Search Results

You searched for all publications:

with publication states 'Published (Approved)'
published in journal 'The Embo Journal'

Search Again...

These results only include publications that have been reviewed and processed by Diamond Light Source. To also view papers that have not yet been processed click here.

You can use the folder icon under Actions to collate papers as required. You can then click on View Folder in the left-hand navigation to review and/or download your folder.

Exact matches
Related results

56 items found (0 items not approved), displaying 1 to 10.[First/Prev] 1, 2, 3, 4, 5, 6 [Next/Last]

Instruments / Technical Area	Publication Details	Published
I04-Macromolecular Crystallography	Structure and functional mapping of the KRAB‐KAP1 repressor complex Guido A. Stoll , Ninoslav Pandiloski , Christopher H. Douse , Yorgo Modis The Embo Journal 55 DOI: 10.15252/embj.2022111179 Diamond Proposal Number(s): [21426] Open Access Show Abstract ▼ Abstract: Transposable elements are a genetic reservoir from which new genes and regulatory elements can emerge. However, expression of transposable elements can be pathogenic and is therefore tightly controlled. KRAB domain-containing zinc finger proteins (KRAB-ZFPs) recruit the co-repressor KRAB-associated protein 1 (KAP1/TRIM28) to regulate many transposable elements, but how KRAB-ZFPs and KAP1 interact remains unclear. Here, we report the crystal structure of the KAP1 tripartite motif (TRIM) in complex with the KRAB domain from a human KRAB-ZFP, ZNF93. Structure-guided mutations in the KAP1-KRAB binding interface abolished repressive activity in an epigenetic transcriptional silencing assay. Deposition of H3K9me3 over thousands of loci is lost genome-wide in cells expressing a KAP1 variant with mutations that abolish KRAB binding. Our work identifies and functionally validates the KRAB-KAP1 molecular interface, which is critical for a central transcriptional control axis in vertebrates. In addition, the structure-based prediction of KAP1 recruitment efficiency will enable optimization of KRABs used in CRISPRi.	Nov 2022
Krios IV-Titan Krios IV at Diamond	Cryo‐EM structure of the human NKCC1 transporter reveals mechanisms of ion coupling and specificity Caroline Neumann , Lena Lindtoft Rosenbæk , Rasmus Kock Flygaard , Michael Habeck , Jesper Lykkegaard Karlsen , Yong Wang , Kresten Lindorff-Larsen , Hans Henrik Gad , Rune Hartmann , Joseph A. Lyons , Robert A. Fenton , Poul Nissen The Embo Journal 8 DOI: 10.15252/embj.2021110169 Diamond Proposal Number(s): [21404] Open Access Show Abstract ▼ Abstract: The sodium–potassium–chloride transporter NKCC1 of the SLC12 family performs Na+-dependent Cl−- and K+-ion uptake across plasma membranes. NKCC1 is important for regulating cell volume, hearing, blood pressure, and regulation of hyperpolarizing GABAergic and glycinergic signaling in the central nervous system. Here, we present a 2.6 Å resolution cryo-electron microscopy structure of human NKCC1 in the substrate-loaded (Na+, K+, and 2 Cl−) and occluded, inward-facing state that has also been observed for the SLC6-type transporters MhsT and LeuT. Cl− binding at the Cl1 site together with the nearby K+ ion provides a crucial bridge between the LeuT-fold scaffold and bundle domains. Cl−-ion binding at the Cl2 site seems to undertake a structural role similar to conserved glutamate of SLC6 transporters and may allow for Cl−-sensitive regulation of transport. Supported by functional studies in mammalian cells and computational simulations, we describe a putative Na+ release pathway along transmembrane helix 5 coupled to the Cl2 site. The results provide insight into the structure–function relationship of NKCC1 with broader implications for other SLC12 family members.	Oct 2022
Krios I-Titan Krios I at Diamond	Cryo‐EM structures of perforin‐2 in isolation and assembled on a membrane suggest a mechanism for pore formation Xiulian Yu , Tao Ni , George Munson , Peijun Zhang , Robert J. C. Gilbert The Embo Journal 11 DOI: 10.15252/embj.2022111857 Diamond Proposal Number(s): [20223, 21004] Open Access Show Abstract ▼ Abstract: Perforin-2 (PFN2, MPEG1) is a key pore-forming protein in mammalian innate immunity restricting intracellular bacteria proliferation. It forms a membrane-bound pre-pore complex that converts to a pore-forming structure upon acidification; but its mechanism of conformational transition has been debated. Here we used cryo-electron microscopy, tomography and subtomogram averaging to determine structures of PFN2 in pre-pore and pore conformations in isolation and bound to liposomes. In isolation and upon acidification, the pre-assembled complete pre-pore rings convert to pores in both flat ring and twisted conformations. On membranes, in situ assembled PFN2 pre-pores display various degrees of completeness; whereas PFN2 pores are mainly incomplete arc structures that follow the same subunit packing arrangements as found in isolation. Both assemblies on membranes use their P2 β-hairpin for binding to the lipid membrane surface. Overall, these structural snapshots suggest a molecular mechanism for PFN2 pre-pore to pore transition on a targeted membrane, potentially using the twisted pore as an intermediate or alternative state to the flat conformation, with the capacity to cause bilayer distortion during membrane insertion.	Oct 2022
I24-Microfocus Macromolecular Crystallography	Structure of cytosine transport protein CodB provides insight into nucleobase‐cation symporter 1 mechanism Caitlin E. Hatton , Deborah H. Brotherton , Mahalah Spencer , Alexander D Cameron The Embo Journal 19 DOI: 10.15252/embj.2021110527 Diamond Proposal Number(s): [19880] Open Access Show Abstract ▼ Abstract: CodB is a cytosine transporter from the Nucleobase-Cation-Symport-1 (NCS1) transporter family, a member of the widespread LeuT superfamily. Previous experiments with the nosocomial pathogen Pseudomonas aeruginosa have shown CodB as also important for the uptake of 5-fluorocytosine, which has been suggested as a novel drug to combat antimicrobial resistance by suppressing virulence. Here we solve the crystal structure of CodB from Proteus vulgaris, at 2.4 Å resolution in complex with cytosine. We show that CodB carries out the sodium-dependent uptake of cytosine and can bind 5-fluorocytosine. Comparison of the substrate-bound structures of CodB and the hydantoin transporter Mhp1, the only other NCS1 family member for which the structure is known, highlight the importance of the hydrogen bonds that the substrates make with the main chain at the breakpoint in the discontinuous helix, TM6. In contrast to other LeuT superfamily members, neither CodB nor Mhp1 makes specific interactions with residues on TM1. Comparison of the structures provides insight into the intricate mechanisms of how these proteins transport substrates across the plasma membrane.	Jul 2022
Krios III-Titan Krios III at Diamond	Cryo‐EM reveals mechanisms of angiotensin I‐converting enzyme allostery and dimerization Lizelle Lubbe , Bryan Trevor Sewell , Jeremy D. Woodward , Edward D. Sturrock The Embo Journal 66 DOI: 10.15252/embj.2021110550 Diamond Proposal Number(s): [24039] Open Access Show Abstract ▼ Abstract: Hypertension (high blood pressure) is a major risk factor for cardiovascular disease, which is the leading cause of death worldwide. The somatic isoform of angiotensin I-converting enzyme (sACE) plays a critical role in blood pressure regulation, and ACE inhibitors are thus widely used to treat hypertension and cardiovascular disease. Our current understanding of sACE structure, dynamics, function, and inhibition has been limited because truncated, minimally glycosylated forms of sACE are typically used for X-ray crystallography and molecular dynamics simulations. Here, we report the first cryo-EM structures of full-length, glycosylated, soluble sACE (sACES1211). Both monomeric and dimeric forms of the highly flexible apo enzyme were reconstructed from a single dataset. The N- and C-terminal domains of monomeric sACES1211 were resolved at 3.7 and 4.1 Å, respectively, while the interacting N-terminal domains responsible for dimer formation were resolved at 3.8 Å. Mechanisms are proposed for intradomain hinging, cooperativity, and homodimerization. Furthermore, the observation that both domains were in the open conformation has implications for the design of sACE modulators.	Jul 2022
Krios I-Titan Krios I at Diamond	Cooperative amyloid fibre binding and disassembly by the Hsp70 disaggregase Joseph G. Beton , Jim Monistrol , Anne Wentink , Erin C. Johnston , Anthony J. Roberts , Bernd G. Bukau , Bart W. Hoogenboom , Helen R. Saibil The Embo Journal 9 DOI: 10.15252/embj.2021110410 Diamond Proposal Number(s): [20287] Open Access Show Abstract ▼ Abstract: Although amyloid fibres are highly stable protein aggregates, a specific combination of human Hsp70 system chaperones can disassemble them, including fibres formed of α-synuclein, huntingtin, or Tau. Disaggregation requires the ATPase activity of the constitutively expressed Hsp70 family member, Hsc70, together with the J domain protein DNAJB1 and the nucleotide exchange factor Apg2. Clustering of Hsc70 on the fibrils appears to be necessary for disassembly. Here we use atomic force microscopy to show that segments of in vitro assembled α-synuclein fibrils are first coated with chaperones and then undergo bursts of rapid, unidirectional disassembly. Cryo-electron tomography and total internal reflection fluorescence microscopy reveal fibrils with regions of densely bound chaperones, preferentially at one end of the fibre. Sub-stoichiometric amounts of Apg2 relative to Hsc70 dramatically increase recruitment of Hsc70 to the fibres, creating localised active zones that then undergo rapid disassembly at a rate of ~ 4 subunits per second. The observed unidirectional bursts of Hsc70 loading and unravelling may be explained by differences between the two ends of the polar fibre structure.	Jun 2022
B21-High Throughput SAXS	Teneurin4 dimer structures reveal a calcium‐stabilized compact conformation supporting homomeric trans‐interactions Dimphna H Meijer , Cátia P Frias , J. Wouter Beugelink , Yanthi N. Deurloo , Bert J. C. Janssen The Embo Journal 66 DOI: 10.15252/embj.2020107505 Diamond Proposal Number(s): [19800] Open Access Show Abstract ▼ Abstract: Establishment of correct synaptic connections is a crucial step during neural circuitry formation. The Teneurin family of neuronal transmembrane proteins promotes cell–cell adhesion via homophilic and heterophilic interactions, and is required for synaptic partner matching in the visual and hippocampal systems in vertebrates. It remains unclear how individual Teneurins form macromolecular cis- and trans-synaptic protein complexes. Here, we present a 2.7 Å cryo-EM structure of the dimeric ectodomain of human Teneurin4. The structure reveals a compact conformation of the dimer, stabilized by interactions mediated by the C-rich, YD-shell, and ABD domains. A 1.5 Å crystal structure of the C-rich domain shows three conserved calcium binding sites, and thermal unfolding assays and SAXS-based rigid-body modeling demonstrate that the compactness and stability of Teneurin4 dimers are calcium-dependent. Teneurin4 dimers form a more extended conformation in conditions that lack calcium. Cellular assays reveal that the compact cis-dimer is compatible with homomeric trans-interactions. Together, these findings support a role for teneurins as a scaffold for macromolecular complex assembly and the establishment of cis- and trans-synaptic interactions to construct functional neuronal circuits.	Jan 2022
Krios IV-Titan Krios IV at Diamond	Helical ordering of envelope‐associated proteins and glycoproteins in respiratory syncytial virus Michaela J Conley , Judith M. Short , Andrew Burns , James Streetley , Joshua Hutchings , Saskia E. Bakker , B. Joanne Power , Hussain Jaffery , Joanne Haney , Giulia Zanetti , Pablo R. Murcia , Murray Stewart , Rachel Fearns , Swetha Vijayakrishnan , David Bhella The Embo Journal 12 DOI: 10.15252/embj.2021109728 Diamond Proposal Number(s): [16637] Open Access Show Abstract ▼ Abstract: Human respiratory syncytial virus (RSV) causes severe respiratory illness in children and the elderly. Here, using cryogenic electron microscopy and tomography combined with computational image analysis and three-dimensional reconstruction, we show that there is extensive helical ordering of the envelope-associated proteins and glycoproteins of RSV filamentous virions. We calculated a 16 Å resolution sub-tomogram average of the matrix protein (M) layer that forms an endoskeleton below the viral envelope. These data define a helical lattice of M-dimers, showing how M is oriented relative to the viral envelope. Glycoproteins that stud the viral envelope were also found to be helically ordered, a property that was coordinated by the M-layer. Furthermore, envelope glycoproteins clustered in pairs, a feature that may have implications for the conformation of fusion (F) glycoprotein epitopes that are the principal target for vaccine and monoclonal antibody development. We also report the presence, in authentic virus infections, of N-RNA rings packaged within RSV virions. These data provide molecular insight into the organisation of the virion and the mechanism of its assembly.	Dec 2021
Krios III-Titan Krios III at Diamond	Cryo‐EM structure of metazoan TRAPPIII, the multi‐subunit complex that activates the GTPase Rab1 Antonio Galindo , Vicente J. Planelles-Herrero , Gianluca Degliesposti , Sean Munro The Embo Journal 40 DOI: 10.15252/embj.2020107608 Diamond Proposal Number(s): [17434] Open Access Show Abstract ▼ Abstract: The TRAPP complexes are nucleotide exchange factors that play essential roles in membrane traffic and autophagy. TRAPPII activates Rab11, and TRAPPIII activates Rab1, with the two complexes sharing a core of small subunits that affect nucleotide exchange but being distinguished by specific large subunits that are essential for activity in vivo. Crystal structures of core subunits have revealed the mechanism of Rab activation, but how the core and the large subunits assemble to form the complexes is unknown. We report a cryo-EM structure of the entire Drosophila TRAPPIII complex. The TRAPPIII-specific subunits TRAPPC8 and TRAPPC11 hold the catalytic core like a pair of tongs, with TRAPPC12 and TRAPPC13 positioned at the joint between them. TRAPPC2 and TRAPPC2L link the core to the two large arms, with the interfaces containing residues affected by disease-causing mutations. The TRAPPC8 arm is positioned such that it would contact Rab1 that is bound to the core, indicating how the arm could determine the specificity of the complex. A lower resolution structure of TRAPPII shows a similar architecture and suggests that the TRAPP complexes evolved from a single ur-TRAPP.	Jun 2021
Krios III-Titan Krios III at Diamond	Structural basis of TRAPPIII‐mediated Rab1 activation Aaron M. N. Joiner , Ben P. Phillips , Kumar Yugandhar , Ethan J Sanford , Marcus B Smolka , Haiyuan Yu , Elizabeth A. Miller , J Christopher Fromme The Embo Journal 74 DOI: 10.15252/embj.2020107607 Diamond Proposal Number(s): [17434] Open Access Show Abstract ▼ Abstract: The GTPase Rab1 is a master regulator of the early secretory pathway and is critical for autophagy. Rab1 activation is controlled by its guanine nucleotide exchange factor, the multisubunit TRAPPIII complex. Here, we report the 3.7 Å cryo-EM structure of the Saccharomyces cerevisiae TRAPPIII complex bound to its substrate Rab1/Ypt1. The structure reveals the binding site for the Rab1/Ypt1 hypervariable domain, leading to a model for how the complex interacts with membranes during the activation reaction. We determined that stable membrane binding by the TRAPPIII complex is required for robust activation of Rab1/Ypt1 in vitro and in vivo, and is mediated by a conserved amphipathic α-helix within the regulatory Trs85 subunit. Our results show that the Trs85 subunit serves as a membrane anchor, via its amphipathic helix, for the entire TRAPPIII complex. These findings provide a structural understanding of Rab activation on organelle and vesicle membranes.	May 2021

Publications Database

Search Results

You searched for all publications:

Search Again...

Subject Areas (check all that apply) select all

Instruments used to collect data (check all that apply) select all

Collaborations involved (check all that apply) select all

Diamond Offline Facilities used

Relevant Technical Areas (check all that apply) select all

Menu for Anonymous User