I24-Microfocus Macromolecular Crystallography
VMXi-Versatile Macromolecular Crystallography in situ
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Open Access
Abstract: Multi-crystal processing of X-ray diffraction data has become highly automated to keep pace with the current high-throughput capabilities afforded by beamlines. A significant challenge, however, is the automated clustering of such data based on subtle differences such as ligand binding or conformational shifts. Intensity-based hierarchical clustering has been shown to be a viable method of identifying such subtle structural differences, but the interpretation of the resulting dendrograms is difficult to automate. Using isomorphous crystals of bovine, porcine and human insulin, the existing clustering methods in the multi-crystal processing software xia2.multiplex were validated and their limits were tested. It was determined that weighting the pairwise correlation coefficient calculations with the intensity uncertainties was required for accurate calculation of the pairwise correlation coefficient matrix (correlation clustering) and dimension optimization was required when expressing this matrix as a set of coordinates representing data sets (cosine-angle clustering). Finally, the introduction of the OPTICS spatial density-based clustering algorithm into DIALS allowed the automatic output of species-pure clusters of bovine, porcine and human insulin data sets.
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Jun 2025
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Metrology
Optics
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Open Access
Abstract: The Optics & Metrology group at Diamond Light Source has recently published a description of a bimorph deformable X-ray mirror operating in closed-loop using multi-beam interferometric feedback. This "adaptive" mirror can make fast and stabilised changes to the X-ray beam profile. Beam shaping at a rate of 1 Hz was achieved, a contrast to the now usual "set and forget" operation of "active" bimorph mirrors at synchrotrons. However, this breakthrough cannot be applied to synchrotron beamlines without a robust control system that allows the mirror to be rapidly and controllably deformed. Diamond has now responded to this need by taking an integrated approach, considering: the bimorph power supplies, the beamline control software, the beam imaging camera, the bimorph mirror optimisation software, and the bimorph mirror itself as part of a single system. In collaboration with CINEL, new HV-ADAPTOS high-voltage power supplies have been made available. The latest models contain new firmware that adds features not previously available, such as piezo-elastic creep compensation. Communication with the HV-ADAPTOS power supplies over Ethernet has been made more reliable by a new EPICS asynPortDriver interface developed at Diamond and rolled out to all appropriate Diamond beamlines. A new Bluesky/Ophyd plan for the measurement of the bimorph mirror's piezo response functions is under development and has undergone its preliminary tests. This plan is expected to be less affected by upgrades of the beamline control software than previous solutions. It relies on beam images produced by Gigabit Ethernet cameras and processed by the EPICS areaDetector driver. Finally, the need for strain-free clamping of the mirror has now been fully recognized and procedures for ensuring it have been put into practice. Although such a system is more complex than that required for a mechanically bent mirror, it gives bimorph mirrors an ability to operate rapidly and repeatably that other optics do not offer, and it lays a foundation for more advanced beam-shaping functions.
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May 2025
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Open Access
Abstract: The latest developments in acoustic technology and their integration of standing waves offers the potential to eject and trap tiny amounts of both liquid and solids with precision and stability in free space. This is appealing for experimenters at Light Sources who are traditionally relying on the use of containers or on the manual attachment of the samples to solid supports for analysis with X rays or IR. Here we present a brief overview of applications of acoustics and levitation for sample manipulation and delivery currently under investigation at Diamond Light source.
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May 2025
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Abstract: In serial crystallography, large numbers of microcrystals are sequentially delivered to an X-ray beam and a diffraction pattern is obtained from each crystal. This serial approach was developed primarily for X-ray Free Electron Lasers (XFELs) where crystals are destroyed by the beam but is increasingly used in synchrotron experiments. The combination of XFEL and synchrotron-based serial crystallography enables time-resolved experiments over an extremely wide range of time domains - from femtoseconds to seconds - and allows intact or pristine structures free of the effects of radiation damage to be obtained. Several approaches have been developed for sample delivery with varying levels of sample efficiency and ease of use. In the fixed target approach, microcrystals are loaded onto a solid support which is then rastered through the X-ray beam. The key advantages of fixed targets are that every crystal loaded can be used for data collection, and that precise control of when crystals are moved into the beam allows for time-resolved experiments over a very wide range of time domains as well as multi-shot experiments characterising the effects of the X-ray beam on the sample. We describe the application of fixed targets for serial crystallography as implemented at beamline I24 at Diamond Light Source and at the SACLA XFEL. We discuss methodologies for time-resolved serial crystallography in fixed targets and describe best practices for obtaining high-quality structures covering sample preparation, data collection strategies and data analysis pipelines.
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Oct 2024
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I24-Microfocus Macromolecular Crystallography
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Diamond Proposal Number(s):
[27314]
Open Access
Abstract: Human gamma-D crystallin (HGD) is a major constituent of the eye lens. Aggregation of HGD contributes to cataract formation, the leading cause of blindness worldwide. It is unique in its longevity, maintaining its folded and soluble state for 50-60 years. One outstanding question is the structural basis of this longevity despite oxidative aging and environmental stressors including ultraviolet radiation (UV). Here we present crystallographic structures evidencing a UV-induced crystallin redox switch mechanism. The room-temperature serial synchrotron crystallographic (SSX) structure of freshly prepared crystallin mutant (R36S) shows no post-translational modifications. After aging for nine months in the absence of light, a thiol-adduct (dithiothreitol) modifying surface cysteines is observed by low-dose SSX. This is shown to be UV-labile in an acutely light-exposed structure. This suggests a mechanism by which a major source of crystallin damage, UV, may also act as a rescuing factor in a finely balanced redox system.
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Apr 2024
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I24-Microfocus Macromolecular Crystallography
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Rachel
Bolton
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Moritz M.
Machelett
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Jack
Stubbs
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Danny
Axford
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Nicolas
Caramello
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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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I24-Microfocus Macromolecular Crystallography
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James
Birch
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Tristan O. C.
Kwan
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Peter J.
Judge
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Danny
Axford
,
Pierre
Aller
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Agata
Butryn
,
Rosana
Reis
,
Juan F.
Bada Juarez
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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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I24-Microfocus Macromolecular Crystallography
VMXm-Versatile Macromolecular Crystallography microfocus
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Jeremy R.
Keown
,
Adam D.
Crawshaw
,
Jose
Trincao
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Loic
Carrique
,
Richard J.
Gildea
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Sam
Horrell
,
Anna J.
Warren
,
Danny
Axford
,
Robin
Owen
,
Gwyndaf
Evans
,
Annie
Bézier
,
Peter
Metcalf
,
Jonathan M.
Grimes
Diamond Proposal Number(s):
[19946, 23570, 27314, 28534]
Open Access
Abstract: Infectious protein crystals are an essential part of the viral lifecycle for double-stranded DNA Baculoviridae and double-stranded RNA cypoviruses. These viral protein crystals, termed occlusion bodies or polyhedra, are dense protein assemblies that form a crystalline array, encasing newly formed virions. Here, using X-ray crystallography we determine the structure of a polyhedrin from Nudiviridae. This double-stranded DNA virus family is a sister-group to the baculoviruses, whose members were thought to lack occlusion bodies. The 70-year-old sample contains a well-ordered lattice formed by a predominantly α-helical building block that assembles into a dense, highly interconnected protein crystal. The lattice is maintained by extensive hydrophobic and electrostatic interactions, disulfide bonds, and domain switching. The resulting lattice is resistant to most environmental stresses. Comparison of this structure to baculovirus or cypovirus polyhedra shows a distinct protein structure, crystal space group, and unit cell dimensions, however, all polyhedra utilise common principles of occlusion body assembly.
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Jul 2023
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Mechanical Engineering
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Abstract: This paper describes collaborative work carried out between engineering and beamline teams at the Diamond Light Source to further develop an automated ‘tractor-beam’ acoustic levitation system for across beamline sample delivery. To date great success has been had utilising the system for protein crystallography which has been detailed in papers published in Nature Scientific reports [1] and SRI conference proceedings [2]. These papers describe levitation of protein crystals in their mother liquor, free from any physical support, and via inherent motions, enables successful room-temperature protein structure determination in relatively short time frames. The latest device described here in detail is highly portable measuring only 100 mm tall, provides unimpeded access to the sample, requires only a few Watts of power, and costs only $100 to build. Additionally, the latest system, has an integrated PolyPico acoustic ejector, which provides the ability to remotely load the levitator with pico litre volumes of liquid, including suspensions of protein crystals. Given that the system can process the sample into the beam line, the positional control and absence of physical support, mixing and light activated experiments are also explored. Thus far, device application has been tailored towards, and experimentation carried out on a Macromolecular Crystallography beamline (I24). Given the inherent flexibility of the system, we are beginning to explore the potential application of the device on other beamlines, including small molecule diffraction, SAXS/WAXS (small/wide angle X ray scattering and XES (X ray emission spectroscopy), VMXI and X-ray Free Electron Laser XFEL applications. These applications are explored and preliminary results given in this paper.
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Jun 2023
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I03-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
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Tamar
Skaist Mehlmam
,
Justin T.
Biel
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Syeda Maryam
Azeem
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Elliot R.
Nelson
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Sakib
Hossain
,
Louise
Dunnett
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Neil G.
Paterson
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Alice
Douangamath
,
Romain
Talon
,
Danny
Axford
,
Helen
Orins
,
Frank
Von Delft
,
Daniel A.
Keedy
Diamond Proposal Number(s):
[15751, 18340, 23570]
Open Access
Abstract: Much of our current understanding of how small-molecule ligands interact with proteins stems from X-ray crystal structures determined at cryogenic (cryo) temperature. For proteins alone, room-temperature (RT) crystallography can reveal previously hidden, biologically relevant alternate conformations. However, less is understood about how RT crystallography may impact the conformational landscapes of protein-ligand complexes. Previously, we showed that small-molecule fragments cluster in putative allosteric sites using a cryo crystallographic screen of the therapeutic target PTP1B (Keedy et al., 2018). Here, we have performed two RT crystallographic screens of PTP1B using many of the same fragments, representing the largest RT crystallographic screens of a diverse library of ligands to date, and enabling a direct interrogation of the effect of data collection temperature on protein-ligand interactions. We show that at RT, fewer ligands bind, and often more weakly – but with a variety of temperature-dependent differences, including unique binding poses, changes in solvation, new binding sites, and distinct protein allosteric conformational responses. Overall, this work suggests that the vast body of existing cryo-temperature protein-ligand structures may provide an incomplete picture, and highlights the potential of RT crystallography to help complete this picture by revealing distinct conformational modes of protein-ligand systems. Our results may inspire future use of RT crystallography to interrogate the roles of protein-ligand conformational ensembles in biological function.
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Mar 2023
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