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Protein structure dynamics and interplay: by single-particle electron microscopy
KTH, School of Technology and Health (STH), Structural Biotechnology.
2008 (English)Doctoral thesis, comprehensive summary (Other scientific)
Abstract [en]

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.

Place, publisher, year, edition, pages
Stockholm: KTH , 2008. , 83 p.
Series
Trita-STH : report, ISSN 1653-3836 ; 2008:1
Keyword [en]
biophysics, biochemistry, molecular
National Category
Physical Chemistry
Identifiers
URN: urn:nbn:se:kth:diva-4669ISBN: 978-91-7178-892-4 (print)OAI: oai:DiVA.org:kth-4669DiVA: diva2:13347
Public defence
2008-04-04, Hörsallen plan 4, Novum, Flemingsberg, Huddinge, 13:00
Opponent
Supervisors
Note
QC 20100719Available from: 2008-03-18 Created: 2008-03-18 Last updated: 2010-10-01Bibliographically approved
List of papers
1. A new cryo-EM single-particle ab initio reconstruction method visualizes secondary structure elements in an ATP-fueled AAA+ motor
Open this publication in new window or tab >>A new cryo-EM single-particle ab initio reconstruction method visualizes secondary structure elements in an ATP-fueled AAA+ motor
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2008 (English)In: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 375, no 4, 934-947 p.Article in journal (Refereed) Published
Abstract [en]

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 similar to 660-kDa ATP-fueled AAA+ motor to 7.5 angstrom 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.

Keyword
electron microscopy; cryo-EM; single particle; reconstruction; alignment
National Category
Chemical Sciences
Identifiers
urn:nbn:se:kth:diva-8111 (URN)10.1016/j.jmb.2007.11.028 (DOI)000253098000005 ()
Note
QC 20100719Available from: 2008-03-18 Created: 2008-03-18 Last updated: 2017-12-14Bibliographically approved
2. Cryo-electron microscopy reveals an ATP-fueled and Integrin-I mediated conformational transition of the AAA+ activation complex in R. capsulatus Mg-chelatase
Open this publication in new window or tab >>Cryo-electron microscopy reveals an ATP-fueled and Integrin-I mediated conformational transition of the AAA+ activation complex in R. capsulatus Mg-chelatase
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(English)Manuscript (Other academic)
Keyword
cryo-EM, single-particle, reconstruction, alignment, AAA, BchI, BchD, BchH, Mg-chelatase
National Category
Chemical Sciences
Identifiers
urn:nbn:se:kth:diva-8112 (URN)
Note
QC 20100719Available from: 2008-03-18 Created: 2008-03-18 Last updated: 2010-10-04Bibliographically approved
3. Visualization of a massive TBP-binding coupled histone-fold domain rearrangement within the general transcription factor IID
Open this publication in new window or tab >>Visualization of a massive TBP-binding coupled histone-fold domain rearrangement within the general transcription factor IID
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(English)Article in journal (Other academic) Submitted
Keyword
transcription; TFIID; TBP; TAF; electron microscopy; single-particle; heterogeneity
National Category
Chemical Sciences
Identifiers
urn:nbn:se:kth:diva-8113 (URN)
Note
QS 20120326Available from: 2008-03-18 Created: 2008-03-18 Last updated: 2012-03-26Bibliographically approved
4. The cyclin-dependent kinase 8 module sterically blocks Mediator interactions with RNA polymerase II
Open this publication in new window or tab >>The cyclin-dependent kinase 8 module sterically blocks Mediator interactions with RNA polymerase II
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2006 (English)In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 103, no 43, 15788-15793 p.Article in journal (Refereed) Published
Abstract [en]

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.

Keyword
electron microscopy; transcription; protein structure; yeast; Schizosaccharomyces pombe
National Category
Natural Sciences Natural Sciences
Identifiers
urn:nbn:se:kth:diva-8114 (URN)10.1073/pnas.0607483103 (DOI)000241568500013 ()2-s2.0-33750481873 (Scopus ID)
Note
QC 20100719Available from: 2008-03-18 Created: 2008-03-18 Last updated: 2017-12-14Bibliographically approved

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