Krios III-Titan Krios III at Diamond
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Zhen
Hou
,
Yao
Shen
,
Stanley
Fronik
,
Juan
Shen
,
Jiong
Shi
,
Jialu
Xu
,
Long
Chen
,
Nathan
Hardenbrook
,
Alan N.
Engelman
,
Christopher
Aiken
,
Peijun
Zhang
Diamond Proposal Number(s):
[29812]
Open Access
Abstract: Lentiviruses, such as HIV-1, infect non-dividing cells by traversing the nuclear pore complex (NPC); however, the detailed molecular processes remain unclear. Here we reconstituted functional HIV-1 nuclear import using permeabilized T cells and isolated HIV-1 cores, which significantly increases import events, and developed an integrated three-dimensional cryo-correlative workflow to specifically target and image 1,489 native HIV-1 cores at 4 distinct nuclear import stages using cryo-electron tomography. We found HIV-1 nuclear import depends on both capsid elasticity and nuclear pore adaptability. The NPC acts as a selective filter, preferentially importing smaller cores, while expanding and deforming to accommodate their passage. Brittle mutant cores fail to enter the NPC, while CPSF6-binding-deficient cores enter but stall within the NPC, leading to impaired nuclear import. This study uncovers the interplay between the HIV-1 core and the NPC and provides a framework to dissect HIV-1 nuclear import and downstream events, such as uncoating and integration.
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Jul 2025
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I04-Macromolecular Crystallography
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Hugo
Belda
,
David
Bradley
,
Evangelos
Christodoulou
,
Stephanie D.
Nofal
,
Malgorzata
Broncel
,
David
Jones
,
Heledd
Davies
,
M. Teresa
Bertran
,
Andrew G.
Purkiss
,
Roksana W.
Ogrodowicz
,
Dhira
Joshi
,
Nicola
O’reilly
,
Louise
Walport
,
Andrew
Powell
,
David
House
,
Svend
Kjaer
,
Antoine
Claessens
,
Christian R.
Landry
,
Moritz
Treeck
Diamond Proposal Number(s):
[25587]
Open Access
Abstract: Of 250 Plasmodium species, 6 infect humans, with P. falciparum causing over 95% of 600,000 annual malaria-related deaths. Its pathology arises from host cell remodelling driven by over 400 exported parasite proteins, including the FIKK kinase family. About one million years ago, a bird-infecting Plasmodium species crossed into great apes and a single non-exported FIKK kinase gained an export element. This led to a rapid expansion into 15–21 atypical, exported Ser/Thr effector kinases. Here, using genomic and proteomic analyses, we demonstrate FIKK differentiation via changes in subcellular localization, expression timing and substrate motifs, which supports an individual important role in host–pathogen interactions. Structural data and AlphaFold2 predictions reveal fast-evolving loops in the kinase domain that probably enabled rapid functional diversification for substrate preferences. One FIKK evolved exclusive tyrosine phosphorylation, previously thought absent in Plasmodium. Despite divergence of substrate preferences, the atypical ATP binding pocket is conserved and we identified a single compound that inhibits all FIKKs. A pan-specific inhibitor could reduce resistance development and improve malaria control strategies.
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Jun 2025
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I03-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
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Thomas C.
Mclean
,
Francisco
Balaguer-Pérez
,
Joshua
Chandanani
,
Christopher M.
Thomas
,
Clara
Aicart-Ramos
,
Sophia
Burick
,
Paul Dominic B.
Olinares
,
Giulia
Gobbato
,
Julia E. A.
Mundy
,
Brian T.
Chait
,
David M.
Lawson
,
Seth A.
Darst
,
Elizabeth A.
Campbell
,
Fernando
Moreno-Herrero
,
Tung
Le
Diamond Proposal Number(s):
[25108]
Open Access
Abstract: Examples of long-range gene regulation in bacteria are rare and generally thought to involve DNA looping. Here, using a combination of biophysical approaches including X-ray crystallography and single-molecule analysis for the KorB–KorA system in Escherichia coli, we show that long-range gene silencing on the plasmid RK2, a source of multi-drug resistance across diverse Gram-negative bacteria, is achieved cooperatively by a DNA-sliding clamp, KorB, and a clamp-locking protein, KorA. We show that KorB is a CTPase clamp that can entrap and slide along DNA to reach distal target promoters up to 1.5 kb away. We resolved the tripartite crystal structure of a KorB–KorA–DNA co-complex, revealing that KorA latches KorB into a closed clamp state. DNA-bound KorA thus stimulates repression by stalling KorB sliding at target promoters to occlude RNA polymerase holoenzymes. Together, our findings explain the mechanistic basis for KorB role switching from a DNA-sliding clamp to a co-repressor and provide an alternative mechanism for long-range regulation of gene expression in bacteria.
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Jan 2025
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I24-Microfocus Macromolecular Crystallography
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Phillip
Nußbaum
,
Danguole
Kureisaite-Ciziene
,
Dom
Bellini
,
Chris
Van Der Does
,
Marko
Kojic
,
Najwa
Taib
,
Anna
Yeates
,
Maxime
Tourte
,
Simonetta
Gribaldo
,
Martin
Loose
,
Jan
Lowe
,
Sonja-Verena
Albers
Diamond Proposal Number(s):
[28677]
Abstract: Cell division in all domains of life requires the orchestration of many proteins, but in Archaea most of the machinery remains poorly characterized. Here we investigate the FtsZ-based cell division mechanism in Haloferax volcanii and find proteins containing photosynthetic reaction centre (PRC) barrel domains that play an essential role in archaeal cell division. We rename these proteins cell division protein B 1 (CdpB1) and CdpB2. Depletions and deletions in their respective genes cause severe cell division defects, generating drastically enlarged cells. Fluorescence microscopy of tagged FtsZ1, FtsZ2 and SepF in CdpB1 and CdpB2 mutant strains revealed an unusually disordered divisome that is not organized into a distinct ring-like structure. Biochemical analysis shows that SepF forms a tripartite complex with CdpB1/2 and crystal structures suggest that these two proteins might form filaments, possibly aligning SepF and the FtsZ2 ring during cell division. Overall our results indicate that PRC-domain proteins play essential roles in FtsZ-based cell division in Archaea.
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Mar 2024
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I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[2452]
Open Access
Abstract: Phages can use a small-molecule communication arbitrium system to coordinate lysis–lysogeny decisions, but the underlying mechanism remains unknown. Here we determined that the arbitrium system in Bacillus subtilis phage phi3T modulates the bacterial toxin–antitoxin system MazE–MazF to regulate the phage life cycle. We show that phi3T expresses AimX and YosL, which bind to and inactivate MazF. AimX also inhibits the function of phi3T_93, a protein that promotes lysogeny by binding to MazE and releasing MazF. Overall, these mutually exclusive interactions promote the lytic cycle of the phage. After several rounds of infection, the phage-encoded AimP peptide accumulates intracellularly and inactivates the phage antiterminator AimR, a process that eliminates aimX expression from the aimP promoter. Therefore, when AimP increases, MazF activity promotes reversion back to lysogeny, since AimX is absent. Altogether, our study reveals the evolutionary strategy used by arbitrium to control lysis–lysogeny by domesticating and fine-tuning a phage-defence mechanism.
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Jan 2024
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I03-Macromolecular Crystallography
I04-Macromolecular Crystallography
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Simon G.
Caulton
,
Carey
Lambert
,
Jess
Tyson
,
Paul
Radford
,
Asmaa
Al-Bayati
,
Samuel
Greenwood
,
Emma J.
Banks
,
Callum
Clark
,
Rob
Till
,
Elisabete
Pires
,
R. Elizabeth
Sockett
,
Andrew L.
Lovering
Diamond Proposal Number(s):
[26803, 19880]
Open Access
Abstract: Predatory bacteria, like the model endoperiplasmic bacterium Bdellovibrio bacteriovorus, show several adaptations relevant to their requirements for locating, entering and killing other bacteria. The mechanisms underlying prey recognition and handling remain obscure. Here we use complementary genetic, microscopic and structural methods to address this deficit. During invasion, the B. bacteriovorus protein CpoB concentrates into a vesicular compartment that is deposited into the prey periplasm. Proteomic and structural analyses of vesicle contents reveal several fibre-like proteins, which we name the mosaic adhesive trimer (MAT) superfamily, and show localization on the predator surface before prey encounter. These dynamic proteins indicate a variety of binding capabilities, and we confirm that one MAT member shows specificity for surface glycans from a particular prey. Our study shows that the B. bacteriovorus MAT protein repertoire enables a broad means for the recognition and handling of diverse prey epitopes encountered during bacterial predation and invasion.
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Jan 2024
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Krios I-Titan Krios I at Diamond
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Diamond Proposal Number(s):
[25452]
Open Access
Abstract: Translational control is an essential process for the cell to adapt to varying physiological or environmental conditions. To survive adverse conditions such as low nutrient levels, translation can be shut down almost entirely by inhibiting ribosomal function. Here we investigated eukaryotic hibernating ribosomes from the microsporidian parasite Spraguea lophii in situ by a combination of electron cryo-tomography and single-particle electron cryo-microscopy. We show that microsporidian spores contain hibernating ribosomes that are locked in a dimeric (100S) state, which is formed by a unique dimerization mechanism involving the beak region. The ribosomes within the dimer are fully assembled, suggesting that they are ready to be activated once the host cell is invaded. This study provides structural evidence for dimerization acting as a mechanism for ribosomal hibernation in microsporidia, and therefore demonstrates that eukaryotes utilize this mechanism in translational control.
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Sep 2023
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I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[28534]
Open Access
Abstract: Rodent-borne hantaviruses are prevalent worldwide and upon spillover to human populations, cause severe disease for which no specific treatment is available. A potent antibody response is key for recovery from hantavirus infection. Here we study a highly neutralizing human monoclonal antibody, termed SNV-42, which was derived from a memory B cell isolated from an individual with previous Sin Nombre virus (SNV) infection. Crystallographic analysis demonstrates that SNV-42 targets the Gn subcomponent of the tetrameric (Gn−Gc)4 glycoprotein assembly that is relevant for viral entry. Integration of our 1.8 Å structure with the (Gn−Gc)4 ultrastructure arrangement indicates that SNV-42 targets the membrane-distal region of the virus envelope. Comparison of the SNV-42 paratope encoding variable genes with inferred germline gene segments reveals high sequence conservation, suggesting that germline-encoded antibodies inhibit SNV. Furthermore, mechanistic assays reveal that SNV-42 interferes with both receptor recognition and fusion during host-cell entry. This work provides a molecular-level blueprint for understanding the human neutralizing antibody response to hantavirus infection.
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Jun 2023
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Krios III-Titan Krios III at Diamond
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Diamond Proposal Number(s):
[23268]
Abstract: In most bacteria, cell division relies on the synthesis of new cell wall material by the multiprotein divisome complex. Thus, at the core of the divisome are the transglycosylase FtsW, which synthesises peptidoglycan strands from its substrate Lipid II, and the transpeptidase FtsI that cross-links these strands to form a mesh, shaping and protecting the bacterial cell. The FtsQ–FtsB–FtsL trimeric complex interacts with the FtsWI complex and is involved in regulating its enzymatic activities; however, the structure of this pentameric complex is unknown. Here, we present the cryogenic electron microscopy structure of the FtsWIQBL complex from Pseudomonas aeruginosa at 3.7 Å resolution. Our work reveals intricate structural details, including an extended coiled coil formed by FtsL and FtsB and the periplasmic interaction site between FtsL and FtsI. Our structure explains the consequences of previously reported mutations and we postulate a possible activation mechanism involving a large conformational change in the periplasmic domain. As FtsWIQBL is central to the divisome, our structure is foundational for the design of future experiments elucidating the precise mechanism of bacterial cell division, an important antibiotic target.
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Jun 2023
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I02-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
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Lorena
Zuliani-Alvarez
,
Morten L.
Govasli
,
Jane
Rasaiyaah
,
Chris
Monit
,
Stephen O.
Perry
,
Rebecca P.
Sumner
,
Simon
Mcalpine-Scott
,
Claire
Dickson
,
K. M.
Rifat Faysal
,
Laura
Hilditch
,
Richard J.
Miles
,
Frederic
Bibollet-Ruche
,
Beatrice H.
Hahn
,
Till
Boecking
,
Nikos
Pinotsis
,
Leo C.
James
,
David A.
Jacques
,
Greg J.
Towers
Diamond Proposal Number(s):
[8547, 11235]
Open Access
Abstract: Of the 13 known independent zoonoses of simian immunodeficiency viruses to humans, only one, leading to human immunodeficiency virus (HIV) type 1(M) has become pandemic, causing over 80 million human infections. To understand the specific features associated with pandemic human-to-human HIV spread, we compared replication of HIV-1(M) with non-pandemic HIV-(O) and HIV-2 strains in myeloid cell models. We found that non-pandemic HIV lineages replicate less well than HIV-1(M) owing to activation of cGAS and TRIM5-mediated antiviral responses. We applied phylogenetic and X-ray crystallography structural analyses to identify differences between pandemic and non-pandemic HIV capsids. We found that genetic reversal of two specific amino acid adaptations in HIV-1(M) enables activation of TRIM5, cGAS and innate immune responses. We propose a model in which the parental lineage of pandemic HIV-1(M) evolved a capsid that prevents cGAS and TRIM5 triggering, thereby allowing silent replication in myeloid cells. We hypothesize that this capsid adaptation promotes human-to-human spread through avoidance of innate immune response activation.
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Nov 2022
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