scholarly journals Serial crystallography captures enzyme catalysis in copper nitrite reductase at atomic resolution from one crystal

IUCrJ ◽  
2016 ◽  
Vol 3 (4) ◽  
pp. 271-281 ◽  
Author(s):  
Sam Horrell ◽  
Svetlana V. Antonyuk ◽  
Robert R. Eady ◽  
S. Samar Hasnain ◽  
Michael A. Hough ◽  
...  
Keyword(s):  
Nitrite Reductase ◽  
Enzyme Catalysis ◽  
Structural Changes ◽  
Enzyme Mechanism ◽  
Nitrite Reduction ◽  
Protein Crystal ◽  
X Ray ◽  
Real Time Analysis ◽  
Multiple Structures ◽  
Serial Crystallography

Relating individual protein crystal structures to an enzyme mechanism remains a major and challenging goal for structural biology. Serial crystallography using multiple crystals has recently been reported in both synchrotron-radiation and X-ray free-electron laser experiments. In this work, serial crystallography was used to obtain multiple structures serially from one crystal (MSOX) to studyin crystalloenzyme catalysis. Rapid, shutterless X-ray detector technology on a synchrotron MX beamline was exploited to perform low-dose serial crystallography on a single copper nitrite reductase crystal, which survived long enough for 45 consecutive 100 K X-ray structures to be collected at 1.07–1.62 Å resolution, all sampled from the same crystal volume. This serial crystallography approach revealed the gradual conversion of the substrate bound at the catalytic type 2 Cu centre from nitrite to nitric oxide, following reduction of the type 1 Cu electron-transfer centre by X-ray-generated solvated electrons. Significant, well defined structural rearrangements in the active site are evident in the series as the enzyme moves through its catalytic cycle, namely nitrite reduction, which is a vital step in the global denitrification process. It is proposed that such a serial crystallography approach is widely applicable for studying any redox or electron-driven enzyme reactions from a single protein crystal. It can provide a `catalytic reaction movie' highlighting the structural changes that occur during enzyme catalysis. The anticipated developments in the automation of data analysis and modelling are likely to allow seamless and near-real-time analysis of such data on-site at some of the powerful synchrotron crystallographic beamlines.

scholarly journals Superoxide reductase fromGiardia intestinalis: structural characterization of the first SOR from a eukaryotic organism shows an iron centre that is highly sensitive to photoreduction

2015 ◽  
Vol 71 (11) ◽  
pp. 2236-2247 ◽  
Author(s):  
Cristiana M. Sousa ◽  
Philippe Carpentier ◽  
Pedro M. Matias ◽  
Fabrizio Testa ◽  
Filipa Pinho ◽  
...  
Keyword(s):  
Iron Reduction ◽  
Structural Changes ◽  
Quaternary Structure ◽  
High Sensitivity ◽  
Protozoan Parasite ◽  
Protein Crystal ◽  
Superoxide Reductase ◽  
X Ray ◽  
Eukaryotic Organism ◽  
High Flexibility

Superoxide reductase (SOR), which is commonly found in prokaryotic organisms, affords protection from oxidative stress by reducing the superoxide anion to hydrogen peroxide. The reaction is catalyzed at the iron centre, which is highly conserved among the prokaryotic SORs structurally characterized to date. Reported here is the first structure of an SOR from a eukaryotic organism, the protozoan parasiteGiardia intestinalis(GiSOR), which was solved at 2.0 Å resolution. By collecting several diffraction data sets at 100 K from the same flash-cooled protein crystal using synchrotron X-ray radiation, photoreduction of the iron centre was observed. Reduction was monitored using an online UV–visible microspectrophotometer, following the decay of the 647 nm absorption band characteristic of the iron site in the glutamate-bound, oxidized state. Similarly to other 1Fe-SORs structurally characterized to date, the enzyme displays a tetrameric quaternary-structure arrangement. As a distinctive feature, the N-terminal loop of the protein, containing the characteristic EKHxP motif, revealed an unusually high flexibility regardless of the iron redox state. At variance with previous evidence collected by X-ray crystallography and Fourier transform infrared spectroscopy of prokaryotic SORs, iron reduction did not lead to dissociation of glutamate from the catalytic metal or other structural changes; however, the glutamate ligand underwent X-ray-induced chemical changes, revealing high sensitivity of theGiSOR active site to X-ray radiation damage.

scholarly journals Serial crystallography using automated drop dispensing

2021 ◽  
Vol 28 (5) ◽  
Author(s):  
Zhen Su ◽  
Joshua Cantlon ◽  
Lacey Douthit ◽  
Max Wiedorn ◽  
Sébastien Boutet ◽  
...  
Keyword(s):  
Free Electron Laser ◽  
Protein Crystal ◽  
Cross Contamination ◽  
X Ray ◽  
On Demand ◽  
Drop On Demand ◽  
Serial Crystallography ◽  
Cycling System ◽  
Serial Femtosecond Crystallography

Automated, pulsed liquid-phase sample delivery has the potential to greatly improve the efficiency of both sample and photon use at pulsed X-ray facilities. In this work, an automated drop on demand (DOD) system that accelerates sample exchange for serial femtosecond crystallography (SFX) is demonstrated. Four different protein crystal slurries were tested, and this technique is further improved here with an automatic sample-cycling system whose effectiveness was verified by the indexing results. Here, high-throughput SFX screening is shown to be possible at free-electron laser facilities with very low risk of cross contamination and minimal downtime. The development of this technique will significantly reduce sample consumption and enable structure determination of proteins that are difficult to crystallize in large quantities. This work also lays the foundation for automating sample delivery.

scholarly journals High-viscosity injector-based pink-beam serial crystallography of microcrystals at a synchrotron radiation source

IUCrJ ◽  
2019 ◽  
Vol 6 (3) ◽  
pp. 412-425 ◽  
Author(s):  
Jose M. Martin-Garcia ◽  
Lan Zhu ◽  
Derek Mendez ◽  
Ming-Yue Lee ◽  
Eugene Chun ◽  
...  
Keyword(s):  
Synchrotron Radiation ◽  
Radiation Source ◽  
Structural Changes ◽  
High Viscosity ◽  
Synchrotron Radiation Source ◽  
Proteinase K ◽  
Moderate Increase ◽  
Time Resolved ◽  
X Ray ◽  
Serial Crystallography

Since the first successful serial crystallography (SX) experiment at a synchrotron radiation source, the popularity of this approach has continued to grow showing that third-generation synchrotrons can be viable alternatives to scarce X-ray free-electron laser sources. Synchrotron radiation flux may be increased ∼100 times by a moderate increase in the bandwidth (`pink beam' conditions) at some cost to data analysis complexity. Here, we report the first high-viscosity injector-based pink-beam SX experiments. The structures of proteinase K (PK) and A2A adenosine receptor (A2AAR) were determined to resolutions of 1.8 and 4.2 Å using 4 and 24 consecutive 100 ps X-ray pulse exposures, respectively. Strong PK data were processed using existing Laue approaches, while weaker A2AAR data required an alternative data-processing strategy. This demonstration of the feasibility presents new opportunities for time-resolved experiments with microcrystals to study structural changes in real time at pink-beam synchrotron beamlines worldwide.

scholarly journals RABDAM: quantifying specific radiation damage in individual protein crystal structures

2018 ◽  
Vol 51 (2) ◽  
pp. 552-559 ◽  
Author(s):  
Kathryn L. Shelley ◽  
Thomas P. E. Dixon ◽  
Jonathan C. Brooks-Bartlett ◽  
Elspeth F. Garman
Keyword(s):  
Radiation Damage ◽  
Open Source Software ◽  
Structural Changes ◽  
Data Bank ◽  
Protein Crystal ◽  
Standard Format ◽  
X Ray ◽  
Extent Of Damage ◽  
Local Packing Density

Radiation damage remains one of the major limitations to accurate structure determination in protein crystallography (PX). Despite the use of cryo-cooling techniques, it is highly probable that a number of the structures deposited in the Protein Data Bank (PDB) have suffered substantial radiation damage as a result of the high flux densities of third generation synchrotron X-ray sources. Whereas the effects of global damage upon diffraction pattern reflection intensities are readily detectable, traditionally the (earlier onset) site-specific structural changes induced by radiation damage have proven difficult to identify within individual PX structures. More recently, however, development of theBDamagemetric has helped to address this problem.BDamageis a quantitative, per-atom metric identifies potential sites of specific damage by comparing the atomicB-factor values of atoms that occupy a similar local packing density environment in the structure. Building upon this past work, this article presents a program,RABDAM, to calculate theBDamagemetric for all selected atoms within any standard-format PDB or mmCIF file.RABDAMprovides several useful outputs to assess the extent of damage suffered by an input PX structure. This free and open-source software will allow assessment and improvement of the quality of PX structures both previously and newly deposited in the PDB.

X-ray Structure and Site-Directed Mutagenesis of a Nitrite Reductase from Alcaligenes Faecalis S-6: Roles of Two Copper Atoms in Nitrite Reduction

Biochemistry ◽  
1994 ◽  
Vol 33 (17) ◽  
pp. 5246-5252 ◽  
Author(s):  
Mutsuko Kukimoto ◽  
Makoto Nishiyama ◽  
Michael E. P. Murphy ◽  
Stewart Turley ◽  
Elinor T. Adman ◽  
...  
Keyword(s):  
Nitrite Reductase ◽  
Alcaligenes Faecalis ◽  
Nitrite Reduction ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
X Ray

scholarly journals X-ray diffraction in temporally and spatially resolved biomolecular science

2015 ◽  
Vol 177 ◽  
pp. 429-441 ◽  
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.

scholarly journals Mix-and-inject XFEL crystallography reveals gated conformational dynamics during enzyme catalysis

2019 ◽  
Vol 116 (51) ◽  
pp. 25634-25640 ◽  
Author(s):  
Medhanjali Dasgupta ◽  
Dominik Budday ◽  
Saulo H. P. de Oliveira ◽  
Peter Madzelan ◽  
Darya Marchany-Rivera ◽  
...  
Keyword(s):  
Active Site ◽  
Enzyme Catalysis ◽  
Conformational Dynamics ◽  
Catalytic Efficiency ◽  
Enzyme Mechanism ◽  
Time Resolved ◽  
X Ray ◽  
Protein Motions ◽  
X Ray Crystallography ◽  
Active Site Cysteine

How changes in enzyme structure and dynamics facilitate passage along the reaction coordinate is a fundamental unanswered question. Here, we use time-resolved mix-and-inject serial crystallography (MISC) at an X-ray free electron laser (XFEL), ambient-temperature X-ray crystallography, computer simulations, and enzyme kinetics to characterize how covalent catalysis modulates isocyanide hydratase (ICH) conformational dynamics throughout its catalytic cycle. We visualize this previously hypothetical reaction mechanism, directly observing formation of a thioimidate covalent intermediate in ICH microcrystals during catalysis. ICH exhibits a concerted helical displacement upon active-site cysteine modification that is gated by changes in hydrogen bond strength between the cysteine thiolate and the backbone amide of the highly strained Ile152 residue. These catalysis-activated motions permit water entry into the ICH active site for intermediate hydrolysis. Mutations at a Gly residue (Gly150) that modulate helical mobility reduce ICH catalytic turnover and alter its pre-steady-state kinetic behavior, establishing that helical mobility is important for ICH catalytic efficiency. These results demonstrate that MISC can capture otherwise elusive aspects of enzyme mechanism and dynamics in microcrystalline samples, resolving long-standing questions about the connection between nonequilibrium protein motions and enzyme catalysis.

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

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.

scholarly journals Serial crystallography using synchrotron radiation - novel strategies for microcrystallography

2014 ◽  
Vol 70 (a1) ◽  
pp. C316-C316
Author(s):  
Cornelius Gati ◽  
Gleb Bourenkov ◽  
Marco Klinge ◽  
Dirk Rehders ◽  
Francesco Stellato ◽  
...  
Keyword(s):  
Structural Model ◽  
Structural Information ◽  
Protein Crystal ◽  
Array Detector ◽  
X Ray ◽  
Pixel Array ◽  
Serial Crystallography ◽  
Diffraction Patterns ◽  
Full Intensity

Protein crystallography continues to be one of the most frequently used techniques to obtain structural information of biomacromolecules to atomic resolution. Since protein crystals of delicate target systems are often limited in size, one of the main goals in the design of modern beamlines is the construction of highly intense X-ray beams with small focal size to obtain high resolution diffraction images of microcrystals. However, this development has led to the situation, that the full intensity of the beam can destroy a protein crystal within fractions of a second. Therefore often only a small number of diffraction patterns can be obtained from one single crystal. Here we describe the adaptation of the serial crystallography approach, which has first been developed at X-ray Free-Electron Lasers (Chapman et al. 2011) to the usage of a microfocus synchrotron beamline, using a standard cryogenic loop for sample delivery. We proved this concept with in vivo grown cathepsinB microcrystals (TbCatB, Koopmann et al. 2012, Redecke et al. 2013) (average of 9 μm3), a medically and pharmaceutically relevant protein, involved in the life cycle of T. brucei. In these experiments it was possible to show that serial crystallography enables the utilization and outcome of the above described bottlenecks and features of modern 3rd generation synchrotron microfocus beamlines. Our strategy exploits the combination of a micron-sized X-ray beam, high precision diffractometry and shutterless data acquisition with a pixel-array detector. By combining the data of 80 TbCatB crystals, it was possible to assemble a dataset to 3.0 Å resolution. The data allow the refinement of a structural model that is consistent with that previously obtained using FEL radiation, providing mutual validation.

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