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Abstract: Dynamic structural biology enables studying biological events at the atomic scale from 10’s of femtoseconds to a few seconds duration. With the advent of X-ray Free Electron Lasers (XFELs) and 4th generation synchrotrons, serial crystallography is becoming a major player for time-resolved experiments in structural biology. Despite significant progress, challenges such as obtaining sufficient amounts of protein to produce homogeneous microcrystal slurry, remain. Given this, it has been paramount to develop instrumentation that reduces the amount of microcrystal slurry required for experiments. Tape-drive systems use a conveyor belt made of X-ray transparent material as a motorized solid-support to steer deposited microcrystals into the beam. For efficient sample consumption on-demand ejectors can be synchronized with the X-ray pulses to expose crystals contained in droplets deposited on the tape. Reactions in the crystals can be triggered via various strategies, including pump-probe, substrate/ligand mixing, or gas incubation in the space between droplet ejection and X-ray illumination. Another challenge in time-resolved serial crystallography is interpreting the resulting electron density maps. This is especially difficult for metalloproteins where the active site metal is intimately involved in catalysis and often proceeds through multiple oxidation states during enzymatic catalysis. The unrestricted space around tape-drive systems can be used to accommodate complementary spectroscopic equipment. Here, we highlight tape-drive sample delivery systems for complementary and simultaneous X-ray diffraction (XRD) and X-ray emission spectroscopy (XES) measurements. We describe how the combination of both XRD and XES is a powerful tool for time-resolved experiments at XFELs and synchrotrons.
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Oct 2024
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I23-Long wavelength MX
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Yishun
Lu
,
Ramona
Duman
,
James
Beilsten-Edmands
,
Graeme
Winter
,
Mark
Basham
,
Gwyndaf
Evans
,
Jos J. A. G.
Kamps
,
Allen M.
Orville
,
Hok-Sau
Kwong
,
Konstantinos
Beis
,
Wesley
Armour
,
Armin
Wagner
Open Access
Abstract: rocessing of single-crystal X-ray diffraction data from area detectors can be separated into two steps. First, raw intensities are obtained by integration of the diffraction images, and then data correction and reduction are performed to determine structure-factor amplitudes and their uncertainties. The second step considers the diffraction geometry, sample illumination, decay, absorption and other effects. While absorption is only a minor effect in standard macromolecular crystallography (MX), it can become the largest source of uncertainty for experiments performed at long wavelengths. Current software packages for MX typically employ empirical models to correct for the effects of absorption, with the corrections determined through the procedure of minimizing the differences in intensities between symmetry-equivalent reflections; these models are well suited to capturing smoothly varying experimental effects. However, for very long wavelengths, empirical methods become an unreliable approach to model strong absorption effects with high fidelity. This problem is particularly acute when data multiplicity is low. This paper presents an analytical absorption correction strategy (implemented in new software AnACor) based on a volumetric model of the sample derived from X-ray tomography. Individual path lengths through the different sample materials for all reflections are determined by a ray-tracing method. Several approaches for absorption corrections (spherical harmonics correction, analytical absorption correction and a combination of the two) are compared for two samples, the membrane protein OmpK36 GD, measured at a wavelength of λ = 3.54 Å, and chlorite dismutase, measured at λ = 4.13 Å. Data set statistics, the peak heights in the anomalous difference Fourier maps and the success of experimental phasing are used to compare the results from the different absorption correction approaches. The strategies using the new analytical absorption correction are shown to be superior to the standard spherical harmonics corrections. While the improvements are modest in the 3.54 Å data, the analytical absorption correction outperforms spherical harmonics in the longer-wavelength data (λ = 4.13 Å), which is also reflected in the reduced amount of data being required for successful experimental phasing.
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Jun 2024
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I24-Microfocus Macromolecular Crystallography
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Rachel
Bolton
,
Moritz M.
Machelett
,
Jack
Stubbs
,
Danny
Axford
,
Nicolas
Caramello
,
Lucrezia
Catapano
,
Martin
Maly
,
Matthew J.
Rodrigues
,
Charlotte
Cordery
,
Graham J.
Tizzard
,
Fraser
Macmillan
,
Sylvain
Engilberge
,
David
Von Stetten
,
Takehiko
Tosha
,
Hiroshi
Sugimoto
,
Jonathan A. R.
Worrall
,
Jeremy S.
Webb
,
Mike
Zubkov
,
Simon
Coles
,
Eric
Mathieu
,
Roberto A.
Steiner
,
Garib
Murshudov
,
Tobias E.
Schrader
,
Allen M.
Orville
,
Antoine
Royant
,
Gwyndaf
Evans
,
Michael A.
Hough
,
Robin L.
Owen
,
Ivo
Tews
Diamond Proposal Number(s):
[15722, 14493, 23570]
Open Access
Abstract: The marine cyanobacterium Prochlorococcus is a main contributor to global photosynthesis, whilst being limited by iron availability. Cyanobacterial genomes generally encode two different types of FutA iron-binding proteins: periplasmic FutA2 ABC transporter subunits bind Fe(III), while cytosolic FutA1 binds Fe(II). Owing to their small size and their economized genome Prochlorococcus ecotypes typically possess a single futA gene. How the encoded FutA protein might bind different Fe oxidation states was previously unknown. Here, we use structural biology techniques at room temperature to probe the dynamic behavior of FutA. Neutron diffraction confirmed four negatively charged tyrosinates, that together with a neutral water molecule coordinate iron in trigonal bipyramidal geometry. Positioning of the positively charged Arg103 side chain in the second coordination shell yields an overall charge-neutral Fe(III) binding state in structures determined by neutron diffraction and serial femtosecond crystallography. Conventional rotation X-ray crystallography using a home source revealed X-ray-induced photoreduction of the iron center with observation of the Fe(II) binding state; here, an additional positioning of the Arg203 side chain in the second coordination shell maintained an overall charge neutral Fe(II) binding site. Dose series using serial synchrotron crystallography and an XFEL X-ray pump–probe approach capture the transition between Fe(III) and Fe(II) states, revealing how Arg203 operates as a switch to accommodate the different iron oxidation states. This switching ability of the Prochlorococcus FutA protein may reflect ecological adaptation by genome streamlining and loss of specialized FutA proteins.
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Mar 2024
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Jack
Stubbs
,
Theo
Hornsey
,
Niall
Hanrahan
,
Luis Blay
Esteban
,
Rachel
Bolton
,
Martin
Maly
,
Shibom
Basu
,
Julien
Orlans
,
Daniele
De Sanctis
,
Jung-Uk
Shim
,
Patrick D.
Shaw Stewart
,
Allen M.
Orville
,
Ivo
Tews
,
Jonathan
West
Open Access
Abstract: Serial crystallography requires large numbers of microcrystals and robust strategies to rapidly apply substrates to initiate reactions in time-resolved studies. Here, we report the use of droplet miniaturization for the controlled production of uniform crystals, providing an avenue for controlled substrate addition and synchronous reaction initiation. The approach was evaluated using two enzymatic systems, yielding 3 µm crystals of lysozyme and 2 µm crystals of Pdx1, an Arabidopsis enzyme involved in vitamin B6 biosynthesis. A seeding strategy was used to overcome the improbability of Pdx1 nucleation occurring with diminishing droplet volumes. Convection within droplets was exploited for rapid crystal mixing with ligands. Mixing times of <2 ms were achieved. Droplet microfluidics for crystal size engineering and rapid micromixing can be utilized to advance time-resolved serial crystallography.
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Mar 2024
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Romie C.
Nguyen
,
Ian
Davis
,
Medhanjali
Dasgupta
,
Yifan
Wang
,
Philipp S.
Simon
,
Agata
Butryn
,
Hiroki
Makita
,
Isabel
Bogacz
,
Kednerlin
Dornevil
,
Pierre
Aller
,
Asmit
Bhowmick
,
Ruchira
Chatterjee
,
In-Sik
Kim
,
Tiankun
Zhou
,
Derek
Mendez
,
Daniel W.
Paley
,
Franklin
Fuller
,
Roberto
Alonso Mori
,
Alexander
Batyuk
,
Nicholas K.
Sauter
,
Aaron S.
Brewster
,
Allen M.
Orville
,
Vittal K.
Yachandra
,
Junko
Yano
,
Jan F.
Kern
,
Aimin
Liu
Abstract: The P450 enzyme CYP121 from Mycobacterium tuberculosis catalyzes a carbon–carbon (C–C) bond coupling cyclization of the dityrosine substrate containing a diketopiperazine ring, cyclo(l-tyrosine-l-tyrosine) (cYY). An unusual high-spin (S = 5/2) ferric intermediate maximizes its population in less than 5 ms in the rapid freeze-quenching study of CYP121 during the shunt reaction with peracetic acid or hydrogen peroxide in acetic acid solution. We show that this intermediate can also be observed in the crystalline state by EPR spectroscopy. By developing an on-demand-rapid-mixing method for time-resolved serial femtosecond crystallography with X-ray free-electron laser (tr-SFX-XFEL) technology covering the millisecond time domain and without freezing, we structurally monitored the reaction in situ at room temperature. After a 200 ms peracetic acid reaction with the cocrystallized enzyme–substrate microcrystal slurry, a ferric-hydroperoxo intermediate is observed, and its structure is determined at 1.85 Å resolution. The structure shows a hydroperoxyl ligand between the heme and the native substrate, cYY. The oxygen atoms of the hydroperoxo are 2.5 and 3.2 Å from the iron ion. The end-on binding ligand adopts a near-side-on geometry and is weakly associated with the iron ion, causing the unusual high-spin state. This compound 0 intermediate, spectroscopically and structurally observed during the catalytic shunt pathway, reveals a unique binding mode that deviates from the end-on compound 0 intermediates in other heme enzymes. The hydroperoxyl ligand is only 2.9 Å from the bound cYY, suggesting an active oxidant role of the intermediate for direct substrate oxidation in the nonhydroxylation C–C bond coupling chemistry.
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Nov 2023
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I24-Microfocus Macromolecular Crystallography
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James
Birch
,
Tristan O. C.
Kwan
,
Peter J.
Judge
,
Danny
Axford
,
Pierre
Aller
,
Agata
Butryn
,
Rosana
Reis
,
Juan F.
Bada Juarez
,
Javier
Vinals
,
Robin L.
Owen
,
Eriko
Nango
,
Rie
Tanaka
,
Kensuke
Tono
,
Yasumasa
Joti
,
Tomoyuki
Tanaka
,
Shigeki
Owada
,
Michihiro
Sugahara
,
So
Iwata
,
Allen M.
Orville
,
Anthony
Watts
,
Isabel
Moraes
Diamond Proposal Number(s):
[19152]
Open Access
Abstract: Serial crystallography has emerged as an important tool for structural studies of integral membrane proteins. The ability to collect data from micrometre-sized weakly diffracting crystals at room temperature with minimal radiation damage has opened many new opportunities in time-resolved studies and drug discovery. However, the production of integral membrane protein microcrystals in lipidic cubic phase at the desired crystal density and quantity is challenging. This paper introduces VIALS (versatile approach to high-density microcrystals in lipidic cubic phase for serial crystallography), a simple, fast and efficient method for preparing hundreds of microlitres of high-density microcrystals suitable for serial X-ray diffraction experiments at both synchrotron and free-electron laser sources. The method is also of great benefit for rational structure-based drug design as it facilitates in situ crystal soaking and rapid determination of many co-crystal structures. Using the VIALS approach, room-temperature structures are reported of (i) the archaerhodopsin-3 protein in its dark-adapted state and 110 ns photocycle intermediate, determined to 2.2 and 1.7 Å, respectively, and (ii) the human A2A adenosine receptor in complex with two different ligands determined to a resolution of 3.5 Å.
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Oct 2023
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Hugo
Lebrette
,
Vivek
Srinivas
,
Juliane
John
,
Oskar
Aurelius
,
Rohit
Kumar
,
Daniel
Lundin
,
Aaron S.
Brewster
,
Asmit
Bhowmick
,
Abhishek
Sirohiwal
,
In-Sik
Kim
,
Sheraz
Gul
,
Cindy
Pham
,
Kyle D.
Sutherlin
,
Philipp
Simon
,
Agata
Butryn
,
Pierre
Aller
,
Allen M.
Orville
,
Franklin D.
Fuller
,
Roberto
Alonso-Mori
,
Alexander
Batyuk
,
Nicholas K.
Sauter
,
Vittal K.
Yachandra
,
Junko
Yano
,
Ville R. I.
Kaila
,
Britt-Marie
Sjöberg
,
Jan
Kern
,
Katarina
Roos
,
Martin
Högbom
Abstract: Aerobic ribonucleotide reductases (RNRs) initiate synthesis of DNA building blocks by generating a free radical within the R2 subunit; the radical is subsequently shuttled to the catalytic R1 subunit through proton-coupled electron transfer (PCET). We present a high-resolution room temperature structure of the class Ie R2 protein radical captured by x-ray free electron laser serial femtosecond crystallography. The structure reveals conformational reorganization to shield the radical and connect it to the translocation path, with structural changes propagating to the surface where the protein interacts with the catalytic R1 subunit. Restructuring of the hydrogen bond network, including a notably short O–O interaction of 2.41 angstroms, likely tunes and gates the radical during PCET. These structural results help explain radical handling and mobilization in RNR and have general implications for radical transfer in proteins.
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Oct 2023
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B23-Circular Dichroism
I24-Microfocus Macromolecular Crystallography
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Ryan M.
Lithgo
,
Marko
Hanževački
,
Gemma
Harris
,
Jos J. A. G.
Kamps
,
Ellie
Holden
,
Tiberiu-Marius
Gianga
,
Justin L. P.
Benesch
,
Christof M.
Jäger
,
Anna K.
Croft
,
Rohanah
Hussain
,
Jon L.
Hobman
,
Allen M.
Orville
,
Andrew
Quigley
,
Stephen B.
Carr
,
David J.
Scott
Open Access
Abstract: The periplasmic chaperone SilF has been identified as part of an Ag(I) detoxification system in Gram negative bacteria. Sil proteins also bind Cu(I), but with reported weaker affinity, therefore leading to the designation of a specific detoxification system for Ag(I). Using isothermal titration calorimetry we show that binding of both ions is not only tighter than previously thought, but of very similar affinities. We investigated the structural origins of ion binding using molecular dynamics and QM/MM simulations underpinned by structural and biophysical experiments. The results of this analysis showed that the binding site adapts to accommodate either ion, with key interactions with the solvent in the case of Cu(I). The implications of this are that Gram negative bacteria do not appear to have evolved a specific Ag(I) efflux system but take advantage of the existing Cu(I) detoxification system. Therefore, there are consequences for how we define a particular metal resistance mechanism and understand its evolution in the environment.
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Oct 2023
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Juliane
John
,
Oskar
Aurelius
,
Vivek
Srinivas
,
Patricia
Saura
,
In-Sik
Kim
,
Asmit
Bhowmick
,
Philipp S.
Simon
,
Medhanjali
Dasgupta
,
Cindy
Pham
,
Sheraz
Gul
,
Kyle D.
Sutherlin
,
Pierre
Aller
,
Agata
Butryn
,
Allen M.
Orville
,
Mun Hon
Cheah
,
Shigeki
Owada
,
Kensuke
Tono
,
Franklin D
Fuller
,
Alexander
Batyuk
,
Aaron S.
Brewster
,
Nicholas K.
Sauter
,
Vittal K
Yachandra
,
Junko
Yano
,
Ville R. I.
Kaila
,
Jan
Kern
,
Hugo
Lebrette
,
Martin
Högbom
Open Access
Abstract: Redox reactions are central to biochemistry and are both controlled by and induce protein structural changes. Here, we describe structural rearrangements and crosstalk within the Bacillus cereus ribonucleotide reductase R2b–NrdI complex, a di-metal carboxylate-flavoprotein system, as part of the mechanism generating the essential catalytic free radical of the enzyme. Femtosecond crystallography at an X-ray free electron laser was utilized to obtain structures at room temperature in defined redox states without suffering photoreduction. Together with density functional theory calculations, we show that the flavin is under steric strain in the R2b–NrdI protein complex, likely tuning its redox properties to promote superoxide generation. Moreover, a binding site in close vicinity to the expected flavin O2 interaction site is observed to be controlled by the redox state of the flavin and linked to the channel proposed to funnel the produced superoxide species from NrdI to the di-manganese site in protein R2b. These specific features are coupled to further structural changes around the R2b–NrdI interaction surface. The mechanistic implications for the control of reactive oxygen species and radical generation in protein R2b are discussed.
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Sep 2022
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Open Access
Abstract: The two SARS-CoV-2 proteases, i.e. the main protease (M pro ) and the papain-like protease (PL pro ), which hydrolyze the viral polypeptide chain giving functional non-structural proteins, are essential for viral replication and are medicinal chemistry targets. We report a high-throughput mass spectrometry (MS)-based assay which directly monitors PL pro catalysis in vitro . The assay was applied to investigate the effect of reported small-molecule PL pro inhibitors and selected M pro inhibitors on PL pro catalysis. The results reveal that some, but not all, PL pro inhibitor potencies differ substantially from those obtained using fluorescence-based assays. Some substrate-competing M pro inhibitors, notably PF-07321332 (nirmatrelvir) which is in clinical development, do not inhibit PL pro . Less selective M pro inhibitors, e.g. auranofin, inhibit PL pro , highlighting the potential for dual PL pro /M pro inhibition. MS-based PL pro assays, which are orthogonal to widely employed fluorescence-based assays, are of utility in validating inhibitor potencies, especially for inhibitors operating by non-covalent mechanisms.
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Jan 2022
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