I24-Microfocus Macromolecular Crystallography
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Diamond Proposal Number(s):
[25301]
Open Access
Abstract: Of the five human antibody isotypes, the function of IgD is the least well-understood, although various studies point to a role for IgD in mucosal immunity. IgD is also the least well structurally characterized isotype. Until recently, when crystal structures were reported for the IgD Fab, the only structural information available was a model for intact IgD based on solution scattering data. We now report the crystal structure of human IgD-Fc solved at 3.0 Å resolution. Although similar in overall architecture to other human isotypes, IgD-Fc displays markedly different orientations of the Cδ3 domains in the Cδ3 domain dimer and the lowest interface area of all the human isotypes. The nature of the residues that form the dimer interface also differs from those conserved in the other isotypes. By contrast, the interface between the Cδ2 and Cδ3 domains in each chain is the largest among the human isotypes. This interface is characterized by two binding pockets, not seen in other isotypes, and points to a potential role for the Cδ2/Cδ3 interface in stabilizing the IgD-Fc homodimer. We investigated the thermal stability of IgD-Fc, alone and in the context of an intact IgD antibody, and found that IgD-Fc unfolds in a single transition. Human IgD-Fc clearly has unique structural features not seen in the other human isotypes, and comparison with other mammalian IgD sequences suggests that these unique features might be widely conserved.
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Nov 2024
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I04-Macromolecular Crystallography
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Open Access
Abstract: A 1.7 Å structure is presented for an active form of the virulence factor ScpB, the C5a peptidase from Streptococcus agalactiae. The previously reported structure of the ScpB active site mutant exhibited a large separation (~20 Å) between the catalytic His and Ser residues. Significant differences are observed in the catalytic domain between the current and mutant ScpB structures resulting with a high RMSDCα (4.6 Å). The fold of the active form of ScpB is nearly identical to ScpA (RMSDCα 0.2 Å), the C5a-peptidase from Streptococcus pyogenes. Both ScpA and ScpB have comparable activity against human C5a, indicating neither enzyme require host proteins for C5a-ase activity. These studies are a first step in resolving reported differences in the specificities of these enzymes.
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Nov 2023
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Rhiju
Das
,
Rachael C.
Kretsch
,
Adam J.
Simpkin
,
Thomas
Mulvaney
,
Phillip
Pham
,
Ramya
Rangan
,
Fan
Bu
,
Ronan M.
Keegan
,
Maya
Topf
,
Daniel J.
Rigden
,
Zhichao
Miao
,
Eric
Westhof
Open Access
Abstract: The prediction of RNA three-dimensional structures remains an unsolved problem. Here, we report assessments of RNA structure predictions in CASP15, the first CASP exercise that involved RNA structure modeling. Forty-two predictor groups submitted models for at least one of twelve RNA-containing targets. These models were evaluated by the RNA-Puzzles organizers and, separately, by a CASP-recruited team using metrics (GDT, lDDT) and approaches (Z-score rankings) initially developed for assessment of proteins and generalized here for RNA assessment. The two assessments independently ranked the same predictor groups as first (AIchemy_RNA2), second (Chen), and third (RNAPolis and GeneSilico, tied); predictions from deep learning approaches were significantly worse than these top ranked groups, which did not use deep learning. Further analyses based on direct comparison of predicted models to cryogenic electron microscopy (cryo-EM) maps and x-ray diffraction data support these rankings. With the exception of two RNA-protein complexes, models submitted by CASP15 groups correctly predicted the global fold of the RNA targets. Comparisons of CASP15 submissions to designed RNA nanostructures as well as molecular replacement trials highlight the potential utility of current RNA modeling approaches for RNA nanotechnology and structural biology, respectively. Nevertheless, challenges remain in modeling fine details such as noncanonical pairs, in ranking among submitted models, and in prediction of multiple structures resolved by cryo-EM or crystallography.
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Oct 2023
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Open Access
Abstract: The results of tertiary structure assessment at CASP15 are reported. For the first time, recognizing the outstanding performance of AlphaFold 2 (AF2) at CASP14, all single-chain predictions were assessed together, irrespective of whether a template was available. At CASP15, there was no single stand-out group, with most of the best-scoring groups—led by PEZYFoldings, UM-TBM, and Yang Server—employing AF2 in one way or another. Many top groups paid special attention to generating deep Multiple Sequence Alignments (MSAs) and testing variant MSAs, thereby allowing them to successfully address some of the hardest targets. Such difficult targets, as well as lacking templates, were typically proteins with few homologues. Local divergence between prediction and target correlated with localization at crystal lattice or chain interfaces, and with regions exhibiting high B-factor factors in crystal structure targets, and should not necessarily be considered as representing error in the prediction. However, analysis of exposed and buried side chain accuracy showed room for improvement even in the latter. Nevertheless, a majority of groups produced high-quality predictions for most targets, which are valuable for experimental structure determination, functional analysis, and many other tasks across biology. These include those applying methods similar to those used to generate major resources such as the AlphaFold Protein Structure Database and the ESM Metagenomic atlas: the confidence estimates of the former were also notably accurate.
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Sep 2023
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VMXm-Versatile Macromolecular Crystallography microfocus
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Leila T.
Alexander
,
Janani
Durairaj
,
Andriy
Kryshtafovych
,
Luciano A.
Abriata
,
Yusupha
Bayo
,
Gira
Bhabha
,
Cécile
Breyton
,
Simon G.
Caulton
,
James
Chen
,
Séraphine
Degroux
,
Damian C.
Ekiert
,
Benedikte S.
Erlandsen
,
Peter L.
Freddolino
,
Dominic
Gilzer
,
Chris
Greening
,
Jonathan M.
Grimes
,
Rhys
Grinter
,
Manickam
Gurusaran
,
Marcus D.
Hartmann
,
Charlie J.
Hitchman
,
Jeremy R.
Keown
,
Ashleigh
Kropp
,
Petri
Kursula
,
Andrew L.
Lovering
,
Bruno
Lemaitre
,
Andrea
Lia
,
Shiheng
Liu
,
Maria
Logotheti
,
Shuze
Lu
,
Sigurbjorn
Markusson
,
Mitchell D.
Miller
,
George
Minasov
,
Hartmut H.
Niemann
,
Felipe
Opazo
,
George N.
Phillips
,
Owen R.
Davies
,
Samuel
Rommelaere
,
Monica
Rosas‐lemus
,
Pietro
Roversi
,
Karla
Satchell
,
Nathan
Smith
,
Mark A.
Wilson
,
Kuan‐lin
Wu
,
Xian
Xia
,
Han
Xiao
,
Wenhua
Zhang
,
Z. Hong
Zhou
,
Krzysztof
Fidelis
,
Maya
Topf
,
John
Moult
,
Torsten
Schwede
Diamond Proposal Number(s):
[19946, 23570, 27314, 28534]
Open Access
Abstract: We present an in-depth analysis of selected CASP15 targets, focusing on their biological and functional significance. The authors of the structures identify and discuss key protein features and evaluate how effectively these aspects were captured in the submitted predictions. While the overall ability to predict three-dimensional protein structures continues to impress, reproducing uncommon features not previously observed in experimental structures is still a challenge. Furthermore, instances with conformational flexibility and large multimeric complexes highlight the need for novel scoring strategies to better emphasize biologically relevant structural regions. Looking ahead, closer integration of computational and experimental techniques will play a key role in determining the next challenges to be unraveled in the field of structural molecular biology.
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Jul 2023
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I24-Microfocus Macromolecular Crystallography
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Diamond Proposal Number(s):
[18069]
Open Access
Abstract: The hypoxia-inducible factor (HIF) prolyl-hydroxylases (human PHD1-3) catalyze prolyl hydroxylation in oxygen-dependent degradation (ODD) domains of HIFα isoforms, modifications that signal for HIFα proteasomal degradation in an oxygen-dependent manner. PHD inhibitors are used for treatment of anemia in kidney disease. Increased erythropoietin (EPO) in patients with familial/idiopathic erythrocytosis and pulmonary hypertension is associated with mutations in EGLN1 (PHD2) and EPAS1 (HIF2α); a drug inhibiting HIF2α activity is used for clear cell renal cell carcinoma (ccRCC) treatment. We report crystal structures of PHD2 complexed with the C-terminal HIF2α-ODD in the presence of its 2-oxoglutarate cosubstrate or N-oxalylglycine inhibitor. Combined with the reported PHD2.HIFα-ODD structures and biochemical studies, the results inform on the different PHD.HIFα-ODD binding modes and the potential effects of clinically observed mutations in HIFα and PHD2 genes. They may help enable new therapeutic avenues, including PHD isoform-selective inhibitors and sequestration of HIF2α by the PHDs for ccRCC treatment.
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Jul 2023
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I03-Macromolecular Crystallography
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Rob
Barringer
,
Alice E.
Parnell
,
Aleix
Lafita
,
Vivian
Monzon
,
Catherine R.
Back
,
Mariusz
Madej
,
Jan
Potempa
,
Angela H.
Nobbs
,
Steven G.
Burston
,
Alex
Bateman
,
Paul R.
Race
Diamond Proposal Number(s):
[23269]
Open Access
Abstract: Bacterial fibrillar adhesins are specialised extracellular polypeptides that promote the attachment of bacteria to the surfaces of other cells or materials. Adhesin-mediated interactions are critical for the establishment and persistence of stable bacterial populations within diverse environmental niches and are important determinants of virulence. The fibronectin (Fn) binding fibrillar adhesin CshA, and its paralogue CshB, play important roles in host colonisation by the oral commensal and opportunistic pathogen Streptococcus gordonii. As paralogues are often catalysts for functional diversification, we have probed the early stages of structural and functional divergence in Csh proteins by determining the X-ray crystal structure of the CshB adhesive domain NR2 and characterising its Fn binding properties in vitro. Despite sharing a common fold, CshB_NR2 displays an ~1.7-fold reduction in Fn binding affinity relative to CshA_NR2. This correlates with reduced electrostatic charge in the Fn binding cleft. Complementary bioinformatic studies reveal that homologues of CshA/B_NR2 domains are widely distributed in both Gram-positive and Gram-negative bacteria, where they are found housed within functionally cryptic multi-domain polypeptides. Our findings are consistent with the classification of Csh adhesins and their relatives as members of the recently defined Polymer Adhesin Domain (PAD) family of bacterial proteins.
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Mar 2023
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I03-Macromolecular Crystallography
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Diamond Proposal Number(s):
[13467]
Open Access
Abstract: Bacteria are equipped with a diverse set of regulatory tools that allow them to quickly adapt to their environment. The RimK system allows for Pseudomonas spp. to adapt through post-transcriptional regulation by altering the ribosomal subunit RpsF. RimK is found in a wide range of bacteria with a conserved amino acid sequence, however the genetic context and the role of this protein is highly diverse. By solving and comparing the structures of RimK homologues from two related but functionally divergent systems, we uncovered key structural differences that likely contribute to the different activity levels of each of these homologues. Moreover, we were able to clearly resolve the active site of this protein for the first time, resolving binding of the glutamate substrate. This work advances our understanding of how subtle differences in protein sequence and structure can have profound effects on protein activity, which can in turn result in widespread mechanistic changes.
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Sep 2022
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I03-Macromolecular Crystallography
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Leila T.
Alexander
,
Rosalba
Lepore
,
Andriy
Kryshtafovych
,
Athanasios
Adamopoulos
,
Markus
Alahuhta
,
Ann M.
Arvin
,
Yannick J.
Bomble
,
Bettina
Böttcher
,
Cécile
Breyton
,
Valerio
Chiarini
,
Naga Babu
Chinnam
,
Wah
Chiu
,
Krzysztof
Fidelis
,
Rhys
Grinter
,
Gagan D.
Gupta
,
Marcus D.
Hartmann
,
Christopher S.
Hayes
,
Tatjana
Heidebrecht
,
Andrea
Ilari
,
Andrzej
Joachimiak
,
Youngchang
Kim
,
Romain
Linares
,
Andrew L.
Lovering
,
Vladimir V.
Lunin
,
Andrei N.
Lupas
,
Cihan
Makbul
,
Karolina
Michalska
,
John
Moult
,
Prasun K.
Mukherjee
,
William
Nutt
,
Stefan L.
Oliver
,
Anastassis
Perrakis
,
Lucy
Stols
,
John A.
Tainer
,
Maya
Topf
,
Susan E.
Tsutakawa
,
Mauricio
Valdivia‐delgado
,
Torsten
Schwede
Open Access
Abstract: The biological and functional significance of selected Critical Assessment of Techniques for Protein Structure Prediction 14 (CASP14) targets are described by the authors of the structures. The authors highlight the most relevant features of the target proteins and discuss how well these features were reproduced in the respective submitted predictions. The overall ability to predict three-dimensional structures of proteins has improved remarkably in CASP14, and many difficult targets were modeled with impressive accuracy. For the first time in the history of CASP, the experimentalists not only highlighted that computational models can accurately reproduce the most critical structural features observed in their targets, but also envisaged that models could serve as a guidance for further studies of biologically-relevant properties of proteins.
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Dec 2021
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Open Access
Abstract: We report here an assessment of the model refinement category of the 14th round of Critical Assessment of Structure Prediction (CASP14). As before, predictors submitted up to five ranked refinements, along with associated residue-level error estimates, for targets that had a wide range of starting quality. The ability of groups to accurately rank their submissions and to predict coordinate error varied widely. Overall only four groups out-performed a “naïve predictor” corresponding to resubmission of the starting model. Among the top groups there are interesting differences of approach and in the spread of improvements seen: some methods are more conservative, others more adventurous. Some targets were “double-barrelled” for which predictors were offered a high-quality AlphaFold 2 (AF2)-derived prediction alongside another of lower quality. The AF2-derived models were largely unimprovable, many of their apparent errors being found to reside at domain and, especially, crystal lattice contacts. Refinement is shown to have a mixed impact overall on structure-based function annotation methods to predict nucleic acid binding, spot catalytic sites and dock protein structures.
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Jul 2021
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