scholarly journals The domain architecture of JBP1 suggests synergy between J-base DNA binding and thymidine hydroxylase activity

2018 ◽  
Author(s):  
Athanassios Adamopoulos ◽  
Tatjana Heidebrecht ◽  
Jeroen Roosendaal ◽  
Wouter G Touw ◽  
Isabelle Phan ◽  
...  
Keyword(s):  
Dna Binding ◽  
Domain Architecture ◽  
Three Dimensional ◽  
Specific Sequence ◽  
X Ray ◽  
Tet Proteins ◽  
X Ray Scattering ◽  
Sequence Patterns ◽  
Three Dimensional Models

JBP1 (J-DNA Binding Protein 1) contributes to biosynthesis and maintenance of base J (β-D-glucosyl-hydroxymethyluracil), a modification of thymidine (T) confined to pathogenic protozoa. JBP1 has two known functional domains: an N-terminal thymidine hydroxylase (TH) homologous to the 5-methylcytosine hydroxylase domain in TET proteins; and a J-DNA binding domain (JDBD) that resides in the middle of JBP1. Here we show that removing JDBD from JBP1 results in a soluble protein (Δ-JDBD) with the N- and C-terminal regions tightly associated together in a well-ordered domain. This Δ-JDBD domain retains thymidine hydroxylation activity in vitro, but displays a fifteen-fold lower apparent rate of hydroxylation compared to JBP1. Small Angle X-ray Scattering (SAXS) experiments on JBP1 and JDBD in the presence and absence of J-DNA, and on Δ-JDBD, allowed us to generate low-resolution three-dimensional models. We conclude that Δ-JDBD, and not the N-terminal region of JBP1 alone, is a distinct folding unit. Our SAXS-based model supports the notion that binding of JDBD specifically to J-DNA can facilitate hydroxylation a T 12-14 bp downstream on the complementary strand of the J-recognition site. We postulate that insertion of the JDBD module in the Δ-JDBD scaffold during evolution provided a mechanism to synergize between J recognition and T hydroxylation, ensuring inheritance of J in specific sequence patterns following DNA replication.

scholarly journals Two practical Java software tools for small-angle X-ray scattering analysis of biomolecules

2014 ◽  
Vol 47 (2) ◽  
pp. 810-815 ◽  
Author(s):  
Andreas Hofmann ◽  
Andrew E. Whitten
Keyword(s):  
Small Angle ◽  
Three Dimensional ◽  
Small Angle Scattering ◽  
Ease Of Use ◽  
Scattering Data ◽  
X Ray ◽  
X Ray Scattering ◽  
Angle Scattering ◽  
Three Dimensional Models ◽  
Ray Scattering

Small-angle X-ray scattering has established itself as a common technique in structural biology research. Here, two novel Java applications to aid modelling of three-dimensional macromolecular structures based on small-angle scattering data are described.MolScatis an application that computes small-angle scattering intensities from user-provided three-dimensional models. The program can fit the theoretical scattering intensities to experimental X-ray scattering data.SAFIRis a program for interactive rigid-body modelling into low-resolution shapes restored from small-angle scattering data. The program has been designed with an emphasis on ease of use and intuitive handling. An embedded version ofMolScatis used to enable quick evaluation of the fit between the model and experimental scattering data.SAFIRalso provides options to refine macromolecular complexes with optional user-specified restraints against scattering data by means of a Monte Carlo approach.

scholarly journals Structural analysis of tRNA(His) guanylyltransferase comlexed with tRNA

2014 ◽  
Vol 70 (a1) ◽  
pp. C1816-C1816
Author(s):  
Akiyoshi Nakamura ◽  
Taiki Nemoto ◽  
Isao Tanaka ◽  
Min Yao
Keyword(s):  
Crystal Structures ◽  
Domain Architecture ◽  
Gel Filtration ◽  
Three Dimensional ◽  
Trna Synthetase ◽  
Dimensional Structure ◽  
Catalytic Core ◽  
X Ray ◽  
Catalytic Activation ◽  
X Ray Scattering

tRNA(His) guanylyltransferase (Thg1) of eukaryote adds a guanylate to the 5' end of immature or incorrectly processed tRNAs (3'-5' polymerization) by three reaction steps: adenylylation; guanylylation and dephosphorylation. This additional guanylate provides the major identity element for histidyl-tRNA synthetase to recognize its cognate substrate tRNA(His) and differentiates tRNA(His) from the pool of tRNAs present in the cell (1). Previous studies indicate that Thg1 is a structural homolog of canonical 5'-3' polymerases in the catalytic core with no obvious conservation of the amino acid sequence(2). However, the substrate binding of Thg1 is unclear and requires information on the three-dimensional structure in complex with tRNA. In this study, we determined the crystal structures of Thg1 from Candida albicans (CaThg1) in tRNA-bound (CaThg1-tRNA), ATP-bound (CaThg1-ATP), and GTP-bound (CaThg1-GTP) form, and elucidated how Thg1 functions as a reverse polymerase to add nucleotide(3). The crystal structures of CaThg1-tRNA complex shows that two tRNAs are bound to tetrameric Thg1 in parallel orientation which is consistent with SAXS (Small angle X-ray scattering) and gel filtration analysis. One tRNA interacts with three monomers for its positioning, anticodon recognition, and catalytic activation. The end of the acceptor stem and the anticodon loop are both recognized by the same sub-domain belonging to the different monomers. Moreover, the structural comparison of Thg1-tRNA with canonical 5'-3' polymerase shows that the domain architecture of Thg1 is reversed to that of canonical 5'-3' polymerase.

Time-Resolved Cryo-Electron Microscopy of Oscillating Microtubules

1990 ◽  
Vol 48 (1) ◽  
pp. 502-503
Author(s):  
Eva-Maria Mandelkow ◽  
Ron Milligan
Keyword(s):  
Electron Microscopy ◽  
Dynamic Instability ◽  
Entire Solution ◽  
Reaction Scheme ◽  
Time Resolved ◽  
X Ray ◽  
X Ray Scattering ◽  
A Chain ◽  
Ray Scattering

Microtubules form part of the cytoskeleton of eukaryotic cells. They are hollow libers of about 25 nm diameter made up of 13 protofilaments, each of which consists of a chain of heterodimers of α-and β-tubulin. Microtubules can be assembled in vitro at 37°C in the presence of GTP which is hydrolyzed during the reaction, and they are disassembled at 4°C. In contrast to most other polymers microtubules show the behavior of “dynamic instability”, i.e. they can switch between phases of growth and phases of shrinkage, even at an overall steady state [1]. In certain conditions an entire solution can be synchronized, leading to autonomous oscillations in the degree of assembly which can be observed by X-ray scattering (Fig. 1), light scattering, or electron microscopy [2-5]. In addition such solutions are capable of generating spontaneous spatial patterns [6].In an earlier study we have analyzed the structure of microtubules and their cold-induced disassembly by cryo-EM [7]. One result was that disassembly takes place by loss of protofilament fragments (tubulin oligomers) which fray apart at the microtubule ends. We also looked at microtubule oscillations by time-resolved X-ray scattering and proposed a reaction scheme [4] which involves a cyclic interconversion of tubulin, microtubules, and oligomers (Fig. 2). The present study was undertaken to answer two questions: (a) What is the nature of the oscillations as seen by time-resolved cryo-EM? (b) Do microtubules disassemble by fraying protofilament fragments during oscillations at 37°C?

scholarly journals Polymorphic Protective Dps–DNA Co-Crystals by Cryo Electron Tomography and Small Angle X-Ray Scattering

Biomolecules ◽  
2019 ◽  
Vol 10 (1) ◽  
pp. 39 ◽  
Author(s):  
Roman Kamyshinsky ◽  
Yury Chesnokov ◽  
Liubov Dadinova ◽  
Andrey Mozhaev ◽  
Ivan Orlov ◽  
...  
Keyword(s):  
Small Angle ◽  
Electron Tomography ◽  
Actual Structure ◽  
X Ray ◽  
Cell Parameters ◽  
X Ray Scattering ◽  
Cryo Electron Tomography ◽  
Dps Protein ◽  
Ray Scattering

Rapid increase of intracellular synthesis of specific histone-like Dps protein that binds DNA to protect the genome against deleterious factors leads to in cellulo crystallization—one of the most curious processes in the area of life science at the moment. However, the actual structure of the Dps–DNA co-crystals remained uncertain in the details for more than two decades. Cryo-electron tomography and small-angle X-ray scattering revealed polymorphous modifications of the co-crystals depending on the buffer parameters. Two different types of the Dps–DNA co-crystals are formed in vitro: triclinic and cubic. Three-dimensional reconstruction revealed DNA and Dps molecules in cubic co-crystals, and the unit cell parameters of cubic lattice were determined consistently by both methods.

scholarly journals Protective Dps–DNA co‐crystallization in stressed cells: an in vitro structural study by small‐angle X‐ray scattering and cryo‐electron tomography

FEBS Letters ◽  
2019 ◽  
Vol 593 (12) ◽  
pp. 1360-1371 ◽  
Author(s):  
Liubov A. Dadinova ◽  
Yurii M. Chesnokov ◽  
Roman A. Kamyshinsky ◽  
Ivan A. Orlov ◽  
Maxim V. Petoukhov ◽  
...  
Keyword(s):  
Structural Study ◽  
Small Angle ◽  
Electron Tomography ◽  
X Ray ◽  
X Ray Scattering ◽  
Cryo Electron Tomography ◽  
Stressed Cells ◽  
Ray Scattering

X-ray imaging with ultra-small-angle X-ray scattering as a contrast mechanism

2004 ◽  
Vol 37 (5) ◽  
pp. 757-765 ◽  
Author(s):  
L. E. Levine ◽  
G. G. Long
Keyword(s):  
Small Angle ◽  
Imaging Technique ◽  
Three Dimensional ◽  
Sample Volume ◽  
Contrast Imaging ◽  
X Ray ◽  
X Ray Scattering ◽  
X Ray Imaging ◽  
Contrast Mechanism ◽  
Ray Scattering

A new transmission X-ray imaging technique using ultra-small-angle X-ray scattering (USAXS) as a contrast mechanism is described. USAXS imaging can sometimes provide contrast in cases where radiography and phase-contrast imaging are unsuccessful. Images produced at different scattering vectors highlight different microstructural features within the same sample volume. When used in conjunction with USAXS scans, USAXS imaging provides substantial quantitative and qualitative three-dimensional information on the sizes, shapes and spatial arrangements of the scattering objects. The imaging technique is demonstrated on metal and biological samples.

scholarly journals Sequence and Structure of Human Rhinoviruses Reveal the Basis of Receptor Discrimination

2003 ◽  
Vol 77 (12) ◽  
pp. 6923-6930 ◽  
Author(s):  
Marketa Vlasak ◽  
Soile Blomqvist ◽  
Tapani Hovi ◽  
Elizabeth Hewat ◽  
Dieter Blaas
Keyword(s):  
Low Density Lipoprotein ◽  
Three Dimensional ◽  
Density Lipoprotein ◽  
Major Group ◽  
Amino Acid Residues ◽  
X Ray ◽  
Vldl Receptor ◽  
Capsid Protein Vp1 ◽  
Three Dimensional Models ◽  
Positively Charged

ABSTRACT The sequences of the capsid protein VP1 of all minor receptor group human rhinoviruses were determined. A phylogenetic analysis revealed that minor group HRVs were not more related to each other than to the nine major group HRVs whose sequences are known. Examination of the surface exposed amino acid residues of HRV1A and HRV2, whose X-ray structures are available, and that of three-dimensional models computed for the remaining eight minor group HRVs indicated a pattern of positively charged residues within the region, which, in HRV2, was shown to be the binding site of the very-low-density lipoprotein (VLDL) receptor. A lysine in the HI loop of VP1 (K224 in HRV2) is strictly conserved within the minor group. It lies in the middle of the footprint of a single repeat of the VLDL receptor on HRV2. Major group virus serotypes exhibit mostly negative charges at the corresponding positions and do not bind the negatively charged VLDL receptor, presumably because of charge repulsion.

scholarly journals Structural Dynamics and Single-Stranded DNA Binding Activity of the Three N-Terminal Domains of the Large Subunit of Replication Protein A from Small Angle X-ray Scattering

Biochemistry ◽  
2010 ◽  
Vol 49 (13) ◽  
pp. 2880-2889 ◽  
Author(s):  
Dalyir I. Pretto ◽  
Susan Tsutakawa ◽  
Chris A. Brosey ◽  
Amalchi Castillo ◽  
Marie-Eve Chagot ◽  
...  
Keyword(s):  
Dna Binding ◽  
Structural Dynamics ◽  
Small Angle ◽  
Large Subunit ◽  
Protein A ◽  
Replication Protein A ◽  
Binding Activity ◽  
X Ray ◽  
Dna Binding Activity ◽  
X Ray Scattering

scholarly journals Cell Cycle Localization, Dimerization, and Binding Domain Architecture of the Telomere Protein cPot1

2004 ◽  
Vol 24 (5) ◽  
pp. 2091-2102 ◽  
Author(s):  
Chao Wei ◽  
Carolyn M. Price
Keyword(s):  
Cell Cycle ◽  
Dna Binding ◽  
Binding Site ◽  
Domain Architecture ◽  
Binding Domain ◽  
Binding Motif ◽  
Dna Binding Domain ◽  
Telomere Protein ◽  
Ob Fold

ABSTRACT Pot1 is a single-stranded-DNA-binding protein that recognizes telomeric G-strand DNA. It is essential for telomere capping in Saccharomyces pombe and regulates telomere length in humans. Human Pot1 also interacts with proteins that bind the duplex region of the telomeric tract. Thus, like Cdc13 from S. cerevisiae, Pot 1 may have multiple roles at the telomere. We show here that endogenous chicken Pot1 (cPot1) is present at telomeres during periods of the cell cycle when t loops are thought to be present. Since cPot1 can bind internal loops and directly adjacent DNA-binding sites, it is likely to fully coat and protect both G-strand overhangs and the displaced G strand of a t loop. The minimum binding site of cPot1 is double that of the S. pombe DNA-binding domain. Although cPot can self associate, dimerization is not required for DNA binding and hence does not explain the binding-site duplication. Instead, the DNA-binding domain appears to be extended to contain a second binding motif in addition to the conserved oligonucleotide-oligosaccharide (OB) fold present in other G-strand-binding proteins. This second motif could be another OB fold. Although dimerization is inefficient in vitro, it may be regulated in vivo and could promote association with other telomere proteins and/or telomere compaction.

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