beam sometimes exhibit serious drawbacks. Samples requiring liquid injection of crystal slurries consume large quantities of crystals (at times up to a gram of protein per data set), may not be compatible with vacuum configurations on beamlines or provide a high background due to additional sheathing liquids present during the injection. Proposed and characterized here is the use of an immiscible inert oil phase to supplement the flow of sample in a hybrid microfluidic 3D-printed co-flow device. Co-flow generation is reported with sample and oil phases flowing in parallel, resulting in stable injection conditions for two different resin materials experimentally. A numerical model is presented that adequately predicts these flow-rate conditions. The co-flow generating devices reduce crystal clogging effects, have the potential to conserve protein crystal samples up to 95% and will allow degradation-free light-induced time-resolved SFX.

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Can Laue catch Maxwell?: observation of short-lived species by Laue X-ray crystallography

Philosophical Transactions of the Royal Society of London Series A Physical and Engineering Sciences ◽

10.1098/rsta.1992.0066 ◽

1992 ◽

Vol 340 (1657) ◽

pp. 273-284 ◽

Cited By ~ 5

Keyword(s):

Real Time ◽

Protein Crystal ◽

Separate Experiment ◽

Enzymatic Reactions ◽

X Ray Diffraction ◽

Single Experiment ◽

Time Resolved ◽

X Ray ◽

X Ray Crystallography ◽

Novel Strategy

Now that the Laue method has been established as a tool for protein crystallography, the main problem involved in any prospective time-resolved X-ray diffraction study is one of chemistry. The reaction or process in question must be initiated on a timescale that is fast compared with its kinetics. For most biochemical events photochemistry is the most suitable trigger, but not all substrates can be caged for photochemical release. This problem can be solved by the novel strategy of caging the enzyme with a photoreversible covalent inhibitor. The logic of this method will be discussed and its application to a time-resolved study of the reaction of a suicide substrate with the protease gamma chymotrypsin shown. The question of real-time crystallographic ‘movies’ of enzymatic reactions can now be considered. It seems likely that following a reaction in real time in a single experiment will be very difficult if not impossible in most cases, in part because even a synchronized process will rapidly become asynchronous in a protein crystal, and also because it will be very difficult to know exactly what species one is observing at any instant unless one has extremely high resolution. It seems that the best use of the Laue technique will be to study unstable species that can be accumulated in the crystal under defined conditions for short periods of time. An entire reaction sequence can then be obtained as a series of individual steps, each of which is obtained from a separate experiment.

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Microtubule nucleation sequence studied by time-resolved x-ray scattering and electron microscopy

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100077402 ◽

1983 ◽

Vol 41 ◽

pp. 766-767

Author(s):

Eva-Maria Mandelkow ◽

Eckhard Mandelkow ◽

Joan Bordas

Keyword(s):

Electron Microscopy ◽

Dynamic Equilibrium ◽

Time Resolved ◽

X Ray ◽

Additional Information ◽

X Ray Scattering ◽

Low Concentrations ◽

Microtubule Growth ◽

Ray Patterns ◽

Ray Scattering

When a solution of microtubule protein is changed from non-polymerising to polymerising conditions (e.g. by temperature jump or mixing with GTP) there is a series of structural transitions preceding microtubule growth. These have been detected by time-resolved X-ray scattering using synchrotron radiation, and they may be classified into pre-nucleation and nucleation events. X-ray patterns are good indicators for the average behavior of the particles in solution, but they are difficult to interpret unless additional information on their structure is available. We therefore studied the assembly process by electron microscopy under conditions approaching those of the X-ray experiment. There are two difficulties in the EM approach: One is that the particles important for assembly are usually small and not very regular and therefore tend to be overlooked. Secondly EM specimens require low concentrations which favor disassembly of the particles one wants to observe since there is a dynamic equilibrium between polymers and subunits.

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Time-Resolved Cryo-Electron Microscopy of Oscillating Microtubules

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100181269 ◽

1990 ◽

Vol 48 (1) ◽

pp. 502-503

Author(s):

Eva-Maria Mandelkow ◽

Ron Milligan

Keyword(s):

Electron Microscopy ◽

Dynamic Instability ◽

Entire Solution ◽

Reaction Scheme ◽

Time Resolved ◽

X Ray ◽

X Ray Scattering ◽

A Chain ◽

Ray Scattering

Microtubules form part of the cytoskeleton of eukaryotic cells. They are hollow libers of about 25 nm diameter made up of 13 protofilaments, each of which consists of a chain of heterodimers of α-and β-tubulin. Microtubules can be assembled in vitro at 37°C in the presence of GTP which is hydrolyzed during the reaction, and they are disassembled at 4°C. In contrast to most other polymers microtubules show the behavior of “dynamic instability”, i.e. they can switch between phases of growth and phases of shrinkage, even at an overall steady state [1]. In certain conditions an entire solution can be synchronized, leading to autonomous oscillations in the degree of assembly which can be observed by X-ray scattering (Fig. 1), light scattering, or electron microscopy [2-5]. In addition such solutions are capable of generating spontaneous spatial patterns [6].In an earlier study we have analyzed the structure of microtubules and their cold-induced disassembly by cryo-EM [7]. One result was that disassembly takes place by loss of protofilament fragments (tubulin oligomers) which fray apart at the microtubule ends. We also looked at microtubule oscillations by time-resolved X-ray scattering and proposed a reaction scheme [4] which involves a cyclic interconversion of tubulin, microtubules, and oligomers (Fig. 2). The present study was undertaken to answer two questions: (a) What is the nature of the oscillations as seen by time-resolved cryo-EM? (b) Do microtubules disassemble by fraying protofilament fragments during oscillations at 37°C?

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