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Open Access
Abstract: Research towards using X-ray free-electron laser (XFEL) data to solve structures using experimental phasing methods such as sulfur single-wavelength anomalous dispersion (SAD) has been hampered by shortcomings in the diffraction models for X-ray diffraction from FELs. Owing to errors in the orientation matrix and overly simple partiality models, researchers have required large numbers of images to converge to reliable estimates for the structure-factor amplitudes, which may not be feasible for all biological systems. Here, data for cytoplasmic polyhedrosis virus type 17 (CPV17) collected at 1.3 Å wavelength at the Linac Coherent Light Source (LCLS) are revisited. A previously published definition of a partiality model for reflections illuminated by self-amplified spontaneous emission (SASE) pulses is built upon, which defines a fraction between 0 and 1 based on the intersection of a reflection with a spread of Ewald spheres modelled by a super-Gaussian wavelength distribution in the X-ray beam. A method of post-refinement to refine the parameters of this model is suggested. This has generated a merged data set with an overall discrepancy (by calculating the Rsplit value) of 3.15% to 1.46 Å resolution from a 7225-image data set. The atomic numbers of C, N and O atoms in the structure are distinguishable in the electron-density map. There are 13 S atoms within the 237 residues of CPV17, excluding the initial disordered methionine. These only possess 0.42 anomalous scattering electrons each at 1.3 Å wavelength, but the 12 that have single predominant positions are easily detectable in the anomalous difference Fourier map. It is hoped that these improvements will lead towards XFEL experimental phase determination and structure determination by sulfur SAD and will generally increase the utility of the method for difficult cases.
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Jun 2015
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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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Helen M. E.
Duyvesteyn
,
Helen M.
Ginn
,
Maija K.
Pietila
,
Armin
Wagner
,
Johan
Hattne
,
Jonathan M.
Grimes
,
Elina
Hirvonen
,
Gwyndaf
Evans
,
Marie-Laure
Parsy
,
Nicholas K.
Sauter
,
Aaron S.
Brewster
,
Juha
Huiskonen
,
David I.
Stuart
,
Geoff
Sutton
,
Dennis H.
Bamford
Open Access
Abstract: Viruses are a significant threat to both human health and the economy, and there is an urgent need for novel anti-viral drugs and vaccines. High-resolution viral structures inform our understanding of the virosphere, and inspire novel therapies. Here we present a method of obtaining such structural information that avoids potentially disruptive handling, by collecting diffraction data from intact infected cells. We identify a suitable combination of cell type and virus to accumulate particles in the cells, establish a suitable time point where most cells contain virus condensates and use electron microscopy to demonstrate that these are ordered crystalline arrays of empty capsids. We then use an X-ray free electron laser to provide extremely bright illumination of sub-micron intracellular condensates of bacteriophage phiX174 inside living Escherichia coli at room temperature. We have been able to collect low resolution diffraction data. Despite the limited resolution and completeness of these initial data, due to a far from optimal experimental setup, we have used novel methodology to determine a putative space group, unit cell dimensions, particle packing and likely maturation state of the particles.
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Feb 2018
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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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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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Open Access
Abstract: As serial femtosecond crystallography expands towards a variety of delivery methods, including chip-based methods, and smaller collected data sets, the requirement to optimize the data analysis to produce maximum structure quality is becoming increasingly pressing. Here cppxfel, a software package primarily written in C++, which showcases several data analysis techniques, is released. This software package presently indexes images using DIALS (diffraction integration for advanced light sources) and performs an initial orientation matrix refinement, followed by post-refinement of individual images against a reference data set. Cppxfel is released with the hope that the unique and useful elements of this package can be repurposed for existing software packages. However, as released, it produces high-quality crystal structures and is therefore likely to be also useful to experienced users of X-ray free-electron laser (XFEL) software who wish to maximize the information extracted from a limited number of XFEL images.
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Jun 2016
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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.
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Sep 2020
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Open Access
Abstract: Drug and fragment screening at X-ray crystallography beamlines has been a huge success. However, it is inevitable that more high-profile biological drug targets will be identified for which high-quality, highly homogenous crystal systems cannot be found. With increasing heterogeneity in crystal systems, the application of current multi-data-set methods becomes ever less sensitive to bound ligands. In order to ease the bottleneck of finding a well behaved crystal system, pre-clustering of data sets can be carried out using cluster4x after data collection to separate data sets into smaller partitions in order to restore the sensitivity of multi-data-set methods. Here, the software cluster4x is introduced for this purpose and validated against published data sets using PanDDA, showing an improved total signal from existing ligands and identifying new hits in both highly heterogenous and less heterogenous multi-data sets. cluster4x provides the researcher with an interactive graphical user interface with which to explore multi-data set experiments.
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Nov 2020
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P.
Mehrabi
,
R.
Bücker
,
G.
Bourenkov
,
H. M.
Ginn
,
D.
Von Stetten
,
H. M.
Müller-Werkmeister
,
A.
Kuo
,
T.
Morizumi
,
B.t.
Eger
,
W.-L.
Ou
,
S.
Oghbaey
,
A.
Sarracini
,
J. E.
Besaw
,
O.
Pare´-Labrosse
,
S.
Meier
,
H.
Schikora
,
F.
Tellkamp
,
A.
Marx
,
D. A.
Sherrell
,
D.
Axford
,
R. I.
Owen
,
O. P.
Ernst
,
E. F.
Pai
,
E. C.
Schulz
,
R. J. D.
Miller
Open Access
Abstract: For the two proteins myoglobin and fluoroacetate dehalogenase, we present a systematic comparison of crystallographic diffraction data collected by serial femtosecond (SFX) and serial synchrotron crystallography (SSX). To maximize comparability, we used the same batch of micron-sized crystals, the same sample delivery device, and the same data analysis software. Overall figures of merit indicate that the data of both radiation sources are of equivalent quality. For both proteins, reasonable data statistics can be obtained with approximately 5000 room-temperature diffraction images irrespective of the radiation source. The direct comparability of SSX and SFX data indicates that the quality of diffraction data obtained from these samples is linked to the properties of the crystals rather than to the radiation source. Therefore, for other systems with similar properties, time-resolved experiments can be conducted at the radiation source that best matches the desired time resolution.
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Mar 2021
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Open Access
Abstract: Structural biology methods have delivered over 150 000 high-resolution structures of macromolecules, which have fundamentally altered our understanding of biology and our approach to developing new medicines. However, the description of molecular flexibility is instrinsically flawed and in almost all cases, regardless of the experimental method used for structure determination, there remains a strong overfitting bias during molecular model building and refinement. In the worst case this can lead to wholly incorrect structures and thus incorrect biological interpretations. Here, by reparametrizing the description of these complex structures in terms of bonds rather than atomic positions, and by modelling flexibility using a deterministic ensemble of structures, it is demonstrated that structures can be described using fewer parameters than in conventional refinement. The current implementation, applied to X-ray diffraction data, significantly reduces the extent of overfitting, allowing the experimental data to reveal more biological information in electron-density maps.
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Apr 2021
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