|
Jaydeep
Patel
,
Adam
Round
,
Raphael
De Wijn
,
Mohammad
Vakili
,
Gabriele
Giovanetti
,
Diogo Filipe Monrroy Vilan E
Melo
,
Juncheng
E
,
Marcin
Sikorski
,
Jayanth
Koliyadu
,
Faisal H. M.
Koua
,
Tokushi
Sato
,
Adrian
Mancuso
,
Andrew
Peele
,
Brian
Abbey
Open Access
Abstract: Automated evaluation of optical microscopy images of liquid jets, commonly used for sample delivery at X-ray free-electron lasers (XFELs), enables real-time tracking of the jet position and liquid jet hit rates, defined here as the proportion of XFEL pulses intersecting with the liquid jet. This method utilizes machine vision for preprocessing, feature extraction, segmentation and jet detection as well as tracking to extract key physical characteristics (such as the jet angle) from optical microscopy images captured during experiments. To determine the effectiveness of these tools in monitoring jet stability and enhancing sample delivery efficiency, we conducted XFEL experiments with various sample compositions (pure water, buffer and buffer with crystals), nozzle designs and jetting conditions. We integrated our real-time analysis algorithm into the Karabo control system at the European XFEL. The results indicate that the algorithm performs well in monitoring the jet angle and provides a quantitative characterization of liquid jet stability through optical image analysis conducted during experiments.
|
Dec 2024
|
|
I19-Small Molecule Single Crystal Diffraction
|
Mariya
Aleksich
,
Yeongsu
Cho
,
Daniel W.
Paley
,
Maggie C.
Willson
,
Hawi N.
Nyiera
,
Patience A.
Kotei
,
Vanessa
Oklejas
,
David W.
Mittan-Moreau
,
Elyse A.
Schriber
,
Kara
Christensen
,
Ichiro
Inoue
,
Shigeki
Owada
,
Kensuke
Tono
,
Michihiro
Sugahara
,
Satomi
Inaba-Inoue
,
Mohammad
Vakili
,
Christopher J.
Milne
,
Fabio
Dallantonia
,
Dmitry
Khakhulin
,
Fernando
Ardana-Lamas
,
Frederico
Lima
,
Joana
Valerio
,
Huijong
Han
,
Tamires
Gallo
,
Hazem
Yousef
,
Oleksii
Turkot
,
Ivette J. Bermudez
Macias
,
Thomas
Kluyver
,
Philipp
Schmidt
,
Luca
Gelisio
,
Adam R.
Round
,
Yifeng
Jiang
,
Doriana
Vinci
,
Yohei
Uemura
,
Marco
Kloos
,
Adrian P.
Mancuso
,
Mark
Warren
,
Nicholas K.
Sauter
,
Jing
Zhao
,
Tess
Smidt
,
Heather J.
Kulik
,
Sahar
Sharifzadeh
,
Aaron S.
Brewster
,
J. Nathan
Hohman
Diamond Proposal Number(s):
[35300]
Abstract: X-ray free electron laser (XFEL) microcrystallography and synchrotron single-crystal crystallography are used to evaluate the role of organic substituent position on the optoelectronic properties of metal–organic chalcogenolates (MOChas). MOChas are crystalline 1D and 2D semiconducting hybrid materials that have varying optoelectronic properties depending on composition, topology, and structure. While MOChas have attracted much interest, small crystal sizes impede routine crystal structure determination. A series of constitutional isomers where the aryl thiol is functionalized by either methoxy or methyl ester are solved by small molecule serial femtosecond X-ray crystallography (smSFX) and single crystal rotational crystallography. While all the methoxy examples have a low quantum yield (0-1%), the methyl ester in the ortho position yields a high quantum yield of 22%. The proximity of the oxygen atoms to the silver inorganic core correlates to a considerable enhancement of quantum yield. Four crystal structures are solved at a resolution range of 0.8–1.0 Å revealing a collapse of the 2D topology for functional groups in the 2- and 3- positions, resulting in needle-like crystals. Further analysis using density functional theory (DFT) and many-body perturbation theory (MBPT) enables the exploration of complex excitonic phenomena within easily prepared material systems.
|
Dec 2024
|
|
B21-High Throughput SAXS
|
Patrick E.
Konold
,
Leonardo
Monrroy
,
Alfredo
Bellisario
,
Diogo
Filipe
,
Patrick
Adams
,
Roberto
Alvarez
,
Richard
Bean
,
Johan
Bielecki
,
Szabolcs
Bódizs
,
Gabriel
Ducrocq
,
Helmut
Grubmueller
,
Richard A.
Kirian
,
Marco
Kloos
,
Jayanath C. P.
Koliyadu
,
Faisal H. M.
Koua
,
Taru
Larkiala
,
Romain
Letrun
,
Fredrik
Lindsten
,
Michael
Maihöfer
,
Andrew
Martin
,
Petra
Mészáros
,
Jennifer
Mutisya
,
Amke
Nimmrich
,
Kenta
Okamoto
,
Adam
Round
,
Tokushi
Sato
,
Joana
Valerio
,
Daniel
Westphal
,
August
Wollter
,
Tej Varma
Yenupuri
,
Tong
You
,
Filipe
Maia
,
Sebastian
Westenhoff
Open Access
Abstract: Detecting microsecond structural perturbations in biomolecules has wide relevance in biology, chemistry and medicine. Here we show how MHz repetition rates at X-ray free-electron lasers can be used to produce microsecond time-series of protein scattering with exceptionally low noise levels of 0.001%. We demonstrate the approach by examining Jɑ helix unfolding of a light-oxygen-voltage photosensory domain. This time-resolved acquisition strategy is easy to implement and widely applicable for direct observation of structural dynamics of many biochemical processes.
|
Jul 2024
|
|
|
Mohammad
Vakili
,
Huijong
Han
,
Christina
Schmidt
,
Agnieszka
Wrona
,
Marco
Kloos
,
Iñaki
De Diego
,
Katerina
Dörner
,
Tian
Geng
,
Chan
Kim
,
Faisal H. M.
Koua
,
Diogo V. M.
Melo
,
Mathieu
Rappas
,
Adam
Round
,
Ekaterina
Round
,
Marcin
Sikorski
,
Joana
Valerio
,
Tiankun
Zhou
,
Kristina
Lorenzen
,
Joachim
Schulz
Open Access
Abstract: Time-resolved crystallography enables the visualization of protein molecular motion during a reaction. Although light is often used to initiate reactions in time-resolved crystallography, only a small number of proteins can be activated by light. However, many biological reactions can be triggered by the interaction between proteins and ligands. The sample delivery method presented here uses a mix-and-extrude approach based on 3D-printed microchannels in conjunction with a micronozzle. The diffusive mixing enables the study of the dynamics of samples in viscous media. The device design allows mixing of the ligands and protein crystals in 2 to 20 s. The device characterization using a model system (fluorescence quenching of iq-mEmerald proteins by copper ions) demonstrated that ligand and protein crystals, each within lipidic cubic phase, can be mixed efficiently. The potential of this approach for time-resolved membrane protein crystallography to support the development of new drugs is discussed.
|
Aug 2023
|
|
|
Mariya
Aleksich
,
Daniel W.
Paley
,
Elyse A.
Schriber
,
Will
Linthicum
,
Vanessa
Oklejas
,
David W.
Mittan-Moreau
,
Ryan P.
Kelly
,
Patience A.
Kotei
,
Anita
Ghodsi
,
Raymond G.
Sierra
,
Andrew
Aquila
,
Frédéric
Poitevin
,
Johannes P.
Blaschke
,
Mohammad
Vakili
,
Christopher J.
Milne
,
Fabio
Dall’antonia
,
Dmitry
Khakhulin
,
Fernando
Ardana-Lamas
,
Frederico
Lima
,
Joana
Valerio
,
Huijong
Han
,
Tamires
Gallo
,
Hazem
Yousef
,
Oleksii
Turkot
,
Ivette J.
Bermudez Macias
,
Thomas
Kluyver
,
Philipp
Schmidt
,
Luca
Gelisio
,
Adam R.
Round
,
Yifeng
Jiang
,
Doriana
Vinci
,
Yohei
Uemura
,
Marco
Kloos
,
Mark
Hunter
,
Adrian P.
Mancuso
,
Bryan D.
Huey
,
Lucas R.
Parent
,
Nicholas K.
Sauter
,
Aaron S.
Brewster
,
J. Nathan
Hohman
Abstract: New synthetic hybrid materials and their increasing complexity have placed growing demands on crystal growth for single-crystal X-ray diffraction analysis. Unfortunately, not all chemical systems are conducive to the isolation of single crystals for traditional characterization. Here, small-molecule serial femtosecond crystallography (smSFX) at atomic resolution (0.833 Å) is employed to characterize microcrystalline silver n-alkanethiolates with various alkyl chain lengths at X-ray free electron laser facilities, resolving long-standing controversies regarding the atomic connectivity and odd–even effects of layer stacking. smSFX provides high-quality crystal structures directly from the powder of the true unknowns, a capability that is particularly useful for systems having notoriously small or defective crystals. We present crystal structures of silver n-butanethiolate (C4), silver n-hexanethiolate (C6), and silver n-nonanethiolate (C9). We show that an odd–even effect originates from the orientation of the terminal methyl group and its role in packing efficiency. We also propose a secondary odd–even effect involving multiple mosaic blocks in the crystals containing even-numbered chains, identified by selected-area electron diffraction measurements. We conclude with a discussion of the merits of the synthetic preparation for the preparation of microdiffraction specimens and compare the long-range order in these crystals to that of self-assembled monolayers.
|
Jul 2023
|
|
|
Austin
Echelmeier
,
Jorvani
Cruz Villarreal
,
Marc
Messerschmidt
,
Daihyun
Kim
,
Jesse D.
Coe
,
Darren
Thifault
,
Sabine
Botha
,
Ana
Egatz-Gomez
,
Sahir
Gandhi
,
Gerrit
Brehm
,
Chelsie E.
Conrad
,
Debra T.
Hansen
,
Caleb
Madsen
,
Saša
Bajt
,
J. Domingo
Meza-Aguilar
,
Dominik
Oberthuer
,
Max O.
Wiedorn
,
Holger
Fleckenstein
,
Derek
Mendez
,
Juraj
Knoška
,
Jose M.
Martin-Garcia
,
Hao
Hu
,
Stella
Lisova
,
Aschkai
Allahgoli
,
Yaroslav
Gevorkov
,
Kartik
Ayyer
,
Steve
Aplin
,
Helen M.
Ginn
,
Heinz
Graafsma
,
Andrew J.
Morgan
,
Dominic
Greiffenberg
,
Alexander
Klujev
,
Torsten
Laurus
,
Jennifer
Poehlsen
,
Ulrich
Trunk
,
Davide
Mezza
,
Bernd
Schmitt
,
Manuela
Kuhn
,
Raimund
Fromme
,
Jolanta
Sztuk-Dambietz
,
Natascha
Raab
,
Steffen
Hauf
,
Alessandro
Silenzi
,
Thomas
Michelat
,
Chen
Xu
,
Cyril
Danilevski
,
Andrea
Parenti
,
Leonce
Mekinda
,
Britta
Weinhausen
,
Grant
Mills
,
Patrik
Vagovic
,
Yoonhee
Kim
,
Henry
Kirkwood
,
Richard
Bean
,
Johan
Bielecki
,
Stephan
Stern
,
Klaus
Giewekemeyer
,
Adam
Round
,
Joachim
Schulz
,
Katerina
Dörner
,
Thomas D.
Grant
,
Valerio
Mariani
,
Anton
Barty
,
Adrian P.
Mancuso
,
Uwe
Weierstall
,
John C. H.
Spence
,
Henry N.
Chapman
,
Nadia
Zatsepin
,
Petra
Fromme
,
Richard A.
Kirian
,
Alexandra
Ros
Open Access
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.
|
Sep 2020
|
|
I03-Macromolecular Crystallography
|
Max O.
Wiedorn
,
Dominik
Oberthuer
,
Richard
Bean
,
Robin
Schubert
,
Nadine
Werner
,
Brian
Abbey
,
Martin
Aepfelbacher
,
Luigi
Adriano
,
Aschkan
Allahgholi
,
Nasser
Al-Qudami
,
Jakob
Andreasson
,
Steve
Aplin
,
Salah
Awel
,
Kartik
Ayyer
,
Saša
Bajt
,
Imrich
Barák
,
Sadia
Bari
,
Johan
Bielecki
,
Sabine
Botha
,
Djelloul
Boukhelef
,
Wolfgang
Brehm
,
Sandor
Brockhauser
,
Igor
Cheviakov
,
Matthew A.
Coleman
,
Francisco
Cruz-Mazo
,
Cyril
Danilevski
,
Connie
Darmanin
,
R. Bruce
Doak
,
Martin
Domaracky
,
Katerina
Dörner
,
Yang
Du
,
Hans
Fangohr
,
Holger
Fleckenstein
,
Matthias
Frank
,
Petra
Fromme
,
Alfonso M.
Gañán-Calvo
,
Yaroslav
Gevorkov
,
Klaus
Giewekemeyer
,
Helen Mary
Ginn
,
Heinz
Graafsma
,
Rita
Graceffa
,
Dominic
Greiffenberg
,
Lars
Gumprecht
,
Peter
Göttlicher
,
Janos
Hajdu
,
Steffen
Hauf
,
Michael
Heymann
,
Susannah
Holmes
,
Daniel A.
Horke
,
Mark S.
Hunter
,
Siegfried
Imlau
,
Alexander
Kaukher
,
Yoonhee
Kim
,
Alexander
Klyuev
,
Juraj
Knoška
,
Bostjan
Kobe
,
Manuela
Kuhn
,
Christopher
Kupitz
,
Jochen
Küpper
,
Janine Mia
Lahey-Rudolph
,
Torsten
Laurus
,
Karoline
Le Cong
,
Romain
Letrun
,
P. Lourdu
Xavier
,
Luis
Maia
,
Filipe R. N. C.
Maia
,
Valerio
Mariani
,
Marc
Messerschmidt
,
Markus
Metz
,
Davide
Mezza
,
Thomas
Michelat
,
Grant
Mills
,
Diana C. F.
Monteiro
,
Andrew
Morgan
,
Kerstin
Mühlig
,
Anna
Munke
,
Astrid
Münnich
,
Julia
Nette
,
Keith A.
Nugent
,
Theresa
Nuguid
,
Allen M.
Orville
,
Suraj
Pandey
,
Gisel
Pena
,
Pablo
Villanueva-Perez
,
Jennifer
Poehlsen
,
Gianpietro
Previtali
,
Lars
Redecke
,
Winnie Maria
Riekehr
,
Holger
Rohde
,
Adam
Round
,
Tatiana
Safenreiter
,
Iosifina
Sarrou
,
Tokushi
Sato
,
Marius
Schmidt
,
Bernd
Schmitt
,
Robert
Schönherr
,
Joachim
Schulz
,
Jonas A.
Sellberg
,
M. Marvin
Seibert
,
Carolin
Seuring
,
Megan L.
Shelby
,
Robert L.
Shoeman
,
Marcin
Sikorski
,
Alessandro
Silenzi
,
Claudiu A.
Stan
,
Xintian
Shi
,
Stephan
Stern
,
Jola
Sztuk-Dambietz
,
Janusz
Szuba
,
Aleksandra
Tolstikova
,
Martin
Trebbin
,
Ulrich
Trunk
,
Patrik
Vagovic
,
Thomas
Ve
,
Britta
Weinhausen
,
Thomas A.
White
,
Krzysztof
Wrona
,
Chen
Xu
,
Oleksandr
Yefanov
,
Nadia
Zatsepin
,
Jiaguo
Zhang
,
Markus
Perbandt
,
Adrian P.
Mancuso
,
Christian
Betzel
,
Henry
Chapman
,
Anton
Barty
Open Access
Abstract: The new European X-ray Free-Electron Laser is the first X-ray free-electron laser capable of delivering X-ray pulses with a megahertz inter-pulse spacing, more than four orders of magnitude higher than previously possible. However, to date, it has been unclear whether it would indeed be possible to measure high-quality diffraction data at megahertz pulse repetition rates. Here, we show that high-quality structures can indeed be obtained using currently available operating conditions at the European XFEL. We present two complete data sets, one from the well-known model system lysozyme and the other from a so far unknown complex of a β-lactamase from K. pneumoniae involved in antibiotic resistance. This result opens up megahertz serial femtosecond crystallography (SFX) as a tool for reliable structure determination, substrate screening and the efficient measurement of the evolution and dynamics of molecular structures using megahertz repetition rate pulses available at this new class of X-ray laser source.
|
Oct 2018
|
|
I04-1-Macromolecular Crystallography (fixed wavelength)
I04-Macromolecular Crystallography
|
Diamond Proposal Number(s):
[59966, 5928]
Open Access
Abstract: Asymmetric cell divisions balance stem cell proliferation and differentiation to sustain tissue morphogenesis and homeostasis. During asymmetric divisions, fate determinants and niche contacts segregate unequally between daughters, but little is known on how this is achieved mechanistically. In Drosophila neuroblasts and murine mammary stem cells, the association of the spindle orientation protein LGN with the stem cell adaptor Inscuteable has been connected to asymmetry. Here we report the crystal structure of Drosophila LGN in complex with the asymmetric domain of Inscuteable, which reveals a tetrameric arrangement of intertwined molecules. We show that Insc:LGN tetramers constitute stable cores of Par3–Insc-LGN-GαiGDP complexes, which cannot be dissociated by NuMA. In mammary stem cells, the asymmetric domain of Insc bound to LGN:GαiGDP suffices to drive asymmetric fate, and reverts aberrant symmetric divisions induced by p53 loss. We suggest a novel role for the Insc-bound pool of LGN acting independently of microtubule motors to promote asymmetric fate specification.
|
Mar 2018
|
|
I04-1-Macromolecular Crystallography (fixed wavelength)
I04-Macromolecular Crystallography
|
Open Access
Abstract: The sarcomeric cytoskeleton is a network of modular proteins that integrate mechanical and signaling roles. Obscurin, or its homolog obscurin-like-1, bridges the giant ruler titin and the myosin crosslinker myomesin at the M-band. Yet, the molecular mechanisms underlying the physical obscurin(-like-1):myomesin connection, important for mechanical integrity of the M-band, remained elusive. Here, using a combination of structural, cellular, and single-molecule force spectroscopy techniques, we decode the architectural and functional determinants defining the obscurin(-like-1):myomesin complex. The crystal structure reveals a trans-complementation mechanism whereby an incomplete immunoglobulin-like domain assimilates an isoform-specific myomesin interdomain sequence. Crucially, this unconventional architecture provides mechanical stability up to forces of ∼135 pN. A cellular competition assay in neonatal rat cardiomyocytes validates the complex and provides the rationale for the isoform specificity of the interaction. Altogether, our results reveal a novel binding strategy in sarcomere assembly, which might have implications on muscle nanomechanics and overall M-band organization.
|
Jan 2017
|
|
B21-High Throughput SAXS
I02-Macromolecular Crystallography
I04-Macromolecular Crystallography
|
Diamond Proposal Number(s):
[7641]
Open Access
Abstract: Filamentous plant pathogens deliver effector proteins to host cells to promote infection. The Phytophthora infestans RXLR-type effector PexRD54 binds potato ATG8 via its ATG8-family interacting motif (AIM) and perturbs host selective autophagy. However, the structural basis of this interaction remains unknown. Here we define the crystal structure of PexRD54, which comprises a modular architecture including five tandem repeat domains, with the AIM sequence presented at the disordered C-terminus. To determine the interface between PexRD54 and ATG8, we solved the crystal structure of potato ATG8CL in complex with a peptide comprising the effector’s AIM sequence, and established a model of the full-length PexRD54/ATG8CL complex using small angle X-ray scattering. Structure-informed deletion of the PexRD54 tandem domains reveals retention of ATG8CL binding in vitro and in planta. This study offers new insights into structure/function relationships of oomycete RXLR effectors and how these proteins engage with host cell targets to promote disease.
|
Jul 2016
|
|