Krios III-Titan Krios III at Diamond
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Diamond Proposal Number(s):
[33230]
Open Access
Abstract: Bacterial conjugation, the process of horizontal gene transfer between bacteria, is initiated by mating pair formation (MPF) via a conjugative pilus. Conjugation of the IncP RP4 plasmid is mediated by short mating pili. Here, we report the cryo-EM structure of the RP4 pilus at 2.74 Å resolution. Uniquely, both the structural and quantitative mass spectral analyses revealed that the cyclic TrbC pilin subunit is not lipidated. Consistently, an E. coli pgsA mutant lacking phosphatidylglycerol (PG) can serve as a donor of RP4 but not of F- (pKpQIL), H- (R27) or W- (R388) pili, whose biogenesis and DNA transfer is PG-dependent. RP4 is the first example of a lipid-independent functional mating pilus. This discovery suggests that an amphipathic lipid moiety is not universally essential for the biogenesis of conjugative pili and MPF, providing an alternative model for their assembly and function. These data expand our understanding of the diverse bacterial mechanisms employ to transfer genetic material.
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Jun 2026
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Krios I-Titan Krios I at Diamond
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Thijs W.
Ettema
,
Satomi
Inaba-Inoue
,
Chancievan
Thangaratnarajah
,
Leticia
Alves Da Silva
,
Nikas
Senning
,
Amy
Clarke
,
Piotr
Stepien
,
Anokhi
Shah
,
Yue
Ma
,
Katie
Hardman
,
Sophia
David
,
Hassane
El Mkami
,
Jonathan G.
Heddle
,
Norimichi
Nomura
,
Satoshi
Ogasawara
,
So
Iwata
,
Dmitry
Ghilarov
,
Christos
Pliotas
,
Thomas
Stockner
,
Dirk J.
Slotboom
,
Konstantinos
Beis
Diamond Proposal Number(s):
[25127]
Open Access
Abstract: SbmA is a membrane transporter from Escherichia coli that imports antimicrobial peptides. SbmA belongs to the SbmA-like peptide transporter (SLiPT) family. Although the protein is a secondary active transporter that is energized by the proton gradient, it is structurally related to the transmembrane domain (TMD) of ATP-binding cassette (ABC) transporters. SbmA therefore bridges the structural divide between primary and secondary transporters. However, it remains unclear, if SbmA also shares the mechanism of alternating access with ABC transporters, because only a single (outward-open) state is resolved. Here, we show by sequence analysis that SbmA is likely evolved from the TMD of an early ancestor of the ABC transporter YddA. We determine the cryogenic electron microscopy structures of SbmA in occluded and inward-facing states. These conformations closely resemble equivalent states found in ABC transporters, indicating a shared structural mechanism of transport. In contrast to ABC transporters, where nucleotide binding, hydrolysis and release steer conformational changes necessary for substrate translocation, electron paramagnetic resonance (EPR) spectroscopy and molecular dynamics (MD) simulations reveal how pH changes induce conformational transitions in SbmA, consistent with a mechanism of substrate internalization that utilizes the transmembrane proton gradient.
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Apr 2026
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Krios I-Titan Krios I at Diamond
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Diamond Proposal Number(s):
[29493]
Open Access
Abstract: During conjugation, plasmid DNA is transferred from donor to recipient bacteria via the plasmid-encoded mating pilus, formed as thin helical assemblies of polymerised pilin subunits. In the IncHI1 R27 plasmid-encoded pilus, the TrhA pilin undergoes cyclisation (via a peptide bond between Gly1 and Asp69), essential for conjugation. Gly1 and Asp69 are exposed on the pilus surface and conserved in all TrhA pilins in the Plascad database. Substituting Asp69 with Asn, Ala, Gly, or Arg does not prevent cyclisation or pilus formation, which remains structurally indistinguishable from the wild type. Conjugation efficiency of the Asp69 substitutions across multiple recipient species correlates with side chain size, in the order Asp69Asn > Asp69Ala > Asp69Gly. However, Asp69Arg, as well as Asp69Lys and Gly1Lys substitutions abolish conjugation, likely due to the positively charged pilus surface (opposite to the wild-type negative charge) forming unfavourable electrostatic interactions with the recipient outer membrane’s inner leaflet, composed solely of zwitterionic phosphatidylethanolamine (PE). Consistently, conjugation is rescued in recipients lacking PE. These findings indicate strong selective pressure to maintain Gly1 and Asp69, as efficient DNA transfer depends on precise electrostatic and steric constraints of the pilus surface.
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Feb 2026
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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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Krios II-Titan Krios II at Diamond
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Naito
Ishimoto
,
Joshua L. C.
Wong
,
Shan
He
,
Sally
Shirran
,
Olivia
Wright-Paramio
,
Chloe
Seddon
,
Nanki
Singh
,
Carlos
Balsalobre
,
Ravi R.
Sonani
,
Abigail
Clements
,
Edward H.
Egelmane
,
Gad
Frankel
,
Konstantinos
Beis
Diamond Proposal Number(s):
[33230]
Open Access
Abstract: Conjugation, the major driver of the spread of antimicrobial resistance genes, relies on a conjugation pilus for DNA transfer. Conjugative pili, such as the F-pilus, are dynamic tubular structures, composed of a polymerized pilin, that mediate the initial donor–recipient interactions, a process known as mating pair formation (MPF). IncH are low-copy-number plasmids, traditionally considered broad host range, which are found in bacteria infecting both humans and animals. The reference IncHI1 plasmid R27, isolated from Salmonella enterica serovar Typhi, encodes the conjugative H-pilus subunit TrhA containing 74 residues after cleavage of the signal sequence. Here, we show that the H-pilus forms long filamentous structures that mediate MPF and describe its cryoelectron-microscopic (cryo-EM) structure at 2.2 Å resolution. Like the F pilus, the H-pilin subunits form helical assemblies with phospholipid molecules at a stoichiometric ratio of 1:1. While there were previous reports that the T-pilus from Agrobacterium tumefaciens was composed of cyclic subunits, three recent cryo-EM structures of the T-pilus found no such cyclization. Here, we report that the H-pilin is cyclic, with a covalent bond connecting the peptide backbone between the N and C termini. Both the cryo-EM map and mass spectrometry revealed cleavage of the last five residues of the pilin, followed by cyclization via condensation of the amine and carboxyl residues. Mutagenesis experiments revealed that loss of cyclization abolished pilus biogenesis and efficient plasmid transfer. The cyclic nature of the pilin could stabilize the pilus and may explain the high incidence of IncH plasmid dissemination.
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Apr 2025
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Krios III-Titan Krios III at Diamond
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Diamond Proposal Number(s):
[25127]
Open Access
Abstract: Conjugation plays a major role in dissemination of antimicrobial resistance genes. Following transfer of IncF-like plasmids, recipients become refractory to a second wave of conjugation with the same plasmid via entry (TraS) and surface (TraT) exclusion mechanisms. Here, we show that TraT from the pKpQIL and F plasmids (TraTpKpQIL and TraTF) exhibits plasmid surface exclusion specificity. The cryo-EM structures of TraTpKpQIL and TraTF reveal that they oligomerise into decameric champagne bottle cork-like structures, which are anchored to the outer membrane via a diacylglycerol and palmitic acid modified α-helical barrel domain. Unexpectedly, we identify chromosomal TraT homologues from multiple Gram-negative phyla which form numerous divergent lineages in a phylogenetic tree of TraT sequences. Plasmid-associated TraT sequences are found in multiple distinct lineages, including two separate clades incorporating TraT from Enterobacteriaceae IncF/F-like and Legionellaceae F-like plasmids. These findings suggest that different plasmid backbones have acquired and co-opted TraT on independent occasions.
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Jan 2025
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I24-Microfocus Macromolecular Crystallography
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Shangwen
Luo
,
Xin-Rong
Li
,
Xiao-Tong
Gong
,
Alexey
Kulikovsky
,
Feng
Qu
,
Konstantinos
Beis
,
Konstantin
Severinov
,
Svetlana
Dubiley
,
Xinxin
Feng
,
Shi-Hui
Dong
,
Satish K.
Nair
Abstract: Infections caused by gram-negative pathogens continue to be a major risk to human health because of the innate antibiotic resistance endowed by their unique cell membrane architecture. Nature has developed an elegant solution to target gram-negative strains, namely by conjugating toxic antibiotic warheads to a suitable carrier to facilitate the active import of the drug to a specific target organism. Microcin C7 (McC) is a Trojan horse peptide–conjugated antibiotic that specifically targets enterobacteria by exploiting active import through oligopeptide transport systems. Here, we characterize the molecular mechanism of McC recognition by YejA, the solute binding protein of the Escherichia coli oligopeptide transporter. Structure-guided mutational and functional analysis elucidates the determinants of substrate recognition. We demonstrate that the peptide carrier can serve as a passport for the entry of molecules that are otherwise not taken into E. coli cells. We show that peptide conjugation can remodel the antibiotic spectrum of clinically relevant parent compounds. Bioinformatics analysis reveals a broad distribution of YejA-like transporters in only the Proteobacteria, underscoring the potential for the development of Trojan horse antibiotics that are actively imported into such gram-negative bacteria.
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Jan 2025
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I23-Long wavelength MX
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Yishun
Lu
,
Ramona
Duman
,
James
Beilsten-Edmands
,
Graeme
Winter
,
Mark
Basham
,
Gwyndaf
Evans
,
Jos J. A. G.
Kamps
,
Allen M.
Orville
,
Hok-Sau
Kwong
,
Konstantinos
Beis
,
Wesley
Armour
,
Armin
Wagner
Open Access
Abstract: rocessing of single-crystal X-ray diffraction data from area detectors can be separated into two steps. First, raw intensities are obtained by integration of the diffraction images, and then data correction and reduction are performed to determine structure-factor amplitudes and their uncertainties. The second step considers the diffraction geometry, sample illumination, decay, absorption and other effects. While absorption is only a minor effect in standard macromolecular crystallography (MX), it can become the largest source of uncertainty for experiments performed at long wavelengths. Current software packages for MX typically employ empirical models to correct for the effects of absorption, with the corrections determined through the procedure of minimizing the differences in intensities between symmetry-equivalent reflections; these models are well suited to capturing smoothly varying experimental effects. However, for very long wavelengths, empirical methods become an unreliable approach to model strong absorption effects with high fidelity. This problem is particularly acute when data multiplicity is low. This paper presents an analytical absorption correction strategy (implemented in new software AnACor) based on a volumetric model of the sample derived from X-ray tomography. Individual path lengths through the different sample materials for all reflections are determined by a ray-tracing method. Several approaches for absorption corrections (spherical harmonics correction, analytical absorption correction and a combination of the two) are compared for two samples, the membrane protein OmpK36 GD, measured at a wavelength of λ = 3.54 Å, and chlorite dismutase, measured at λ = 4.13 Å. Data set statistics, the peak heights in the anomalous difference Fourier maps and the success of experimental phasing are used to compare the results from the different absorption correction approaches. The strategies using the new analytical absorption correction are shown to be superior to the standard spherical harmonics corrections. While the improvements are modest in the 3.54 Å data, the analytical absorption correction outperforms spherical harmonics in the longer-wavelength data (λ = 4.13 Å), which is also reflected in the reduced amount of data being required for successful experimental phasing.
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Jun 2024
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Krios I-Titan Krios I at Diamond
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Diamond Proposal Number(s):
[25127]
Open Access
Abstract: Multidrug resistance-associated protein 2 (MRP2/ABCC2) is a polyspecific efflux transporter of organic anions expressed in hepatocyte canalicular membranes. MRP2 dysfunction, in Dubin-Johnson syndrome or by off-target inhibition, for example by the uricosuric drug probenecid, elevates circulating bilirubin glucuronide and is a cause of jaundice. Here, we determine the cryo-EM structure of rat Mrp2 (rMrp2) in an autoinhibited state and in complex with probenecid. The autoinhibited state exhibits an unusual conformation for this class of transporter in which the regulatory domain is folded within the transmembrane domain cavity. In vitro phosphorylation, mass spectrometry and transport assays show that phosphorylation of the regulatory domain relieves this autoinhibition and enhances rMrp2 transport activity. The in vitro data is confirmed in human hepatocyte-like cells, in which inhibition of endogenous kinases also reduces human MRP2 transport activity. The drug-bound state reveals two probenecid binding sites that suggest a dynamic interplay with autoinhibition. Mapping of the Dubin-Johnson mutations onto the rodent structure indicates that many may interfere with the transition between conformational states.
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Mar 2024
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I03-Macromolecular Crystallography
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Diamond Proposal Number(s):
[23620]
Open Access
Abstract: Conjugation is the process by which plasmids, including those that carry antibiotic-resistance genes, are mobilized from one bacterium (the donor) to another (the recipient). The conjugation efficiency of IncF-like plasmids relies on the formation of mating-pair stabilization via intimate interactions between outer membrane proteins on the donor (a plasmid-encoded TraN isoform) and recipient bacteria. Conjugation of the R100-1 plasmid into Escherichia coli and Klebsiella pneumoniae (KP) recipients relies on pairing between the plasmid-encoded TraNα in the donor and OmpW in the recipient. Here, the crystal structure of K. pneumoniae OmpW (OmpWKP) is reported at 3.2 Å resolution. OmpWKP forms an eight-stranded β-barrel flanked by extracellular loops. The structures of E. coli OmpW (OmpWEC) and OmpWKP show high conservation despite sequence variability in the extracellular loops.
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Jan 2024
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