Publications by authors named "Karen Manalastas-Cantos"

5 Publications

  • Page 1 of 1

Structural analysis of the SRP Alu domain from Plasmodium falciparum reveals a non-canonical open conformation.

Commun Biol 2021 May 20;4(1):600. Epub 2021 May 20.

Heidelberg University Biochemistry Center (BZH), Heidelberg, Germany.

The eukaryotic signal recognition particle (SRP) contains an Alu domain, which docks into the factor binding site of translating ribosomes and confers translation retardation. The canonical Alu domain consists of the SRP9/14 protein heterodimer and a tRNA-like folded Alu RNA that adopts a strictly 'closed' conformation involving a loop-loop pseudoknot. Here, we study the structure of the Alu domain from Plasmodium falciparum (PfAlu), a divergent apicomplexan protozoan that causes human malaria. Using NMR, SAXS and cryo-EM analyses, we show that, in contrast to its prokaryotic and eukaryotic counterparts, the PfAlu domain adopts an 'open' Y-shaped conformation. We show that cytoplasmic P. falciparum ribosomes are non-discriminative and recognize both the open PfAlu and closed human Alu domains with nanomolar affinity. In contrast, human ribosomes do not provide high affinity binding sites for either of the Alu domains. Our analyses extend the structural database of Alu domains to the protozoan species and reveal species-specific differences in the recognition of SRP Alu domains by ribosomes.
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http://dx.doi.org/10.1038/s42003-021-02132-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8137916PMC
May 2021

Anomalous SAXS at P12 beamline EMBL Hamburg: instrumentation and applications.

J Synchrotron Radiat 2021 May 14;28(Pt 3):812-823. Epub 2021 Apr 14.

European Molecular Biology Laboratory (EMBL), Hamburg Outstation c/o DESY, Notkestrasse 85, 22607 Hamburg, Germany.

Small-angle X-ray scattering (SAXS) is an established method for studying nanostructured systems and in particular biological macromolecules in solution. To obtain element-specific information about the sample, anomalous SAXS (ASAXS) exploits changes of the scattering properties of selected atoms when the energy of the incident X-rays is close to the binding energy of their electrons. While ASAXS is widely applied to condensed matter and inorganic systems, its use for biological macromolecules is challenging because of the weak anomalous effect. Biological objects are often only available in small quantities and are prone to radiation damage, which makes biological ASAXS measurements very challenging. The BioSAXS beamline P12 operated by the European Molecular Biology Laboratory (EMBL) at the PETRA III storage ring (DESY, Hamburg) is dedicated to studies of weakly scattering objects. Here, recent developments at P12 allowing for ASAXS measurements are presented. The beamline control, data acquisition and data reduction pipeline of the beamline were adapted to conduct ASAXS experiments. Modelling tools were developed to compute ASAXS patterns from atomic models, which can be used to analyze the data and to help designing appropriate data collection strategies. These developments are illustrated with ASAXS experiments on different model systems performed at the P12 beamline.
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http://dx.doi.org/10.1107/S1600577521003404DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8127372PMC
May 2021

: expanded functionality and new tools for small-angle scattering data analysis.

J Appl Crystallogr 2021 Feb 1;54(Pt 1):343-355. Epub 2021 Feb 1.

European Molecular Biology Laboratory, Hamburg Site, Notkestrasse 85, Building 25 A, Hamburg, 22607, Germany.

The software suite encompasses a number of programs for the processing, visualization, analysis and modelling of small-angle scattering data, with a focus on the data measured from biological macromolecules. Here, new developments in the package are described. They include , for simulating isotropic 2D scattering patterns; , to perform operations on 2D images and masks; , a method for variance estimation of structural invariants through parametric resampling; , which computes the pair distance distribution function by a direct Fourier transform of the scattering data; , to compute the scattering data from a pair distance distribution function, allowing comparison with the experimental data; a new module in for Bayesian consensus-based concentration-independent molecular weight estimation; , an shape analysis method that optimizes the search model directly against the scattering data; , an application to set up the initial search volume for multiphase modelling of membrane proteins; , to perform quasi-atomistic modelling of liposomes with elliptical shapes; , which models conformational changes in nucleic acid structures through normal mode analysis in torsion angle space; , which reconstructs the shape of an unknown intermediate in an evolving system; and and , for modelling multilamellar and asymmetric lipid vesicles, respectively. In addition, technical updates were deployed to facilitate maintainability of the package, which include porting the graphical interface to Qt5, updating - a plugin to run a subset of tools - to be both Python 2 and 3 compatible, and adding utilities to facilitate mmCIF compatibility in future releases. All these features are implemented in , freely available for academic users at https://www.embl-hamburg.de/biosaxs/software.html.
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http://dx.doi.org/10.1107/S1600576720013412DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7941305PMC
February 2021

Solution structure and flexibility of the condensin HEAT-repeat subunit Ycg1.

J Biol Chem 2019 09 26;294(37):13822-13829. Epub 2019 Jul 26.

European Molecular Biology Laboratory, Hamburg Unit, Hamburg 22607, Germany

High-resolution structural analysis of flexible proteins is frequently challenging and requires the synergistic application of different experimental techniques. For these proteins, small-angle X-ray scattering (SAXS) allows for a quantitative assessment and modeling of potentially flexible and heterogeneous structural states. Here, we report SAXS characterization of the condensin HEAT-repeat subunit Ycg1 in solution, complementing currently available high-resolution crystallographic models. We show that the free Ycg1 subunit is flexible in solution but becomes considerably more rigid when bound to its kleisin-binding partner protein Brn1 The analysis of SAXS and dynamic and static multiangle light scattering data furthermore reveals that Ycg1 tends to oligomerize with increasing concentrations in the absence of Brn1. Based on these data, we present a model of the free Ycg1 protein constructed by normal mode analysis, as well as tentative models of Ycg1 dimers and tetramers. These models enable visualization of the conformational transitions that Ycg1 has to undergo to adopt a closed rigid shape and thereby create a DNA-binding surface in the condensin complex.
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http://dx.doi.org/10.1074/jbc.RA119.008661DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6746440PMC
September 2019

Whole-genome sequencing and single nucleotide polymorphisms in multidrug-resistant clinical isolates of Mycobacterium tuberculosis from the Philippines.

J Glob Antimicrob Resist 2018 12 18;15:239-245. Epub 2018 Aug 18.

Philippine Genome Center, University of the Philippines, Diliman, Quezon City 1101, Philippines; Marine Science Institute, University of the Philippines, Diliman, Quezon City 1101, Philippines. Electronic address:

Objectives: Thousands of cases of multidrug-resistant tuberculosis (TB) have been observed in the Philippines, but studies on the Mycobacterium tuberculosis (MTB) genotypes that underlie the observed drug resistance profiles are lacking. This study aimed to analyse the whole genomes of clinical MTB isolates representing various resistance profiles to identify single nucleotide polymorphisms (SNPs) in resistance-associated genes.

Methods: The genomes of ten MTB isolates cultured from banked sputum sources were sequenced. Bioinformatics analysis consisted of assembly, annotation and SNP identification in genes reported to be associated with resistance to isoniazid (INH), rifampicin (RIF), ethambutol (ETH), streptomycin, pyrazinamide (PZA) and fluoroquinolones (FQs).

Results: The draft assemblies covered an average of 97.08% of the expected genome size. Seven of the ten isolates belonged to the Indo-Oceanic lineage/EA12-Manila clade. Two isolates were classified into the Euro-American lineage, whilst the pre-XDR (pre-extensively drug-resistant) isolate was classified under the East Asian/Beijing clade. The SNPs katG Ser315Thr, rpoB Ser450Leu and embB Met306Val were found in INH- (4/7), RIF- (3/6) and ETH-resistant (2/6) isolates, respectively, but not in susceptible isolates. Mutations in the inhA promoter and in the pncA and gyrA genes known to be involved in resistance to INH, PZA and FQs, respectively, were also identified.

Conclusions: This study represents the first effort to investigate the whole genomes of Philippine clinical strains of MTB exhibiting various multidrug resistance profiles. Whole-genome data can provide valuable insights to the mechanistic and epidemiological qualities of TB in a high-burden setting such as the Philippines.
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http://dx.doi.org/10.1016/j.jgar.2018.08.009DOI Listing
December 2018