Change search
CiteExportLink to record
Permanent link

Direct link
Cite
Citation style
  • apa
  • harvard1
  • ieee
  • modern-language-association-8th-edition
  • vancouver
  • Other style
More styles
Language
  • de-DE
  • en-GB
  • en-US
  • fi-FI
  • nn-NO
  • nn-NB
  • sv-SE
  • Other locale
More languages
Output format
  • html
  • text
  • asciidoc
  • rtf
Towards unbiased 3D reconstruction: in single-particle cryo-electron microscopy
KTH, School of Technology and Health (STH), Structural Biotechnology.
2010 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Cryo-electron microscopy of freestanding molecules (single-particles) plays a pivotal role in the difficult and pressing challenge of determining the structures of large macromolecular complexes. Molecular volumes are generated by aligning large sets of randomly oriented two-dimensional (2D) projection images in three dimensions (3D) before reconstruction is performed using tomographic techniques. The increasing popularity of the single-particle method is highly correlated with technical advances in instrumentation and computation. This thesis introduces new computational methods for 3D structure determination from electron microscopic projection images of single molecules. The algorithms have been developed to fill a gap in the single particle methodology – the lack of methods for ab initio 3D reconstruction of asymmetrical or low-symmetry molecules co-existing in different functional states. The proposed approach does not rely on a priori information about the structure or the character of the sample heterogeneity, which minimizes template dependence and makes the methods applicable to a wide range of single molecules. The presented algorithms constitute the basis of a new open source software package - SIMPLE (Single-particle IMage Processing Linux Engine). SIMPLE is an efficient and easy-to-use image processing system for semi-automated ab initio 3D reconstruction from challenging single-particle data sets (asymmetrical particles, significant degree of heterogeneity).

Place, publisher, year, edition, pages
Stockholm: KTH , 2010. , 38 p.
Series
Trita-STH : report, ISSN 1653-3836 ; 2010:7
National Category
Industrial Biotechnology
Identifiers
URN: urn:nbn:se:kth:diva-27612ISBN: 978-91-7415-837-3 (print)OAI: oai:DiVA.org:kth-27612DiVA: diva2:377649
Public defence
2011-01-14, Föreläsningssalen, Novum, Hälsovägen 7, Huddinge, 10:00 (English)
Opponent
Supervisors
Note
QC 20101214Available from: 2010-12-14 Created: 2010-12-14 Last updated: 2010-12-16Bibliographically approved
List of papers
1. Ab initio structure determination from electron microscopic images of single molecules coexisting in different functional states
Open this publication in new window or tab >>Ab initio structure determination from electron microscopic images of single molecules coexisting in different functional states
2010 (English)In: Structure, ISSN 0969-2126, E-ISSN 1878-4186, Vol. 18, no 7, 777-786 p.Article in journal (Refereed) Published
Abstract [en]

We have developed methods for ab initio three-dimensional (3D) structure determination from projection images of randomly oriented single molecules coexisting in multiple functional states, to aid the study of complex samples of macromolecules and nanoparticles by electron microscopy (EM). New algorithms for the determination of relative 3D orientations and conformational state assignment of single-molecule projection images are combined with well-established techniques for alignment and statistical image analysis. We describe how the methodology arrives at homogeneous groups of images aligned in 3D and discuss application to experimental EM data sets of the Escherichia coli ribosome and yeast RNA polymerase II.

Keyword
Proteins, RNA-POLYMERASE-II, CRYO-EM REVEALS, COMMON-LINES, 3-DIMENSIONAL RECONSTRUCTION, CONFORMATIONAL FLEXIBILITY, ORIENTATION REFINEMENT, BIOLOGICAL PARTICLES, 3-D RECONSTRUCTION, PROJECTIONS, RESOLUTION
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:kth:diva-27194 (URN)10.1016/j.str.2010.06.001 (DOI)000280046300005 ()20637414 (PubMedID)2-s2.0-77954629363 (Scopus ID)
Funder
Swedish Research Council
Note
QC 20101209Available from: 2010-12-09 Created: 2010-12-09 Last updated: 2017-12-11Bibliographically approved
2. SIMPLE: an image processing system for ab initio 3D reconstruction in single-particleelectron microscopy
Open this publication in new window or tab >>SIMPLE: an image processing system for ab initio 3D reconstruction in single-particleelectron microscopy
(English)Manuscript (preprint) (Other academic)
Identifiers
urn:nbn:se:kth:diva-27328 (URN)
Note
QC 20101210Available from: 2010-12-09 Created: 2010-12-09 Last updated: 2010-12-14Bibliographically approved
3. High-resolution single-particle orientation refinement based on spectrally self-adapting common lines
Open this publication in new window or tab >>High-resolution single-particle orientation refinement based on spectrally self-adapting common lines
2009 (English)In: Journal of Structural Biology, ISSN 1047-8477, E-ISSN 1095-8657, Vol. 167, no 1, 83-94 p.Article in journal (Refereed) Published
Abstract [en]

Three-dimensional (3D) structure determination from electron microscopic images of single molecules can be difficult for particles with low or no internal symmetry, and for images with low signal-to-noise ratio (SNR), due to the existence of false maxima in the scoring function used for orientation search. In attempt to improve robustness of orientation parameter refinement towards noise and poor starting reconstruction quality, we have developed a method for common lines-based orientation search in Fourier space. The Fourier-space formulation enables inclusion of resolution (spatial frequency of the low-pass limit) as a variable that is adjusted in a particle-dependent, self-adaptive manner. The method allows for the underlying 3D structure to be estimated to high resolution, and requires only a crude, low-resolution reconstruction as starting-point for refinement. Benchmarking of the method is performed on experimental and synthetic data.

Keyword
Electron microscopy, Single-particle, Refinement, Correlation coefficient, Common line
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:kth:diva-27333 (URN)10.1016/j.jsb.2009.04.009 (DOI)000266819300011 ()19410651 (PubMedID)
Note
QC 20101210Available from: 2010-12-09 Created: 2010-12-09 Last updated: 2017-12-11Bibliographically approved
4. The AAA(+) motor complex of subunits CobS and CobT of cobaltochelatase visualized by single particle electron microscopy
Open this publication in new window or tab >>The AAA(+) motor complex of subunits CobS and CobT of cobaltochelatase visualized by single particle electron microscopy
Show others...
2009 (English)In: Journal of Structural Biology, ISSN 1047-8477, E-ISSN 1095-8657, Vol. 167, no 3, 227-234 p.Article in journal (Refereed) Published
Abstract [en]

Cobalamins belong to the tetrapyrrole family of prosthetic groups. The presence of a metal ion is a key feature of these compounds. In the oxygen-dependent (aerobic) cobalamin biosynthetic pathway, cobalt is inserted into a ring-contracted tetrapyrrole called hydrogenobyrinic acid a,c-diamide (HBAD) by a cobaltochelatase that is constituted by three subunits, CobN, CobS and COT, with molecular masses of 137, 37 and 71 kDa, respectively. Based on the similarities with magnesium chelatase, cobaltochelatase has been suggested to belong to the AAA(+) superfamily of proteins. In this paper we present the cloning of the Brucella melitensis cobN, cobS and cobT, the purification of the encoded protein products, and a single-particle reconstruction of the macromolecular assembly formed between CobS and COT from negatively stained electron microscopy images of the complex. The results show for the first time that subunits CobS and COT form a chaperone-like complex, characteristic for the AAA(+) class of proteins. The molecules are arranged in a two-tiered ring structure with the six subunits in each ring organized as a trimer of dimers. The similarity between this structure and that of magnesium chelatase, as well as analysis of the amino acid sequences confirms the suggested evolutionary relationship between the two enzymes.

Keyword
Metallation, Catalysis, Tetrapyrrole, Vitamin B-12, Cobalt
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:kth:diva-27349 (URN)10.1016/j.jsb.2009.06.013 (DOI)000268775700006 ()19545636 (PubMedID)
Note
QC 20101213Available from: 2010-12-13 Created: 2010-12-13 Last updated: 2017-12-11Bibliographically approved
5. ATP-Induced Conformational Dynamics in the AAA plus Motor Unit of Magnesium Chelatase
Open this publication in new window or tab >>ATP-Induced Conformational Dynamics in the AAA plus Motor Unit of Magnesium Chelatase
Show others...
2010 (English)In: Structure, ISSN 0969-2126, E-ISSN 1878-4186, Vol. 18, no 3, 354-365 p.Article in journal (Refereed) Published
Abstract [en]

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.

Keyword
protoporphyrin ix chelatase, rhodobacter-sphaeroides, chlorophyll, biosynthesis, synechocystis pcc6803, electron-microscopy, barley, mutants, mg-chelatase, swiss-model, subunit-d, protein
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:kth:diva-19306 (URN)10.1016/j.str.2010.01.001 (DOI)000275492000012 ()
Note
QC 20110114Available from: 2010-08-05 Created: 2010-08-05 Last updated: 2017-12-12Bibliographically approved
6. NADPH-dependent thioredoxin reductase C and its possible involvement in the cyclase reaction of the chlorophyll biosynthetic pathway
Open this publication in new window or tab >>NADPH-dependent thioredoxin reductase C and its possible involvement in the cyclase reaction of the chlorophyll biosynthetic pathway
Show others...
(English)Manuscript (preprint) (Other academic)
Identifiers
urn:nbn:se:kth:diva-27331 (URN)
Note
QC 20101210Available from: 2010-12-09 Created: 2010-12-09 Last updated: 2010-12-14Bibliographically approved

Open Access in DiVA

fulltext(1162 kB)761 downloads
File information
File name FULLTEXT01.pdfFile size 1162 kBChecksum SHA-512
1f29f9b6ccb09a944e8fcffa02641c61e34a39e288e6b6c0a9b89aabec0e87f5662e639660c3e4a740ae77932596e088b0ed6a4f2bcb7c9a2cb7d47bb300a2c8
Type fulltextMimetype application/pdf

Search in DiVA

By author/editor
Elmlund, Dominika
By organisation
Structural Biotechnology
Industrial Biotechnology

Search outside of DiVA

GoogleGoogle Scholar
Total: 761 downloads
The number of downloads is the sum of all downloads of full texts. It may include eg previous versions that are now no longer available

isbn
urn-nbn

Altmetric score

isbn
urn-nbn
Total: 642 hits
CiteExportLink to record
Permanent link

Direct link
Cite
Citation style
  • apa
  • harvard1
  • ieee
  • modern-language-association-8th-edition
  • vancouver
  • Other style
More styles
Language
  • de-DE
  • en-GB
  • en-US
  • fi-FI
  • nn-NO
  • nn-NB
  • sv-SE
  • Other locale
More languages
Output format
  • html
  • text
  • asciidoc
  • rtf