I03-Macromolecular Crystallography
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Open Access
Abstract: The bacteriophages with single-stranded RNA (ssRNA) genomes (class Leviviricetes) are among the simplest known viruses that encode only three core proteins: a receptor-binding protein, a capsid protein, and an RNA-dependent RNA polymerase. The number of isolated ssRNA phages has remained very low, but the accumulating RNA metagenome data have uncovered a large variety of these viruses in many environments. Besides the core proteins, many of these genomes putatively encode additional proteins, which up to now have remained uncharacterized. We looked for non-conserved open reading frames (ORFs) in Leviviricetes sequences from the IMG/VR virus metagenome database and used sequence- and structure-based clustering to organize them into similarity groups. Potential ORFs were found throughout the ssRNA phage genomes but almost exclusively on the positive-sense RNA strand, suggestive of their protein-coding potential. The prevalence of the non-conserved ORFs varied in various phage lineages, and their distribution among different genome positions was markedly uneven. Most of the identified ORFs encode all-α proteins, a portion of which contain transmembrane segments that resemble a group of known ssRNA phage lysis proteins, while many others represent previously uncharacterized families of globular or semi-globular α-helical proteins. We additionally uncovered a major class of globular α/β proteins and experimentally determined the structure of a representative protein of this group. These results pave the way for further functional studies of novel ssRNA phage proteins for a better understanding of this diverse virus group.
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May 2026
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B21-High Throughput SAXS
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Diamond Proposal Number(s):
[36130]
Open Access
Abstract: Shwachman-Diamond syndrome (SDS) is a rare genetic disorder characterized by pancreatic insufficiency and neutropenia. While most cases are linked to mutations in the SBDS gene, some involve mutations in the GTPase EFL1. This protein works with SBDS to release the anti-association factor eIF6 from the 60S ribosomal subunit during ribosome biogenesis. The pathogenic EFL1 R1095Q mutant (R1086Q in yeast) exhibits altered guanine nucleotide recognition and impaired eIF6 release, prompting an investigation into its structural consequences. Using the yeast Efl1 orthologue in X-ray hydroxyl radical footprinting experiments, we tracked changes in solvent accessibility caused by the mutation. Although the mutation is situated in domain IV, widespread conformational changes were observed across the protein, particularly in domain I, suggesting a long-range intramolecular communication. Strikingly, the growth defect caused by the pathogenic mutation was rescued by a second mutation located in a allosteric pathway that spans from the nucleotide-binding pocket to domain IV. This compensatory mutation restored proper nuclear localization of eIF6 (Tif6 in yeast). These findings reveal that the R1086Q mutation disrupts a structural communication network within Efl1, impairing the conformational dynamics required for its activity. The loss of this coordination likely underlies the ribosome maturation defects observed in SDS cases linked to EFL1, offering new insights into the molecular basis of the disease.
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Apr 2026
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I03-Macromolecular Crystallography
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Olesia
Werbowy
,
Maria
Håkansson
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Sebastian
Dorawa
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Aleksandra
Stefańska-Kaźmierczak
,
L. Anders
Svensson
,
Salam
Al-Karadaghi
,
Agata
Jurczak-Kurek
,
Karolina
Kwiatkowska-Semrau
,
Magdalena
Plotka
,
Olafur H.
Fridjonsson
,
Gudmundur O.
Hreggvidsson
,
Arnthór
Aevarsson
,
Sławomir
Dąbrowski
,
Anna‐karina
Kaczorowska
,
Tadeusz
Kaczorowski
Diamond Proposal Number(s):
[23282]
Abstract: We present the structural and functional characterization of a single-stranded DNA-binding protein (SSB-M5) identified from a hot spring metagenome in Vatnajökull National Park, Iceland. This small protein (136 aa; 15,695 Da) shares 100% amino acid sequence identity with two previously uncharacterized SSBs from hyperthermophilic Fervidobacterium species. Functional complementation assay demonstrated that SSB-M5 can substitute for Escherichia coli SSB in an ssb− mutant strain, confirming its biological activity. A recombinant C-terminally His-tagged SSB-M5 was overproduced, purified to homogeneity, and subjected to structural, biochemical, and biophysical analysis. The crystal structure revealed that SSB-M5 forms a dimer through a crystallographic twofold axis, with each monomer contributing to a large antiparallel β-sheet. The flat surfaces of the β-sheets from the two dimers are packed together via a second crystallographic twofold axis, forming a tetramer that serves as the functional unit of the SSB-M5. Electrophoretic mobility shift assays showed that SSB-M5, after heat treatment up to 100°C, forms stable DNA-protein complexes with the (dT)40 oligo. Quantitative analyses revealed that SSB-M5 binds (dT)70 oligonucleotide with very high affinity (KD = 72 ± 6 pM). Hill analysis indicated cooperative binding, yielding an EC50 of 141 pM and a Hill coefficient of 2. Moreover, inclusion of SSB-M5 in PCR reactions significantly enhanced amplification by eliminating non-specific products. Together, these findings identify SSB-M5 as a hyperthermostable, high-affinity single-stranded DNA-binding protein with potential applications in molecular biology and biotechnology.
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Apr 2026
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I24-Microfocus Macromolecular Crystallography
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Francesca
Coscia
,
Ioannis
Riziotis
,
Antonina
Andreeva
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Delhi
Kalwan
,
Jennifer
De Jong
,
Philip
Hinchliffe
,
Fabio
Parmeggiani
,
Paul R.
Race
,
Steven G.
Burston
,
Alex
Bateman
,
Rob
Barringer
Diamond Proposal Number(s):
[31440]
Open Access
Abstract: Many proteins harbor covalent intramolecular bonds that enhance their stability and resistance to thermal, mechanical, and proteolytic insults. Intramolecular isopeptide bonds represent one such covalent interaction, yet their distribution across protein domains and organisms has been largely unexplored. Here, we sought to address this by employing a large-scale prediction of intramolecular isopeptide bonds in the AlphaFold database using the structural template-based software Isopeptor. Our findings reveal an extensive phyletic distribution in bacterial and archaeal surface proteins resembling fibrillar adhesins and pilins. All identified intramolecular isopeptide bonds are found in two structurally distinct folds, CnaA-like or CnaB-like, from a relatively small set of related Pfam families, including 10 novel families that we predict to contain intramolecular isopeptide bonds. One CnaA-like domain of unknown function, DUF11 (renamed here to “CLIPPER”) is broadly distributed in cell-surface proteins from Gram-positive bacteria, Gram-negative bacteria, and archaea, and is structurally and biophysically characterized in this work. Using x-ray crystallography, we resolve a CLIPPER domain from a Gram-negative fibrillar adhesin that contains an intramolecular isopeptide bond and further demonstrate that it imparts thermostability and resistance to proteolysis. Our findings demonstrate the extensive distribution of intramolecular isopeptide bond-containing protein domains in nature and structurally resolve the previously cryptic CLIPPER domain.
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Dec 2025
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I03-Macromolecular Crystallography
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Diamond Proposal Number(s):
[26779]
Open Access
Abstract: NTF2-like proteins are compact α + β fold domains with cone-shaped architectures and internal pockets, making them attractive scaffolds for the de novo design of small-molecule binders and enzymes. However, creating ligand-binding pockets often compromises folding stability, posing a key challenge in de novo protein design. Here, we introduce strategies to stabilize NTF2-like domains while preserving pocket geometry and accessibility. By expanding the hydrophobic core through computationally designed α-helical subdomains or homodimer interfaces buttressing the β-sheet's convex face, we enhance structural stability without blocking pocket access on the concave face. Biochemical, biophysical, and crystallographic analyses confirm that the designed buttressing elements maintain the intended fold and support diverse, well-formed hydrophobic ligand-binding pockets with increased preorganization. Our results demonstrate that structural stabilization and pocket optimization need not be mutually exclusive, providing a generalizable approach to create robust ligand-binding proteins. This framework addresses a major bottleneck in protein design and should fuel the development of NTF2-based scaffolds for applications in small-molecule biosensing and enzyme catalysis.
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Nov 2025
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I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[25413]
Open Access
Abstract: In directed evolution, enzyme activity is improved in successive generations of laboratory evolution, which can be described by a simple stepwise climb toward a peak in the fitness landscape. In a naive model of evolution, it can be assumed that each enzyme variant along this path is in a single, well-defined state that differs slightly from the previous one. We analyzed the structural changes in mutants of the β-lactamase BlaC from Mycobacterium tuberculosis obtained via directed evolution for increased ceftazidime hydrolysis activity. Crystal structures of three successive mutants only show an increase in the dynamics of a loop that lines the active site (Ω-loop), enabling better access of the large substrate. However, NMR spectra of wild type and nine mutants of different branches of the directed evolution experiment show a much more diverse and complex picture of the conformational effects. Many mutants show micro-millisecond dynamics for certain regions and most show peak doubling, indicative of two or more conformations being populated. Thus, the straightforward climb to increased ceftazidime activity in the fitness landscape masks a complex trajectory in the conformational landscape, emphasizing the complex and epistatic interplay that single mutations can have on the structure and dynamics of enzymes.
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Nov 2025
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I24-Microfocus Macromolecular Crystallography
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Diamond Proposal Number(s):
[25108]
Open Access
Abstract: The Gram-negative Shewanellaceae family is well known for its ability to transfer catabolically derived electrons to extracellular terminal electron acceptors through electron conduits that permeate the outer membrane. The primary conduit is MtrCAB, a trimeric porin-cytochrome complex that contains the cell surface exposed decaheme cytochrome MtrC. This donates electrons to extracellular substrates, including OmcA, soluble metals, organic electron shuttles, and insoluble metal oxides. However, it is not clear whether this broad substrate specificity requires specific sites for binding and reduction, or whether reduction occurs through non-specific interactions near exposed hemes on the cytochrome surface. Shewanella oneidensis MtrC is composed of four domains, with the hemes closely packed and distributed evenly between domains II and IV. The domains are arranged to allow electron transport across the cytochrome via interdomain electron transfer, but the significance of this conserved feature is not understood. Here we use site-directed mutagenesis to generate an MtrC variant that is comprised only of domains I and II (MtrCDI,II). The properties of this MtrCDI,II are effectively identical to domains I and II of full-length MtrC. Whole-cell assays revealed that S. oneidensis cells replacing full-length MtrC with MtrCDI,II had significantly lower rates of OmcA, flavin mononucleotide, and Fe(III) citrate reduction. Our results demonstrate that MtrC domains III and IV contain sites for association of specific substrates, enabling the reduction of extracellular electron acceptors in S. oneidensis.
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Aug 2025
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I03-Macromolecular Crystallography
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Yasushi
Kondo
,
Caitlin
Hatton
,
Robert
Cheng
,
Matilde
Trabuco
,
Hannah
Glover
,
Quentin
Bertrand
,
Fabienne
Stierli
,
Hans-Peter
Seidel
,
Thomas
Mason
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Sivathmika
Sarma
,
Friedjof
Tellkamp
,
Michal
Kepa
,
Florian
Dworkowski
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Pedram
Mehrabi
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Michael
Hennig
,
Joerg
Standfuss
Diamond Proposal Number(s):
[34035]
Open Access
Abstract: Metabotropic glutamate receptor 5 (mGlu5) is implicated in various neurodegenerative disorders, making it an attractive drug target. Although several ligand-bound crystal structures of mGlu5 exist, their apo-state crystal structure remains unknown. Here, we study mGlu5 structural changes using the photochemical affinity switch, alloswitch-1, in combination with time-resolved freeze-trapping methods. By X-ray crystallography, we demonstrated that isomerizing alloswitch-1 leads to its release from the binding pocket within a few seconds. The apo structure, determined at a resolution of 2.9 Å, is more comparable to the inactive state than to the active state. Our approach presents an accessible alternative to time-resolved serial crystallography for capturing thermodynamically stable transient intermediates. The mGlu5 apo-structure provides molecular insights into the ligand-free allosteric pocket, which can guide the design of new allosteric modulators.
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Jul 2025
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I03-Macromolecular Crystallography
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Abstract: Outer membrane β-barrel proteins (OMPs) are channels found in the outer membranes of Gram-negative bacteria characterized by a stable and diverse barrel architecture, which has made them attractive for nanopore sensing applications. Here, we systematically investigated the feasibility of expanding outer membrane protein G (OmpG) from its native 14-stranded β-barrel to an enhanced conductance variant by independently duplicating each of its seven hairpin units and inserting them downstream of their endogenous positions. Most combinations did not increase pore diameter, but duplication of the terminal seventh hairpin exhibited a rare population of pores with enhanced conductance, suggesting barrel enlargement. Further engineering efforts to optimize the terminal β-turn sequence have resulted in up to 50% of pores with increased conductance. Importantly, the enlarged pores retained the sensing functionality of the original scaffold, highlighting the potential of this approach for developing β-barrel OMP sensors with tunable dimensions.
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Jul 2025
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I04-Macromolecular Crystallography
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Open Access
Abstract: We have recently demonstrated a novel anaerobic NADH-dependent haem breakdown reaction, which is carried out by a range of haemoproteins. The Yersinia enterocolitica protein, HemS, is the focus of further research presented in the current paper. Using conventional experimental methods, bioinformatics, and energy landscape theory (ELT), we provide new insight into the mechanism of the novel breakdown process. Of particular interest is the behavior of a double phenylalanine gate, which opens and closes according to the relative situations of haem and NADH within the protein pocket. This behavior suggests that the double phe-gate fulfills a regulatory role within the pocket, controlling the access of NADH to haem. Additionally, stopped-flow spectroscopy results provide kinetic comparisons between the wild-type and the selected mutants. We also present a fully resolved crystal structure for the F104AF199A HemS monomer, including its extensive loop, the first such structure to be completely resolved for HemS or any of its close homologues. The energy landscapes approach provided key information regarding the gating strategy employed by HemS, compensating for current limitations with conventional biophysical and molecular dynamics approaches. We propose that ELT become more widely used in the field, particularly in the investigation of the dynamics and interactions of weak-binding ligands, and for gating features, within protein cavities.
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Feb 2025
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