Krios III-Titan Krios III at Diamond
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Diamond Proposal Number(s):
[18477]
Open Access
Abstract: Traditionally, molecular assembly pathways for viruses are inferred from high resolution structures of purified stable intermediates, low resolution images of cell sections and genetic approaches. Here, we directly visualise an unsuspected ‘single shelled’ intermediate for a mammalian orthoreovirus in cryo-preserved infected cells, by cryo-electron tomography of cellular lamellae. Particle classification and averaging yields structures to 5.6 Å resolution, sufficient to identify secondary structural elements and produce an atomic model of the intermediate, comprising 120 copies each of protein λ1 and σ2. This λ1 shell is ‘collapsed’ compared to the mature virions, with molecules pushed inwards at the icosahedral fivefolds by ~100 Å, reminiscent of the first assembly intermediate of certain prokaryotic dsRNA viruses. This supports the supposition that these viruses share a common ancestor, and suggests mechanisms for the assembly of viruses of the Reoviridae. Such methodology holds promise for dissecting the replication cycle of many viruses.
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Sep 2020
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I04-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
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Open Access
Abstract: Developing methods to determine high-resolution structures from micrometre- or even submicrometre-sized protein crystals has become increasingly important in recent years. This applies to both large protein complexes and membrane proteins, where protein production and the subsequent growth of large homogeneous crystals is often challenging, and to samples which yield only micro- or nanocrystals such as amyloid or viral polyhedrin proteins. The versatile macromolecular crystallography microfocus (VMXm) beamline at Diamond Light Source specializes in X-ray diffraction measurements from micro- and nanocrystals. Because of the possibility of measuring data from crystalline samples that approach the resolution limit of visible-light microscopy, the beamline design includes a scanning electron microscope (SEM) to visualize, locate and accurately centre crystals for X-ray diffraction experiments. To ensure that scanning electron microscopy is an appropriate method for sample visualization, tests were carried out to assess the effect of SEM radiation on diffraction quality. Cytoplasmic polyhedrosis virus polyhedrin protein crystals cryocooled on electron-microscopy grids were exposed to SEM radiation before X-ray diffraction data were collected. After processing the data with DIALS, no statistically significant difference in data quality was found between datasets collected from crystals exposed and not exposed to SEM radiation. This study supports the use of an SEM as a tool for the visualization of protein crystals and as an integrated visualization tool on the VMXm beamline.
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May 2020
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Helen M. E.
Duyvesteyn
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Helen M.
Ginn
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Maija K.
Pietila
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Armin
Wagner
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Johan
Hattne
,
Jonathan M.
Grimes
,
Elina
Hirvonen
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Gwyndaf
Evans
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Marie-Laure
Parsy
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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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Philip
Roedig
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Helen M.
Ginn
,
Tim
Pakendorf
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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
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Nicholas K.
Sauter
,
Abhay
Kotecha
,
James
Kelly
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David J.
Rowlands
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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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I24-Microfocus Macromolecular Crystallography
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Open Access
Abstract: Fringes and speckles occur within diffraction spots when a crystal is illuminated
with coherent radiation during X-ray diffraction. The additional information in
these features provides insight into the imperfections in the crystal at the submicrometre
scale. In addition, these features can provide more accurate
intensity measurements (e.g. by model-based profile fitting), detwinning (by
distinguishing the various components), phasing (by exploiting sampling of the
molecular transform) and refinement (by distinguishing regions with different
unit-cell parameters). In order to exploit these potential benefits, the features
due to coherent diffraction have to be recorded and any change due to radiation
damage properly modelled. Initial results from recording coherent diffraction at
cryotemperatures from polyhedrin crystals of approximately 2 mm in size are
described. These measurements allowed information about the type of crystal
imperfections to be obtained at the sub-micrometre level, together with the
changes due to radiation damage
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Jan 2016
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I24-Microfocus Macromolecular Crystallography
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Diamond Proposal Number(s):
[6387]
Open Access
Abstract: Polyhedra represent an ancient system used by a number of insect viruses to protect virions during long periods of environmental exposure. We present high resolution crystal structures of polyhedra for seven previously uncharacterised types of cypoviruses, four using ab initio selenomethionine phasing (two of these required over 100 selenomethionine crystals each). Approximately 80% of residues are structurally equivalent between all polyhedrins (pairwise rmsd ⩽1.5Å), whilst pairwise sequence identities, based on structural alignment, are as little as 12%. These illustrate the effect of 400 million years of evolution on a system where the crystal lattice is the functionally conserved feature in the face of massive sequence variability. The conservation of crystal contacts is maintained across most of the molecular surface, except for a dispensable virus recognition domain. By spreading the contacts over so much of the protein surface the lattice remains robust in the face of many individual changes. Overall these unusual structural constraints seem to have skewed the molecule’s evolution so that surface residues are almost as conserved as the internal residues.
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Aug 2015
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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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I02-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
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P.
Roedig
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I.
Vartiainen
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R.
Duman
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S.
Panneerselvam
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N.
Stübe
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O.
Lorbeer
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M.
Warmer
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G.
Sutton
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D. I.
Stuart
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E.
Weckert
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C.
David
,
A.
Wagner
,
A.
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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Helen M.
Ginn
,
Marc
Messerschmidt
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Xiaoyun
Ji
,
Hanwen
Zhang
,
Danny
Axford
,
Richard J
Gildea
,
Graeme
Winter
,
Aaron S.
Brewster
,
Johan
Hattne
,
Armin
Wagner
,
Jonathan M.
Grimes
,
Gwyndaf
Evans
,
Nicholas K.
Sauter
,
Geoff
Sutton
,
David I.
Stuart
Open Access
Abstract: The X-ray free-electron laser (XFEL) allows the analysis of small weakly diffracting protein crystals, but has required very many crystals to obtain good data. Here we use an XFEL to determine the room temperature atomic structure for the smallest cytoplasmic polyhedrosis virus polyhedra yet characterized, which we failed to solve at a synchrotron. These protein microcrystals, roughly a micron across, accrue within infected cells. We use a new physical model for XFEL diffraction, which better estimates the experimental signal, delivering a high-resolution XFEL structure (1.75 Å), using fewer crystals than previously required for this resolution. The crystal lattice and protein core are conserved compared with a polyhedrin with less than 10% sequence identity. We explain how the conserved biological phenotype, the crystal lattice, is maintained in the face of extreme environmental challenge and massive evolutionary divergence. Our improved methods should open up more challenging biological samples to XFEL analysis.
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Mar 2015
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I04-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
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Open Access
Abstract: A new indexing method is presented which is capable of
indexing multiple crystal lattices from narrow wedges of
diffraction data. The method takes advantage of a simplification
of Fourier transform-based methods that is applicable
when the unit-cell dimensions are known a priori. The efficacy
of this method is demonstrated with both semi-synthetic multilattice
data and real multi-lattice data recorded from crystals
of 1 mm in size, where it is shown that up to six lattices can
be successfully indexed and subsequently integrated from a
1 wedge of data. Analysis is presented which shows that
improvements in data-quality indicators can be obtained
through accurate identification and rejection of overlapping
reflections prior to scaling.
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Oct 2014
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