I03-Macromolecular Crystallography
|
Kunhua
Li
,
Olena S.
Tokareva
,
Ty M.
Thomson
,
Sebastian C. T.
Wahl
,
Tara L.
Travaline
,
Jessica D.
Ramirez
,
Santosh K.
Choudary
,
Sorabh
Agarwal
,
Ward G.
Walkup
,
Tivoli J.
Olsen
,
Matthew J.
Brennan
,
Gregory L.
Verdine
,
John H.
Mcgee
Diamond Proposal Number(s):
[29353]
Open Access
Abstract: The α-helix is one of the most common protein surface recognition motifs found in nature, and its unique amide-cloaking properties also enable α-helical polypeptide motifs to exist in membranes. Together, these properties have inspired the development of α-helically constrained (Helicon) therapeutics that can enter cells and bind targets that have been considered “undruggable”, such as protein–protein interactions. To date, no general method for discovering α-helical binders to proteins has been reported, limiting Helicon drug discovery to only those proteins with previously characterized α-helix recognition sites, and restricting the starting chemical matter to those known α-helical binders. Here, we report a general and rapid screening method to empirically map the α-helix binding sites on a broad range of target proteins in parallel using large, unbiased Helicon phage display libraries and next-generation sequencing. We apply this method to screen six structurally diverse protein domains, only one of which had been previously reported to bind isolated α-helical peptides, discovering 20 families that collectively comprise several hundred individual Helicons. Analysis of 14 X-ray cocrystal structures reveals at least nine distinct α-helix recognition sites across these six proteins, and biochemical and biophysical studies show that these Helicons can block protein–protein interactions, inhibit enzymatic activity, induce conformational rearrangements, and cause protein dimerization. We anticipate that this method will prove broadly useful for the study of protein recognition and for the development of both biochemical tools and therapeutics for traditionally challenging protein targets.
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Dec 2022
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I03-Macromolecular Crystallography
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Timothy P. C.
Rooney
,
Gregory G.
Aldred
,
Helen K.
Boffey
,
Henriëtte M. G.
Willems
,
Simon
Edwards
,
Stephen J.
Chawner
,
Duncan E.
Scott
,
Christopher
Green
,
David
Winpenny
,
John
Skidmore
,
Jonathan H.
Clarke
,
Stephen P.
Andrews
Diamond Proposal Number(s):
[20026]
Open Access
Abstract: Owing to their central role in regulating cell signaling pathways, the phosphatidylinositol 5-phosphate 4-kinases (PI5P4Ks) are attractive therapeutic targets in diseases such as cancer, neurodegeneration, and immunological disorders. Until now, tool molecules for these kinases have been either limited in potency or isoform selectivity, which has hampered further investigation of biology and drug development. Herein we describe the virtual screening workflow which identified a series of thienylpyrimidines as PI5P4Kγ-selective inhibitors, as well as the medicinal chemistry optimization of this chemotype, to provide potent and selective tool molecules for further use. In vivo pharmacokinetics data are presented for exemplar tool molecules, along with an X-ray structure for ARUK2001607 (15) in complex with PI5P4Kγ, along with its selectivity data against >150 kinases and a Cerep safety panel.
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Dec 2022
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I11-High Resolution Powder Diffraction
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Abstract: A high-resolution synchrotron X-ray diffraction study of a single-crystal YCrO3 compound was employed to obtain its crystallographic information, such as lattice parameters, atomic positions, bond lengths and angles, and local crystalline distortion size and mode. The measurements were taken at 120 K (below the antiferromagnetic transition temperature TN ≃ 141.5 K), 300 K (between TN and the ferroelectric transition temperature TC ≃ 473 K) and 500 K (above TC). Using the high intensity of synchrotron X-rays, it was possible to refine collected patterns with the previously proposed noncentrosymmetric monoclinic structural model (P1211, No. 4) and determine detailed structural parameters. Meanwhile, for a controlled study, the data were refined with the centrosymmetric orthorhombic space group (Pmnb, No. 62). The lattice constants a, b and c and the unit-cell volume increased nearly linearly upon heating. With the P1211 space group, the distributions of bond lengths and angles, as well as local distortion strengths, were observed to be more dispersed. This implies that (i) the local distortion mode of Cr2O6 at 120 K correlates with the formation of canted antiferromagnetic order by Cr1–Cr2 spin interactions, primarily via intermediate O3 and O4 ions; and (ii) the previously reported dielectric anomaly may have a microscopic origin in the strain-balanced Cr1—O3(O4) and Cr2—O5(O6) bonds as well as the local distortion modes of Cr1O6 and Cr2O6 octahedra at 300 K. Local crystalline distortion is shown to be an important factor in the formation of ferroelectric order. The comprehensive set of crystallographic information reported here allows for a complete understanding of the unique magnetic and ferroelectric properties of YCrO3.
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Dec 2022
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I10-Beamline for Advanced Dichroism
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Margaret R.
Mccarter
,
Kook Tae
Kim
,
Vladimir A.
Stoica
,
Sujit
Das
,
Christoph
Klewe
,
Elizabeth P.
Donoway
,
David M.
Burn
,
Padraic
Shafer
,
Fanny
Rodolakis
,
Mauro A. p.
Gonçalves
,
Fernando
Gómez-Ortiz
,
Jorge
Íñiguez
,
Pablo
García-Fernández
,
Javier
Junquera
,
Stephen W.
Lovesey
,
Gerrit
Van Der Laan
,
Se Young
Park
,
John W.
Freeland
,
Lane W.
Martin
,
Dong Ryeol
Lee
,
Ramamoorthy
Ramesh
Diamond Proposal Number(s):
[24797]
Abstract: An escalating challenge in condensed-matter research is the characterization of emergent order-parameter nanostructures such as ferroelectric and ferromagnetic skyrmions. Their small length scales coupled with complex, three-dimensional polarization or spin structures makes them demanding to trace out fully. Resonant elastic x-ray scattering (REXS) has emerged as a technique to study chirality in spin textures such as skyrmions and domain walls. It has, however, been used to a considerably lesser extent to study analogous features in ferroelectrics. Here, we present a framework for modeling REXS from an arbitrary arrangement of charge quadrupole moments, which can be applied to nanostructures in materials such as ferroelectrics. With this, we demonstrate how extended reciprocal space scans using REXS with circularly polarized x rays can probe the three-dimensional structure and chirality of polar skyrmions. Measurements, bolstered by quantitative scattering calculations, show that polar skyrmions of mixed chirality coexist, and that REXS allows valuation of relative fractions of right- and left-handed skyrmions. Our quantitative analysis of the structure and chirality of polar skyrmions highlights the capability of REXS for establishing complex topological structures toward future application exploits.
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Dec 2022
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I12-JEEP: Joint Engineering, Environmental and Processing
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Diamond Proposal Number(s):
[28603]
Open Access
Abstract: Small-diameter, thin-walled pipes have applications in a wide range of industries including high-energy physics, heat transfer, nuclear, medical and communications. There are no standards that exist for welds less than 0.5 mm in width, and as such it is difficult to determine the likely performance of a thin-walled pipe weld. Porosity is largely inevitable in fusion welded joints and is a determining factor in the performance of a connection.
This study focused on characterisation of the evolution of strains in soldered welds less than 0.5 mm in width, by incrementally tensile loading samples and studying them in-situ with Synchrotron X-Ray Computed Tomography and X-Ray Diffraction. Two sample geometries were studied, and porosity defects were present in both, although the levels of porosity size, number and area varied dramatically between the two samples.
Lattice strain interpretation showed that crack propagation for such samples is not driven by porosity but that crack evolution occurs at the same location and load levels irrespective of the presence of pores. Residual stresses of up to 0.3% from the fusion welding process were seen in both samples and appear to have a greater impact on locations of failure than porosity. Porosity does cause differences in strains across directions, however high strains alone did not appear to cause premature failure. Hence, efforts to improve weld strength should in future focus more on reducing residual stresses than reducing porosity.
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Dec 2022
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I10-Beamline for Advanced Dichroism
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N.-J.
Steinke
,
S. L.
Zhang
,
P. J.
Baker
,
L. B.
Duffy
,
F.
Kronast
,
J.
Krieger
,
Z.
Salman
,
T.
Prokscha
,
A.
Suter
,
S.
Langridge
,
Gerrit
Van Der Laan
,
T.
Hesjedal
Diamond Proposal Number(s):
[11503]
Abstract: Chromium-doped
Sb
2
Te
3
is a magnetic topological insulator (MTI), which belongs to the
(
Sb
,
Bi
)
2
(
Se
,
Te
)
3
family. When doped with the transition metals V, Cr, and Mn this family displays long-range ferromagnetic order above liquid nitrogen temperature and is currently intensely explored for quantum device applications. Despite the large magnetic ordering temperature, the experimental observation of dissipationless electrical transport channels, i.e., the quantum anomalous Hall effect, is limited in these materials to temperatures below
≈
2
K. Inhomogeneities in the MTI have been identified as a major concern, affecting the coupling between the Dirac states and the magnetic dopants. Nevertheless, details on the local magnetic order in these materials are not well understood. Here, we report the study of the magnetic correlations in thin films using a combination of muon spin relaxation
(
μ
SR
)
, and magnetic soft x-ray spectroscopy and imaging.
μ
SR
provides two key quantities for understanding the microscopic magnetic behavior: The magnetic volume fraction, i.e., the percentage of the material that is ferromagnetically ordered, and the relaxation rate, which is sensitive to the magnetic static
(
≈
μ
s
)
and dynamic disorder. By choosing different implantation depths for the muons, one can further discriminate between near-surface and bulk properties. No evidence for a surface enhancement of the magnetic ordering is observed, but, instead, we find evidence of small magnetically ordered clusters in a paramagnetic background, which are coupled. The significant magnetic field shift that is present in all samples indicates a percolation transition that proceeds through the formation and growth of magnetically ordered spin clusters. We further find that fluctuations are present even at low temperatures, and that there appears to be a transition between superparamagnetism and superferromagnetism.
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Dec 2022
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I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[26803]
Abstract: Background: The platelet–signaling receptor glycoprotein VI (GPVI) is a promising antithrombotic target. We have previously raised a series of high-affinity nanobodies (Nbs) against GPVI and identified Nb2, Nb21, and Nb35 as potent GPVI inhibitors. The Nb2 binding site has been mapped to the D1 domain, which is directly adjacent to the CRP binding site. Ligand–binding complementary determining region 3 has only 15% conservation between all 3 Nbs. Objectives: To map the binding sites of Nb21 and Nb35 on GPVI. Methods: We determined the X-ray crystal structure of the D1 and D2 extracellular domains of the GPVI-Nb35 complex. We then looked at the effects of various GPVI mutations on the ability of Nbs to inhibit collagen binding and GPVI signaling using surface binding assays and transfected cell lines. Results: The crystal structure of GPVI bound to Nb35 was solved. GPVI was present as a monomer, and the D1+D2 conformation was comparable to that in the dimeric structure. Arg46, Tyr47, and Ala57 are common residues on GPVI targeted by both Nb2 and Nb35. Mutating Arg46 to an Ala abrogated the ability of Nb2, Nb21, and Nb35 to inhibit collagen–induced GPVI signaling and blocked the binding of all 3 Nbs. In addition, Arg60 was found to reduce Nb21 inhibition but not the inhibition Nb2 or Nb35. Conclusions: These findings reveal key residues involved in the high-affinity binding of GPVI inhibitors and negate the idea that GPVI dimerization induces a conformational change required for ligand binding.
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Dec 2022
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B21-High Throughput SAXS
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Diamond Proposal Number(s):
[207821]
Open Access
Abstract: Collagen triple helices are critical in the function of mannan-binding lectin (MBL), an oligomeric recognition molecule in complement activation. The MBL collagen regions form complexes with the serine proteases MASP-1 and MASP-2 in order to activate complement, and mutations lead to common immunodeficiencies. To evaluate their structure-function properties, we studied the solution structures of four MBL-like collagen peptides. The thermal stability of the MBL collagen region was much reduced by the presence of a GQG interruption in the typical (X-Y-Gly)n repeat compared to controls. Experimental solution structural data were collected using analytical ultracentrifugation and small angle X-ray and neutron scattering. As controls, we included two standard Pro-Hyp-Gly collagen peptides (POG)10-13, as well as three more peptides with diverse (X-Y-Gly)n sequences that represented other collagen features. These data were quantitatively compared with atomistic linear collagen models derived from crystal structures and 12,000 conformations obtained from molecular dynamics (MD) simulations. All four MBL peptides were bent to varying degrees up to 85o in the best-fit MD models. The best-fit benchmark peptides (POG)n were more linear but exhibited a degree of conformational flexibility. The remaining three peptides showed mostly linear solution structures. In conclusion, the collagen helix is not strictly linear, the degree of flexibility in the triple helix depends on its sequence, and the triple helix with the GQG interruption showed a pronounced bend. The bend in MBL GQG peptides resembles the bend in the collagen of complement C1q and may be key for lectin pathway activation.
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Dec 2022
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I10-Beamline for Advanced Dichroism
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Diamond Proposal Number(s):
[10207]
Open Access
Abstract: X-ray magnetic circular dichroism (XMCD), which by virtue of the sum rules provides element-specific spin and orbital moments, is obtained from the difference between two polarized spectra by reversing the direction of either the light helicity or the applied magnetic field. Usually, it is tacitly assumed that these two spectra are obtained using the same absolute degree of light and magnetic polarization. This is, however, not always possible and depends on circumstances that can be beyond control. First, we recapitulate the conventional XMCD sum rule method to obtain the values of the moments and emphasize some of the complications in the case of the rare-earth
M
4
,
5
edges, such as the presence of strong core-hole
j
j
overlap, linear dichroism, and magnetic dipole term
⟨
T
z
⟩
. Instead, we propose an alternative method. Using the individual polarized x-ray absorption spectra obtained at the Ho and Dy
M
5
edges, where each of the
Δ
J
=
−
1
,
0
, and
+
1
transitions are separated by
∼
2
eV in photon energy, we are able to determine independently the degree of circular dichroism in a single spectrum. Since light is a transverse wave, we need to include, apart from the circular dichroism, also a linear dichroism contribution in order to fit the circularly polarized spectra. In the measurements on paramagnetic rare-earth dopants it was found that reversing the field produces the same degree of circular dichroism, while reversing the helicity yields a
∼
20% difference in the degree of circular dichroism.
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Dec 2022
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Open Access
Abstract: Determination of protein structures typically entails building a model that satisfies the collected experimental observations and its deposition in the Protein Data Bank. Experimental limitations can lead to unavoidable uncertainties during the process of model building, which result in the introduction of errors into the deposited model. Many metrics are available for model validation, but most are limited to consideration of the physico-chemical aspects of the model or its match to the experimental data. The latest advances in the field of deep learning have enabled the increasingly accurate prediction of inter-residue distances, an advance which has played a pivotal role in the recent improvements observed in the field of protein ab initio modelling. Here, new validation methods are presented based on the use of these precise inter-residue distance predictions, which are compared with the distances observed in the protein model. Sequence-register errors are particularly clearly detected and the register shifts required for their correction can be reliably determined. The method is available in the ConKit package (https://www.conkit.org).
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Dec 2022
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