FragMAX: the fragment-screening platform at the MAX IV Laboratory

Lima, G.M.A.; Talibov, V.; Jagudin, E.; Sele, C.; Nyblom, Maria; Knecht, Wolfgang; Logan, D.T.; Sjögren, Tove; Muêller, U.

doi:10.1107/s205979832000889x

Cited by 43 publications

(40 citation statements)

References 23 publications

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“…For the fragment screening of IMT M pro , we used the settled plates fragment libraries of FragMAXlib (Talibov et al, to be published) and F2XEntry (35,36). In those plates, the content of each drop-well was resuspended in 1.0 µL of 0.1 M MES pH 6.7, 5% DMSO (v/v), 8% PEG 4,000 (w/v), 30% PEG 400 (w/v), and crystals were added afterwards.…”

Section: Fragment Screening Of Imt M Promentioning

confidence: 99%

A Crystallographic Snapshot of SARS-CoV-2 Main Protease Maturation Process

Noske

Nakamura

Gawriljuk

et al. 2020

Preprint

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SARS-CoV-2 is the causative agent of COVID-19. The dimeric form of the viral main protease is responsible for the cleavage of the viral polyprotein in 11 sites, including its own N and C-terminus. Although several mechanisms of self-cleavage had been proposed for SARS-CoV, the lack of structural information for each step is a setback to the understanding of this process. Herein, we used X-ray crystallography to characterize an immature form of the main protease, which revealed major conformational changes in the positioning of domain-three over the active site, hampering the dimerization and diminishing its activity. We propose that this form preludes the cis-cleavage of N-terminal residues within the dimer, leading to the mature active site. Using fragment screening, we probe new cavities in this form which can be used to guide therapeutic development. Furthermore, we characterized a serine site-directed mutant of the main protease bound to its endogenous N and C-terminal residues during the formation of the tetramer. This quaternary form is also present in solution, suggesting a transitional state during the C-terminal trans-cleavage. This data sheds light in the structural modifications of the SARS-CoV-2 main protease during maturation, which can guide the development of new inhibitors targeting its intermediary states.

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Section: Fragment Screening Of Imt M Promentioning

confidence: 99%

A Crystallographic Snapshot of SARS-CoV-2 Main Protease Maturation Process

Noske

Nakamura

Gawriljuk

et al. 2020

Preprint

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“…In contrast, synchrotrons achieved order of magnitude improvements in robotics, automation 9 , 10 , 11 and detector technology 12 , 13 , and combined with equally accelerated computing power and algorithms of data processing 14 , 15 , 16 , complete diffraction datasets can be measured in seconds and large numbers of them entirely unattended, as pioneered at LillyCAT 7 and later MASSIF 17 , 18 (European Synchrotron Radiation Facility (ESRF)). This led synchrotrons to develop highly streamlined platforms to make crystal-based fragment screening as primary screen accessible to a wide user community (XChem at Diamond; CrystalDirect at EMBL/ESRF 19 ; BESSY at Helmholtz-Zentrum Berlin 20 ; FragMax at MaxIV 21 ). This paper documents the protocols that constitute the XChem platform for fragment screening by X-ray crystallography, from sample preparation to the final structural results of 3D-modeled hits.…”

Section: Introductionmentioning

confidence: 99%

“…The pipeline (Figure 1) required developing new approaches to crystal identification 22 , soaking 23 , and harvesting 24 , as well as data management software 25 and an algorithmic approach to identifying fragments 26 that is now widely used in the community. The crystal harvesting technology is now sold by a vendor (see allowed other synchrotrons to adapt them to set up equivalent platforms 21 . Ongoing projects address data analysis, model completion, and data dissemination through the Fragalysis platform 27 .…”

Section: Introductionmentioning

confidence: 99%

Achieving Efficient Fragment Screening at XChem Facility at Diamond Light Source

Douangamath

Powell

Fearon

et al. 2021

JoVE

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In fragment-based drug discovery, hundreds or often thousands of compounds smaller than ~300 Da are tested against the protein of interest to identify chemical entities that can be developed into potent drug candidates. Since the compounds are small, interactions are weak, and the screening method must therefore be highly sensitive; moreover, structural information tends to be crucial for elaborating these hits into leadlike compounds. Therefore, protein crystallography has always been a gold-standard technique, yet historically too challenging to find widespread use as a primary screen.Initial XChem experiments were demonstrated in 2014 and then trialed with academic and industrial collaborators to validate the process. Since then, a large research effort and significant beamtime have streamlined sample preparation, developed a fragment library with rapid follow-up possibilities, automated and improved the capability of I04-1 beamline for unattended data collection, and implemented new tools for data management, analysis and hit identification.XChem is now a facility for large-scale crystallographic fragment screening, supporting the entire crystals-to-deposition process, and accessible to academic and industrial users worldwide. The peer-reviewed academic user program has been actively developed since 2016, to accommodate projects from as broad a scientific scope as possible, including well-validated as well as exploratory projects. Academic access is allocated through biannual calls for peer-reviewed proposals, and proprietary work is arranged by Diamond's Industrial Liaison group. This workflow has already been routinely applied to over a hundred targets from diverse therapeutic areas, and effectively identifies weak binders (1%-30% hit rate), which both serve as high-quality Copyright © 2021 JoVE Creative Commons Attribution-NonCommercial-NoDerivs 3.0 Unported License jove.com May 2021 • 171 • e62414 • Page 2 of 22starting points for compound design and provide extensive structural information on binding sites. The resilience of the process was demonstrated by continued screening of SARS-CoV-2 targets during the COVID-19 pandemic, including a 3-week turnaround for the main protease.

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“…post-crystallization treatments where the crystals remain inside the drop, can involve heavy-metal derivatization for phasing experiments, dehydration to improve diffraction quality or soaking experiments with ligands (Heras & Martin, 2005;Rould, 2007). In particular for drug discovery projects the crystallographic screening of small-molecule compounds called fragments has recently been established as a high-throughput technique that requires the harvesting and preparation of a very large number of samples (Schiebel et al, 2016;Krojer et al, 2017;Lima et al, 2020).…”

Section: Introductionmentioning

confidence: 99%

Facilitated crystal handling using a simple device for evaporation reduction in microtiter plates

Barthel¹,

Huschmann²,

Wallacher³

et al. 2021

J Appl Cryst

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In the past two decades, most of the steps in a macromolecular crystallography experiment have undergone tremendous development with respect to speed, feasibility and increase of throughput. The part of the experimental workflow that is still a bottleneck, despite significant efforts, involves the manipulation and harvesting of the crystals for the diffraction experiment. Here, a novel low-cost device is presented that functions as a cover for 96-well crystallization plates. This device enables access to the individual experiments one at a time by its movable parts, while minimizing evaporation of all other experiments of the plate. In initial tests, drops of many typically used crystallization cocktails could be successfully protected for up to 6 h. Therefore, the manipulation and harvesting of crystals is straightforward for the experimenter, enabling significantly higher throughput. This is useful for many macromolecular crystallography experiments, especially multi-crystal screening campaigns.

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FragMAX: the fragment-screening platform at the MAX IV Laboratory

Cited by 43 publications

References 23 publications

A Crystallographic Snapshot of SARS-CoV-2 Main Protease Maturation Process

A Crystallographic Snapshot of SARS-CoV-2 Main Protease Maturation Process

Achieving Efficient Fragment Screening at XChem Facility at Diamond Light Source

Facilitated crystal handling using a simple device for evaporation reduction in microtiter plates

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