Doctor of Philosophy, Kungliga Tekniska Hogskolan (2011)
Roger Kornberg, Postdoctoral Faculty Sponsor
I am interested in the structural biology of TFIID-dependent transcription. My investigations are focused toward determining the roles of TBP-lacking and TBP-containing TFIID states in the dynamic transcription initiation process. I am also involved in development of new computational methods for 3D structure determination from electron microscopic projection images of single-particles. The collection of algorithms developed so far constitutes a new open source software package SIMPLE (Single-particle IMage Processing Linux Engine. SIMPLE is an easy-to-use image processing system for semi-automated ab initio 3D reconstruction from very challenging single-particle data sets, involving asymmetrical and structurally heterogeneous particles. Structures determined so far include the AAA+ assemblies of cobalt and magnesium chelatase, different conformations of the EF-G bound ribosome, different states of RNA polymerase II, as well as TBP-containing and TBP-lacking states of TFIID. Download SIMPLE at: http://simple.stanford.edu
Thioredoxin and thioredoxin reductase can regulate cell metabolism through redox regulation of disulfide bridges or through removal of H(2)O(2). These two enzymatic functions are combined in NADPH-dependent thioredoxin reductase C (NTRC), which contains an N-terminal thioredoxin reductase domain fused with a C-terminal thioredoxin domain. Rice NTRC exists in different oligomeric states, depending on the absence or presence of its NADPH cofactor. It has been suggested that the different oligomeric states may have diverse activity. Thus, the redox status of the chloroplast could influence the oligomeric state of NTRC and thereby its activity. We have characterized the oligomeric states of NTRC from barley (Hordeum vulgare L.). This also includes a structural model of the tetrameric NTRC derived from cryo-electron microscopy and single-particle reconstruction. We conclude that the tetrameric NTRC is a dimeric arrangement of two NTRC homodimers. Unlike that of rice NTRC, the quaternary structure of barley NTRC complexes is unaffected by addition of NADPH. The activity of NTRC was tested with two different enzyme assays. The N-terminal part of NTRC was tested in a thioredoxin reductase assay. A peroxide sensitive Mg-protoporphyrin IX monomethyl ester (MPE) cyclase enzyme system of the chlorophyll biosynthetic pathway was used to test the catalytic ability of both the N- and C-terminal parts of NTRC. The different oligomeric assembly states do not exhibit significantly different activities. Thus, it appears that the activities are independent of the oligomeric state of barley NTRC.
View details for DOI 10.1021/bi200058a
View details for Web of Science ID 000290056000014
View details for PubMedID 21456578
Unfolding proteins are prevented from irreversible aggregation by small heat shock proteins (sHsps) through interactions that depend on a dynamic equilibrium between sHsp subunits and sHsp oligomers. A chloroplast-localized sHsp, Hsp21, provides protection to client proteins to increase plant stress resistance. Structural information is lacking concerning the oligomeric conformation of this sHsp. We here present a structure model of Arabidopsis thaliana Hsp21, obtained by homology modeling, single-particle electron microscopy, and lysine-specific chemical crosslinking. The model shows that the Hsp21 subunits are arranged in two hexameric discs, similar to a cytosolic plant sHsp homolog that has been structurally determined after crystallization. However, the two hexameric discs of Hsp21 are rotated by 25° in relation to each other, suggesting a role for global dynamics in dodecamer function.
View details for DOI 10.1002/pro.560
View details for Web of Science ID 000286963300006
View details for PubMedID 21280121
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.
View details for DOI 10.1016/j.str.2010.06.001
View details for Web of Science ID 000280046300005
View details for PubMedID 20637414
Mg-chelatase catalyzes the first committed step of the chlorophyll biosynthetic pathway, the ATP-dependent insertion of Mg(2+) 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 A, 14 A, and 13 A 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 ATP 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.
View details for DOI 10.1016/j.str.2010.01.001
View details for Web of Science ID 000275492000012
View details for PubMedID 20223218
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 CobT, with molecular masses of 137, 37 and 71kDa, 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 CobT from negatively stained electron microscopy images of the complex. The results show for the first time that subunits CobS and CobT 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.
View details for DOI 10.1016/j.jsb.2009.06.013
View details for Web of Science ID 000268775700006
View details for PubMedID 19545636
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.
View details for DOI 10.1016/j.jsb.2009.04.009
View details for Web of Science ID 000266819300011
View details for PubMedID 19410651