| 1. |
- Duan, Ming-Rui, et al.
(författare)
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DNA binding mechanism revealed by high resolution crystal structure of Arabidopsis thaliana WRKY1 protein
- 2007
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Ingår i: Nucleic Acids Research. - : Oxford University Press (OUP). - 1362-4962 .- 0305-1048. ; 35:4, s. 54-1145
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Tidskriftsartikel (refereegranskat)abstract
- WRKY proteins, defined by the conserved WRKYGQK sequence, are comprised of a large superfamily of transcription factors identified specifically from the plant kingdom. This superfamily plays important roles in plant disease resistance, abiotic stress, senescence as well as in some developmental processes. In this study, the Arabidopsis WRKY1 was shown to be involved in the salicylic acid signaling pathway and partially dependent on NPR1; a C-terminal domain of WRKY1, AtWRKY1-C, was constructed for structural studies. Previous investigations showed that DNA binding of the WRKY proteins was localized at the WRKY domains and these domains may define novel zinc-binding motifs. The crystal structure of the AtWRKY1-C determined at 1.6 A resolution has revealed that this domain is composed of a globular structure with five beta strands, forming an antiparallel beta-sheet. A novel zinc-binding site is situated at one end of the beta-sheet, between strands beta4 and beta5. Based on this high-resolution crystal structure and site-directed mutagenesis, we have defined and confirmed that the DNA-binding residues of AtWRKY1-C are located at beta2 and beta3 strands. These results provided us with structural information to understand the mechanism of transcriptional control and signal transduction events of the WRKY proteins.
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| 2. |
- Fu, Tian-Min, et al.
(författare)
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Protein preparation, crystallization and preliminary X-ray analysis of the C-terminal domain of human RSK1 serine/threonine protein kinase
- 2007
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Ingår i: Acta Crystallographica. Section F: Structural Biology and Crystallization Communications. - 2053-230X. ; 63:Pt 12, s. 8-1026
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Tidskriftsartikel (refereegranskat)abstract
- As a substrate of extracellular signal-related kinase (ERK), the p90 ribosome S6 kinase 1 (RSK1) is at the terminus of the Ras/ERK pathway. Residues 411-735 of human RSK1, covering the C-terminal serine/threonine kinase catalytic domain and the functionally important tail, were cloned into an Escherichia coli expression vector. The protein was expressed, purified and crystallized. The crystals diffracted to 2.7 A and belonged to space group P2(1), with unit-cell parameters a = 39.8, b = 143.8, c = 59.9 A, beta = 95.7 degrees.
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| 3. |
- Li, Lanfen, et al.
(författare)
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Structural genomics studies of human caries pathogen Streptococcus mutans
- 2014
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Ingår i: Journal of Structural and Functional Genomics. - : Springer Science and Business Media LLC. - 1345-711X .- 1570-0267. ; 15:3, s. 9-91
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Tidskriftsartikel (refereegranskat)abstract
- Gram-positive bacterium Streptococcus mutans is the primary causative agent of human dental caries. To better understand this pathogen at the atomic structure level and to establish potential drug and vaccine targets, we have carried out structural genomics research since 2005. To achieve the goal, we have developed various in-house automation systems including novel high-throughput crystallization equipment and methods, based on which a large-scale, high-efficiency and low-cost platform has been establish in our laboratory. From a total of 1,963 annotated open reading frames, 1,391 non-membrane targets were selected prioritized by protein sequence similarities to unknown structures, and clustered by restriction sites to allow for cost-effective high-throughput conventional cloning. Selected proteins were over-expressed in different strains of Escherichia coli. Clones expressed soluble proteins were selected, expanded, and expressed proteins were purified and subjected to crystallization trials. Finally, protein crystals were subjected to X-ray analysis and structures were determined by crystallographic methods. Using the previously established procedures, we have so far obtained more than 200 kinds of protein crystals and 100 kinds of crystal structures involved in different biological pathways. In this paper we demonstrate and review a possibility of performing structural genomics studies at moderate laboratory scale. Furthermore, the techniques and methods developed in our study can be widely applied to conventional structural biology research practice.
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| 4. |
- Liu, Cong, et al.
(författare)
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Preparation, crystallization and preliminary X-ray analysis of protein YtlP from Bacillus subtilis
- 2006
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Ingår i: Acta Crystallographica. Section F: Structural Biology and Crystallization Communications. - 2053-230X. ; 62:10, s. 967-969
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Tidskriftsartikel (refereegranskat)abstract
- Bacillus subtilis YtlP is a protein that is predicted to belong to the bacterial and archael 2'-5' RNA-ligase family. It contains 183 residues and two copies of the HXTX sequence motif conserved among proteins belonging to this family. In order to determine the structure of YtlP and to compare it with the paralogue YjcG and identified 2'-5' RNA ligases, the gene ytlP was amplified from B. subtilis genomic DNA and cloned into expression vector pET-21a. The soluble protein was produced in Escherichia coli, purified to homogeneity and crystals suitable for X-ray analysis were obtained. The crystal diffracted to 2.0 angstrom and belonged to space group P2(1)2(1)2(1), with unit-cell parameters a = 34.16, b = 48.54, c = 105.75 angstrom.
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| 5. |
- Ren, Hui, et al.
(författare)
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The crystal structure of human adenylate kinase 6 : An adenylate kinase localized to the cell nucleus
- 2005
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Ingår i: Proceedings of the National Academy of Sciences. - : Proceedings of the National Academy of Sciences. - 0027-8424 .- 1091-6490. ; 102:2, s. 8-303
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Tidskriftsartikel (refereegranskat)abstract
- Adenylate kinases (AKs) play important roles in nucleotide metabolism in all organisms and in cellular energetics by means of phosphotransfer networks in eukaryotes. The crystal structure of a human AK named AK6 was determined by in-house sulfur single-wavelength anomalous dispersion phasing methods and refined to 2.0-A resolution with a free R factor of 21.8%. Sequence analyses revealed that human AK6 belongs to a distinct subfamily of AKs present in all eukaryotic organisms sequenced so far. Enzymatic assays show that human AK6 has properties similar with other AKs, particularly with AK5. Fluorescence microscopy showed that human AK6 is localized predominantly to the nucleus of HeLa cells. The identification of a nuclear-localized AK sheds light on nucleotide metabolism in the nucleus and the energetic communication between mitochondria and nucleus by means of phosphotransfer networks.
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| 6. |
- Su, Xiao Dong, et al.
(författare)
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A large-scale, high-efficiency and low-cost platform for structural genomics studies
- 2006
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Ingår i: Acta Crystallographica. Section D: Biological Crystallography. - 0907-4449. ; 62:Pt 8, s. 51-843
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Tidskriftsartikel (refereegranskat)abstract
- A large-scale, high-efficiency and low-cost platform based on a Beckman Coulter Biomek FX and custom-made automation systems for structural genomics has been set up at Peking University, Beijing, People's Republic of China. This platform has the capacity to process up to 2000 genes per year for structural and functional analyses. Bacillus subtilis, a model organism for Gram-positive bacteria, and Streptococcus mutans, a major pathogen of dental caries, were selected as the main targets. To date, more than 470 B. subtilis and 1200 S. mutans proteins and hundreds of proteins from other sources, including human liver proteins, have been selected as targets for this platform. The selected genes are mainly related to important metabolism pathways and/or have potential relevance for drug design. To date, 40 independent structures have been determined; of these 11 are in the category of novel structures by the criterion of having less than 30% sequence identity to known structures. More than 13 structures were determined by SAD/MAD phasing. The macromolecular crystallography beamline at the Beijing Synchrotron Radiation Facility and modern phasing programs have been crucial components of the operation of the platform. The idea and practice of the genomic approach have been successfully adopted in a moderately funded structural biology program and it is believed this adaptation will greatly improve the production of protein structures. The goal is to be able to solve a protein structure of moderate difficulty at a cost about US 10,000 dollars.
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| 7. |
- Wang, Kai Tuo, et al.
(författare)
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Purification, crystallization and preliminary X-ray crystallographic analysis of 23S RNA m(2)G2445 methyltransferase RlmL from Escherichia coli
- 2010
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Ingår i: Acta Crystallographica. Section F: Structural Biology and Crystallization Communications. - 2053-230X. ; 66:Pt 11, s. 6-1484
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Tidskriftsartikel (refereegranskat)abstract
- The RlmL (YcbY) protein in Escherichia coli is an rRNA methyltransferase that is specific for m(2)G2445 modification of 23S RNA. The rlmL gene was cloned into the expression vector pET28a and expressed in the host E. coli strain BL21 (DE3). Recombinant protein with a six-histidine tag was purified by Ni(2+)-affinity chromatography followed by gel filtration. Crystals were grown using the hanging-drop vapour-diffusion method and a detergent was used as an additive to improve diffraction quality. The final crystals diffracted to 2.2 Å resolution. The crystals belonged to space group P2(1), with unit-cell parameters a = 73.6, b = 140.8, c = 102.9 Å, β = 102.3°. The crystal has a most probable solvent content of 62.8% with two molecules in the asymmetric unit.
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