| 1. |
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| 2. |
- Cheng, Kimberley, et al.
(författare)
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Rapana thomasiana hemocyanin (RtH): Comparison of the two isoforms, RtH1 and RtH2, at 19 angstrom 16 angstrom resolution
- 2006
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Ingår i: Micron. - : Elsevier BV. - 0968-4328 .- 1878-4291. ; 37:6, s. 566-576
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Forskningsöversikt (refereegranskat)abstract
- Three-dimensional (3D) reconstructions of the two 8.4 MDa Rapana thomasiana hemocyanin isoforms, RtH1 and RtH2, have been obtained by cryoelectron microscopy of molecules embedded in vitreous ice and single particle image processing. The final 3D structures of the RtH1 and RtH2 didecamers at 19 angstrom and 16 angstrom resolution, respectively, are very similar to earlier reconstructions of gastropodan hemocyanins, revealing structural features such as the obliquely oriented subunits, the five- and two-fold symmetrical axes. Three new interactions are defined; two of them connecting the arch and the wall while the third is formed between the collar and the wall. The collar-wall connection and one of the arch-wall connections are positioned between two individual subunit dimers, while the second arch-wall connection is located between two subunits within the subunit dimer. All three interactions establish connections to the first tier of the wall. Furthermore, for each interaction we have allocated two first tier functional units most likely involved in forming the connections. (
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| 3. |
- Elmlund, Hans, et al.
(författare)
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A new cryo-EM single-particle Ab initio reconstruction method visualizes secondary structure elements in an ATP-Fueled AAA+ motor
- 2008
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Ingår i: Journal of Molecular Biology. - : Elsevier BV. - 1089-8638 .- 0022-2836. ; 375:4, s. 934-947
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Tidskriftsartikel (refereegranskat)abstract
- The generation of ab initio three-dimensional (3D) models is a bottleneck in the studies of large macromolecular assemblies by single-particle cryo-electron microscopy. We describe here a novel method, in which established methods for two-dimensional image processing are combined with newly developed programs for joint rotational 3D alignment of a large number of class averages (RAD) and calculation of 3D volumes from aligned projections (VolRec). We demonstrate the power of the method by reconstructing an 660-kDa ATP-fueled AAA+ motor to 7.5 Å resolution, with secondary structure elements identified throughout the structure. We propose the method as a generally applicable automated strategy to obtain 3D reconstructions from unstained single particles imaged in vitreous ice.
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| 4. |
- Elmlund, Hans, et al.
(författare)
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Cryo-EM Reveals Promoter DNA Binding and Conformational Flexibility of the General Transcription Factor TFIID
- 2009
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Ingår i: Structure. - : Elsevier BV. - 0969-2126 .- 1878-4186. ; 17:11, s. 1442-1452
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Tidskriftsartikel (refereegranskat)abstract
- The general transcription factor IID (TFIID) is required for initiation of RNA polymerase II-dependent transcription at many eukaryotic promoters. TFIID comprises the TATA-binding protein (TBP) and several conserved TBP-associated factors (TAFs). Recognition of the core promoter by TFIID assists assembly of the preinitiation complex. Using cryo-electron microscopy in combination with methods for ab initio single-particle reconstruction and heterogeneity analysis, we have produced density maps of two conformational states of Schizosaccharomyces pombe TFIID, containing and lacking TBP. We report that TBP-binding is coupled to a massive histone-fold domain rearrangement. Moreover, docking of the TBP-TAF1(N-terminus) atomic structure to the THID map and reconstruction of a TAF-promoter DNA complex helps to account for TAF-dependent regulation of promoter-TBP and promoter-TAF interactions.
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| 5. |
- Elmlund, Hans, 1978-
(författare)
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Protein structure dynamics and interplay : by single-particle electron microscopy
- 2008
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Single-particle cryo-electron microscopy (cryo-EM) is a method capable of obtaining information about the structural organization and dynamics of large macromolecular assemblies. In the late nineties, the method was suggested to have the potential of generating “atomic resolution” reconstructions of particles above a certain mass. However, visualization of secondary structure elements in cryo-EM reconstructions has so far been achieved mainly for highly symmetrical macromolecular assemblies or by using previously existing X-ray structures to solve the initial alignment problem. A factor that severely limits the resolution for low-symmetry (point group symmetry Cn) particles is the problem of ab initio three-dimensional alignment of cryo-EM projection images of proteins in vitreous ice. A more general problem in the field of molecular biology is the study of heterogeneous structural properties of particles in preparations of purified macromolecular complexes. If not resolved, structural heterogeneity limits the achievable resolution of a cryo-EM reconstruction and makes correct biological interpretation difficult. If resolved, the heterogeneity instead offers a tremendous biological insight into the dynamic behaviour of a structure, and statistical information about partitioning over subpopulations with distinct structural features within the ensemble of particles may be gained. This thesis adds to the existing body of methods in the field of single-particle cryo-EM by addressing the problem of ab initio rotational alignment and the problem of resolving structural heterogeneity without using a priori information about the structural variability within large populations of cryo-EM projections of unstained proteins. The thesis aims at making the single-particle cryo-EM method a generally applicable tool for generating subnanometer resolution reconstructions and perform heterogeneity analysis of biological macromolecules.
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| 6. |
- Elmlund, Hans, et al.
(författare)
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The cyclin-dependent kinase 8 module sterically blocks Mediator interactions with RNA polymerase II
- 2006
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Ingår i: Proceedings of the National Academy of Sciences of the United States of America. - : Proceedings of the National Academy of Sciences. - 0027-8424 .- 1091-6490. ; 103:43, s. 15788-15793
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Tidskriftsartikel (refereegranskat)abstract
- CDK8 (cyclin-dependent kinase 8), along with CycC, Med12, and Med13, form a repressive module (the Cdk8 module) that prevents RNA polymerase II (pol II) interactions with Mediator. Here, we report that the ability of the Cdk8 module to prevent pol II interactions is independent of the Cdk8-dependent kinase activity. We use electron microscopy and single-particle reconstruction to demonstrate that the Cdk8 module forms a distinct structural entity that binds to the head and middle region of Mediator, thereby sterically blocking interactions with pol II.
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| 7. |
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| 8. |
- Kronqvist, Nina, et al.
(författare)
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Efficient protein production inspired by how spiders make silk
- 2017
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Ingår i: Nature Communications. - : Nature Publishing Group. - 2041-1723. ; 8
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Tidskriftsartikel (refereegranskat)abstract
- Membrane proteins are targets of most available pharmaceuticals, but they are difficult to produce recombinantly, like many other aggregation-prone proteins. Spiders can produce silk proteins at huge concentrations by sequestering their aggregation-prone regions in micellar structures, where the very soluble N-terminal domain (NT) forms the shell. We hypothesize that fusion to NT could similarly solubilize non-spidroin proteins, and design a charge-reversed mutant (NT star) that is pH insensitive, stabilized and hypersoluble compared to wildtype NT. NT star-transmembrane protein fusions yield up to eight times more of soluble protein in Escherichia coli than fusions with several conventional tags. NT star enables transmembrane peptide purification to homogeneity without chromatography and manufacture of low-cost synthetic lung surfactant that works in an animal model of respiratory disease. NT star also allows efficient expression and purification of non-transmembrane proteins, which are otherwise refractory to recombinant production, and offers a new tool for reluctant proteins in general.
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| 9. |
- Lundqvist, Joakim, et al.
(författare)
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ATP-Induced Conformational Dynamics in the AAA plus Motor Unit of Magnesium Chelatase
- 2010
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Ingår i: Structure. - : Elsevier BV. - 0969-2126 .- 1878-4186. ; 18:3, s. 354-365
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Tidskriftsartikel (refereegranskat)abstract
- Mg-chelatase catalyzes the first committed step of the chlorophyll biosynthetic pathway, the ATP-dependent insertion of Mg2+ into protoporphyrin IX (PPIX). Here we report the reconstruction using single-particle cryo-electron microscopy of the complex between subunits BchD and BchI of Rhodobacter capsulatus Mg-chelatase in the presence of ADP, the nonhydrolyzable ATP analog AMPPNP, and ATP at 7.5 angstrom, 14 angstrom, and 13 angstrom resolution, respectively. We show that the two AAA+ modules of the subunits form a unique complex of 3 dimers related by a three-fold axis. The reconstructions demonstrate substantial differences between the conformations of the complex in the presence of ATIP and ADP, and suggest that the C-terminal integrin-I domains of the BchD subunits play a central role in transmitting conformational changes of BchI to BchD. Based on these data a model for the function of magnesium chelatase is proposed.
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| 10. |
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