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Open Access
Abstract: The crystallization of recombinant proteins in living cells is an exciting new approach in structural biology. Recent success has highlighted the need for fast and efficient diffraction data collection, optimally directly exposing intact crystal-containing cells to the X-ray beam, thus protecting the in cellulo crystals from environmental challenges. Serial femtosecond crystallography (SFX) at free-electron lasers (XFELs) allows the collection of detectable diffraction even from tiny protein crystals, but requires very fast sample exchange to utilize each XFEL pulse. Here, an efficient approach is presented for high-resolution structure elucidation using serial femtosecond in cellulo diffraction of micometre-sized crystals of the protein HEX-1 from the fungus Neurospora crassa on a fixed target. Employing the fast and highly accurate Roadrunner II translation-stage system allowed efficient raster scanning of the pores of micro-patterned, single-crystalline silicon chips loaded with living, crystal-containing insect cells. Compared with liquid-jet and LCP injection systems, the increased hit rates of up to 30% and reduced background scattering enabled elucidation of the HEX-1 structure. Using diffraction data from only a single chip collected within 12 min at the Linac Coherent Light Source, a 1.8 Å resolution structure was obtained with significantly reduced sample consumption compared with previous SFX experiments using liquid-jet injection. This HEX-1 structure is almost superimposable with that previously determined using synchrotron radiation from single HEX-1 crystals grown by sitting-drop vapour diffusion, validating the approach. This study demonstrates that fixed-target SFX using micro-patterned silicon chips is ideally suited for efficient in cellulo diffraction data collection using living, crystal-containing cells, and offers huge potential for the straightforward structure elucidation of proteins that form intracellular crystals at both XFELs and synchrotron sources.
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Jul 2021
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Sebastian
Gunther
,
Patrick Y. A.
Reinke
,
Yaiza
Fernández-García
,
Julia
Lieske
,
Thomas J.
Lane
,
Helen M.
Ginn
,
Faisal H. M.
Koua
,
Christiane
Ehrt
,
Wiebke
Ewert
,
Dominik
Oberthuer
,
Oleksandr
Yefanov
,
Susanne
Meier
,
Kristina
Lorenzen
,
Boris
Krichel
,
Janine-Denise
Kopicki
,
Luca
Gelisio
,
Wolfgang
Brehm
,
Ilona
Dunkel
,
Brandon
Seychell
,
Henry
Gieseler
,
Brenna
Norton-Baker
,
Beatriz
Escudero-Pérez
,
Martin
Domaracky
,
Sofiane
Saouane
,
Alexandra
Tolstikova
,
Thomas A.
White
,
Anna
Hänle
,
Michael
Groessler
,
Holger
Fleckenstein
,
Fabian
Trost
,
Marina
Galchenkova
,
Yaroslav
Gevorkov
,
Chufeng
Li
,
Salah
Awel
,
Ariana
Peck
,
Miriam
Barthelmess
,
Frank
Schluenzen
,
Paulraj
Lourdu Xavier
,
Nadine
Werner
,
Hina
Andaleeb
,
Najeeb
Ullah
,
Sven
Falke
,
Vasundara
Srinivasan
,
Bruno Alves
França
,
Martin
Schwinzer
,
Hévila
Brognaro
,
Cromarte
Rogers
,
Diogo
Melo
,
Joanna J.
Zaitseva-Doyle
,
Juraj
Knoska
,
Gisel E.
Peña-Murillo
,
Aida Rahmani
Mashhour
,
Vincent
Hennicke
,
Pontus
Fischer
,
Johanna
Hakanpää
,
Jan
Meyer
,
Philip
Gribbon
,
Bernhard
Ellinger
,
Maria
Kuzikov
,
Markus
Wolf
,
Andrea R.
Beccari
,
Gleb
Bourenkov
,
David
Von Stetten
,
Guillaume
Pompidor
,
Isabel
Bento
,
Saravanan
Panneerselvam
,
Ivars
Karpics
,
Thomas R.
Schneider
,
Maria Marta
Garcia-Alai
,
Stephan
Niebling
,
Christian
Günther
,
Christina
Schmidt
,
Robin
Schubert
,
Huijong
Han
,
Juliane
Boger
,
Diana C. F.
Monteiro
,
Linlin
Zhang
,
Xinyuanyuan
Sun
,
Jonathan
Pletzer-Zelgert
,
Jan
Wollenhaupt
,
Christian G.
Feiler
,
Manfred S.
Weiss
,
Eike-Christian
Schulz
,
Pedram
Mehrabi
,
Katarina
Karničar
,
Aleksandra
Usenik
,
Jure
Loboda
,
Henning
Tidow
,
Ashwin
Chari
,
Rolf
Hilgenfeld
,
Charlotte
Uetrecht
,
Russell
Cox
,
Andrea
Zaliani
,
Tobias
Beck
,
Matthias
Rarey
,
Stephan
Günther
,
Dusan
Turk
,
Winfried
Hinrichs
,
Henry N.
Chapman
,
Arwen R.
Pearson
,
Christian
Betzel
,
Alke
Meents
Open Access
Abstract: The coronavirus disease (COVID-19) caused by SARS-CoV-2 is creating tremendous human suffering. To date, no effective drug is available to directly treat the disease. In a search for a drug against COVID-19, we have performed a high-throughput X-ray crystallographic screen of two repurposing drug libraries against the SARS-CoV-2 main protease (Mpro), which is essential for viral replication. In contrast to commonly applied X-ray fragment screening experiments with molecules of low complexity, our screen tested already approved drugs and drugs in clinical trials. From the three-dimensional protein structures, we identified 37 compounds that bind to Mpro. In subsequent cell-based viral reduction assays, one peptidomimetic and six non-peptidic compounds showed antiviral activity at non-toxic concentrations. We identified two allosteric binding sites representing attractive targets for drug development against SARS-CoV-2.
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Apr 2021
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Carolin
Seuring
,
Kartik
Ayyer
,
Eleftheria
Filippaki
,
Miriam
Barthelmess
,
Jean-Nicolas
Longchamp
,
Philippe
Ringler
,
Tommaso
Pardini
,
David H.
Wojtas
,
Matthew A.
Coleman
,
Katerina
Dörner
,
Silje
Fuglerud
,
Greger
Hammarin
,
Birgit
Habenstein
,
Annette E.
Langkilde
,
Antoine
Loquet
,
Alke
Meents
,
Roland
Riek
,
Henning
Stahlberg
,
Sébastien
Boutet
,
Mark S.
Hunter
,
Jason
Koglin
,
Mengning
Liang
,
Helen M.
Ginn
,
Rick P.
Millane
,
Matthias
Frank
,
Anton
Barty
,
Henry N.
Chapman
Open Access
Abstract: Here we present a new approach to diffraction imaging of amyloid fibrils, combining a free-standing graphene support and single nanofocused X-ray pulses of femtosecond duration from an X-ray free-electron laser. Due to the very low background scattering from the graphene support and mutual alignment of filaments, diffraction from tobacco mosaic virus (TMV) filaments and amyloid protofibrils is obtained to 2.7 Å and 2.4 Å resolution in single diffraction patterns, respectively. Some TMV diffraction patterns exhibit asymmetry that indicates the presence of a limited number of axial rotations in the XFEL focus. Signal-to-noise levels from individual diffraction patterns are enhanced using computational alignment and merging, giving patterns that are superior to those obtainable from synchrotron radiation sources. We anticipate that our approach will be a starting point for further investigations into unsolved structures of filaments and other weakly scattering objects.
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May 2018
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Philip
Roedig
,
Helen M.
Ginn
,
Tim
Pakendorf
,
Geoff
Sutton
,
Karl
Harlos
,
Thomas S.
Walter
,
Jan
Meyer
,
Pontus
Fischer
,
Ramona
Duman
,
Ismo
Vartiainen
,
Bernd
Reime
,
Martin
Warmer
,
Aaron S.
Brewster
,
Iris D.
Young
,
Tara
Michels-Clark
,
Nicholas K.
Sauter
,
Abhay
Kotecha
,
James
Kelly
,
David J.
Rowlands
,
Marcin
Sikorsky
,
Silke
Nelson
,
Daniel S.
Damiani
,
Roberto
Alonso-Mori
,
Jingshan
Ren
,
Elizabeth E.
Fry
,
Christian
David
,
David I.
Stuart
,
Armin
Wagner
,
Alke
Meents
Abstract: We report a method for serial X-ray crystallography at X-ray free-electron lasers (XFELs), which allows for full use of the current 120-Hz repetition rate of the Linear Coherent Light Source (LCLS). Using a micropatterned silicon chip in combination with the high-speed Roadrunner goniometer for sample delivery, we were able to determine the crystal structures of the picornavirus bovine enterovirus 2 (BEV2) and the cytoplasmic polyhedrosis virus type 18 polyhedrin, with total data collection times of less than 14 and 10 min, respectively. Our method requires only micrograms of sample and should therefore broaden the applicability of serial femtosecond crystallography to challenging projects for which only limited sample amounts are available. By synchronizing the sample exchange to the XFEL repetition rate, our method allows for most efficient use of the limited beam time available at XFELs and should enable a substantial increase in sample throughput at these facilities.
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Jun 2017
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Open Access
Abstract: The indexing methods currently used for serial femtosecond crystallography were originally developed for experiments in which crystals are rotated in the X-ray beam, providing significant three-dimensional information. On the other hand, shots from both X-ray free-electron lasers and serial synchrotron crystallography experiments are still images, in which the few three-dimensional data available arise only from the curvature of the Ewald sphere. Traditional synchrotron crystallography methods are thus less well suited to still image data processing. Here, a new indexing method is presented with the aim of maximizing information use from a still image given the known unit-cell dimensions and space group. Efficacy for cubic, hexagonal and orthorhombic space groups is shown, and for those showing some evidence of diffraction the indexing rate ranged from 90% (hexagonal space group) to 151% (cubic space group). Here, the indexing rate refers to the number of lattices indexed per image.
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Aug 2016
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I03-Macromolecular Crystallography
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Abstract: Recent success at X-ray free-electron lasers has led to serial crystallography experiments staging a comeback at synchrotron sources as well. With crystal lifetimes typically in the millisecond range and the latest-generation detector technologies with high framing rates up to 1 kHz, fast sample exchange has become the bottleneck for such experiments. A micro-patterned chip has been developed from single-crystalline silicon, which acts as a sample holder for up to several thousand microcrystals at a very low background level. The crystals can be easily loaded onto the chip and excess mother liquor can be efficiently removed. Dehydration of the crystals is prevented by keeping them in a stream of humidified air during data collection. Further sealing of the sample holder, for example with Kapton, is not required. Room-temperature data collection from insulin crystals loaded onto the chip proves the applicability of the chip for macromolecular crystallography. Subsequent structure refinements reveal no radiation-damage-induced structural changes for insulin crystals up to a dose of 565.6 kGy, even though the total diffraction power of the crystals has on average decreased to 19.1% of its initial value for the same dose. A decay of the diffracting power by half is observed for a dose of D1/2 = 147.5 ± 19.1 kGy, which is about 1/300 of the dose before crystals show a similar decay at cryogenic temperatures.
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Jun 2016
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I02-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
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Philip
Roedig
,
Ismo
Vartiainen
,
Ramona
Duman
,
S.
Panneerselvam
,
N.
Stübe
,
Olga
Lorbeer
,
Martin
Warmer
,
Geoff
Sutton
,
Dave
Stuart
,
E.
Weckert
,
C.
David
,
Armin
Wagner
,
Alke
Meents
Open Access
Abstract: At low emittance synchrotron sources it has become possible to perform structure determinations from the measurement of multiple microcrystals which were previously considered too small for diffraction experiments. Conventional mounting techniques do not fulfill the requirements of these new experiments. They significantly contribute to background scattering and it is difficult to locate the crystals, making them incompatible with automated serial crystallography. We have developed a micro-fabricated sample holder from single crystalline silicon with micropores, which carries up to thousands of crystals and significantly reduces the background scattering level. For loading, the suspended microcrystals are pipetted onto the chip and excess mother liquor is subsequently soaked off through the micropores. Crystals larger than the pore size are retained and arrange themselves according to the micropore pattern. Using our chip we were able to collect 1.5 Å high resolution diffraction data from protein microcrystals with sizes of 4 micrometers and smaller.
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May 2015
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I24-Microfocus Macromolecular Crystallography
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Abstract: Successful cryogenic X-ray structure determination from a single high-pressure-frozen bovine enterovirus 2 crystal is reported. The presented high-pressure-freezing procedure is based on a commercially available device and allows the cryocooling of macromolecular crystals directly in their mother liquor without the time- and crystal-consuming search for optimal cryoconditions. The method is generally applicable and will allow cryogenic data collection from all types of macromolecular crystals.
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Feb 2013
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I04-1-Macromolecular Crystallography (fixed wavelength)
I24-Microfocus Macromolecular Crystallography
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Abstract: High-pressure freezing (HPF) is a method which allows sample vitrification without cryoprotectants. In the present work, protein crystals were cooled to cryogenic temperatures at a pressure of 210 MPa. In contrast to other HPF methods published to date in the field of cryocrystallography, this protocol involves rapid sample cooling using a standard HPF device. The fast cooling rates allow HPF of protein crystals directly in their mother liquor without the need for cryoprotectants or external reagents. HPF was first attempted with hen egg-white lysozyme and cubic insulin crystals, yielding good to excellent diffraction quality. Non-cryoprotected crystals of the membrane protein photosystem II have been successfully cryocooled for the first time. This indicates that the presented HPF method is well suited to the vitrification of challenging systems with large unit cells and weak crystal contacts.
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Apr 2012
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Abstract: Radiation damage is the major impediment for obtaining structural information from biological samples by using ionizing radiation such as x-rays or electrons. The knowledge of underlying processes especially at cryogenic temperatures is still fragmentary, and a consistent mechanism has not been found yet. By using a combination of single-crystal x-ray diffraction, small-angle scattering, and qualitative and quantitative radiolysis experiments, we show that hydrogen gas, formed inside the sample during irradiation, rather than intramolecular bond cleavage between non-hydrogen atoms, is mainly responsible for the loss of high-resolution information and contrast in diffraction experiments and microscopy. The experiments that are presented in this paper cover a temperature range between 5 and 160 K and reveal that the commonly used temperature in x-ray crystallography of 100 K is not optimal in terms of minimizing radiation damage and thereby increasing the structural information obtainable in a single experiment. At 50 K, specific radiation damage to disulfide bridges is reduced by a factor of 4 compared to 100 K, and samples can tolerate a factor of 2.6 and 3.9 higher dose, as judged by the increase of Rfree values of elastase and cubic insulin crystals, respectively.
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Jan 2010
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