ORCID Profile
0000-0002-1910-8017
Does something not look right? The information on this page has been harvested from data sources that may not be up to date. We continue to work with information providers to improve coverage and quality. To report an issue, use the Feedback Form.
In Research Link Australia (RLA), "Research Topics" refer to ANZSRC FOR and SEO codes. These topics are either sourced from ANZSRC FOR and SEO codes listed in researchers' related grants or generated by a large language model (LLM) based on their publications.
Characterisation of Biological Macromolecules | Biochemistry and Cell Biology | Structural Biology (incl. Macromolecular Modelling) | Chemical Characterisation of Materials
Expanding Knowledge in the Physical Sciences | Expanding Knowledge in the Biological Sciences |
Publisher: Elsevier BV
Date: 11-2018
DOI: 10.1016/J.JSB.2018.08.005
Abstract: Cryogenic electron microscopy (cryo-EM) and single-particle analysis enables determination of near-atomic resolution structures of biological molecules. However, large computational requirements limit throughput and rapid testing of new image processing tools. We developed PRIME, an algorithm part of the SIMPLE software suite, for determination of the relative 3D orientations of single-particle projection images. PRIME has primarily found use for generation of an initial ab initio 3D reconstruction. Here we show that the strategy behind PRIME, iterative estimation of per-particle orientation distributions with stochastic hill climbing, provides a competitive approach to near-atomic resolution single-particle 3D reconstruction. A number of mathematical techniques for accelerating the convergence rate are introduced, leading to a speedup of nearly two orders of magnitude. We benchmarked our developments on numerous publicly available data sets and conclude that near-atomic resolution ab initio 3D reconstructions can be obtained with SIMPLE in a matter of hours, using standard over-the-counter CPU workstations.
Publisher: Elsevier BV
Date: 03-2010
DOI: 10.1016/J.STR.2010.01.001
Abstract: 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.
Publisher: American Association for the Advancement of Science (AAAS)
Date: 08-11-2013
Abstract: The regulation of gene expression is critical for almost every aspect of biology. Transcription—generating an RNA copy of a gene—requires the assembly of a large pre-initiation complex (PIC) at every RNA polymerase II (pol II) promoter. Roughly 32 proteins—the subunits of pol II and the general transcription factors—form a PIC that can recognize a minimal TATA-box promoter, select a transcription start site, and synthesize a nascent transcript. Murakami et al. (p. 10.1126/science.1238724 , published online 26 September see the Perspective by Malik and Roeder ) determined the three-dimensional map of the Saccharomyces cerevisiae 30-subunit PIC using cryo-electron microscopy. The saddle-shaped TATA binding protein, the boot-shaped transcription factor IIA (TFIIA), and promoter DNA ∼27 bp downstream of the TATA-box could all be seen. Cross-linking and mass spectrometry was used to determine the spatial proximity of the 30 subunits, revealing that the PIC forms two lobes with TFIIF forming a bridge between them.
Publisher: Elsevier BV
Date: 07-2009
DOI: 10.1016/J.JSB.2009.04.009
Abstract: 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.
Publisher: Elsevier BV
Date: 12-2012
DOI: 10.1016/J.JSB.2012.07.010
Abstract: The open source software suite SIMPLE: Single-particle IMage Processing Linux Engine provides data analysis methods for single-particle cryo-electron microscopy (cryo-EM). SIMPLE addresses the problem of obtaining 3D reconstructions from 2D projections only, without using an input reference volume for approximating orientations. The SIMPLE reconstruction algorithm is tailored to asymmetrical and structurally heterogeneous single-particles. Its basis is global optimization with the use of Fourier common lines. The advance that enables ab initio reconstruction and heterogeneity analysis is the separation of the tasks of in-plane alignment and projection direction determination via bijective orientation search - a new concept in common lines-based strategies. Bijective orientation search ides the configuration space into two groups of paired parameters that are optimized separately. The first group consists of the rotations and shifts in the plane of the projection the second group consists of the projection directions and state assignments. In SIMPLE, ab initio reconstruction is feasible because the 3D in-plane alignment is approximated using reference-free 2D rotational alignment. The subsequent common lines-based search hence searches projection directions and states only. Thousands of class averages are analyzed simultaneously in a matter of hours. Novice SIMPLE users get a head start via the well documented front-end. The structured, object-oriented back-end invites advanced users to develop new alignment and reconstruction algorithms. An overview of the package is presented together with benchmarks on simulated data. Executable binaries, source code, and documentation are available at simple.stanford.edu.
Publisher: American Chemical Society (ACS)
Date: 15-04-2011
DOI: 10.1021/BI200058A
Abstract: 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 erse 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.
Publisher: Elsevier BV
Date: 08-2013
DOI: 10.1016/J.STR.2013.07.002
Abstract: Low-dose electron microscopy of cryo-preserved in idual biomolecules (single-particle cryo-EM) is a powerful tool for obtaining information about the structure and dynamics of large macromolecular assemblies. Acquiring images with low dose reduces radiation damage, preserves atomic structural details, but results in low signal-to-noise ratio of the in idual images. The projection directions of the two-dimensional images are random and unknown. The grand challenge is to achieve the precise three-dimensional (3D) alignment of many (tens of thousands to millions) noisy projection images, which may then be combined to obtain a faithful 3D map. An accurate initial 3D model is critical for obtaining the precise 3D alignment required for high-resolution (<10 Å) map reconstruction. We report a method (PRIME) that, in a single step and without prior structural knowledge, can generate an accurate initial 3D map directly from the noisy images.
Publisher: Elsevier BV
Date: 10-2017
DOI: 10.1016/J.SBI.2017.03.003
Abstract: Cryogenic electron microscopy (cryo-EM) and single-particle analysis now enables the determination of high-resolution structures of macromolecular assemblies that have resisted X-ray crystallography and other approaches. Successful high-resolution structure determination by cryo-EM always depends on the quality of the protein s le. While structural heterogeneity remains a key challenge for cryo-EM, it also represents a rare opportunity to study the intrinsic conformational flexibility of macromolecular assemblies. Here, we review the key technological advancements that have made this 'resolution revolution' possible and give a concise overview of the technical challenges that needed to be overcome to allow high-resolution structure determination.
Publisher: Annual Reviews
Date: 02-06-2015
DOI: 10.1146/ANNUREV-BIOCHEM-060614-034226
Abstract: About 20 years ago, the first three-dimensional (3D) reconstructions at subnanometer ( -Å) resolution of an icosahedral virus assembly were obtained by cryogenic electron microscopy (cryo-EM) and single-particle analysis. Since then, thousands of structures have been determined to resolutions ranging from 30 Å to near atomic ( Å). Almost overnight, the recent development of direct electron detectors and the attendant improvement in analysis software have advanced the technology considerably. Near-atomic-resolution reconstructions can now be obtained, not only for megadalton macromolecular complexes or highly symmetrical assemblies but also for proteins of only a few hundred kilodaltons. We discuss the developments that led to this breakthrough in high-resolution structure determination by cryo-EM and point to challenges that lie ahead.
Publisher: Elsevier BV
Date: 06-2016
DOI: 10.1016/J.STR.2016.04.006
Abstract: A critical step in the analysis of novel cryogenic electron microscopy (cryo-EM) single-particle datasets is the identification of homogeneous subsets of images. Methods for solving this problem are important for data quality assessment, ab initio 3D reconstruction, and analysis of population ersity due to the heterogeneous nature of macromolecules. Here we formulate a stochastic algorithm for identification of homogeneous subsets of images. The purpose of the method is to generate improved 2D class averages that can be used to produce a reliable 3D starting model in a rapid and unbiased fashion. We show that our method overcomes inherent limitations of widely used clustering approaches and proceed to test the approach on six publicly available experimental cryo-EM datasets. We conclude that, in each instance, ab initio 3D reconstructions of quality suitable for initialization of high-resolution refinement are produced from the cluster centers.
Publisher: MDPI AG
Date: 04-07-2019
DOI: 10.3390/IJMS20133290
Abstract: The general transcription factor TFIID is a core promoter selectivity factor that recognizes DNA sequence elements and nucleates the assembly of a pre-initiation complex (PIC). The mechanism by which TFIID recognizes the promoter is poorly understood. The TATA-box binding protein (TBP) is a subunit of the multi-protein TFIID complex believed to be key in this process. We reconstituted transcription from highly purified components on a ribosomal protein gene (RPS5) and discovered that TFIIDΔTBP binds and rearranges the promoter DNA topology independent of TBP. TFIIDΔTBP binds ~200 bp of the promoter and changes the DNA topology to a larger extent than the nucleosome core particle. We show that TBP inhibits the DNA binding activities of TFIIDΔTBP and conclude that the complete TFIID complex may represent an auto-inhibited state. Furthermore, we show that the DNA binding activities of TFIIDΔTBP are required for assembly of a PIC poised to select the correct transcription start site (TSS).
Publisher: Elsevier BV
Date: 12-2020
Publisher: Wiley
Date: 06-09-2017
DOI: 10.1002/PRO.3266
Publisher: Elsevier BV
Date: 07-2010
DOI: 10.1016/J.STR.2010.06.001
Abstract: 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 s les 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.
Location: United States of America
Start Date: 2017
End Date: 12-2019
Amount: $354,224.00
Funder: Australian Research Council
View Funded Activity