Publications by authors named "Marcus Fislage"

10 Publications

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Assessing the JEOL CRYO ARM 300 for high-throughput automated single-particle cryo-EM in a multiuser environment.

IUCrJ 2020 Jul 11;7(Pt 4):707-718. Epub 2020 Jun 11.

Center for Structural Biology, Vlaams Instituut voor Biotechnologie, Pleinlaan 2, Brussels 1050, Belgium.

Single-particle cryo-EM has become an indispensable structural biology method. It requires regular access to high-resolution electron cryogenic microscopes. To fully utilize the capacity of the expensive high-resolution instruments, the time used for data acquisition and the rate of data collection have to be maximized. This in turn requires high stability and high uptime of the instrument. One of the first 300 kV JEOL CRYO ARM 300 microscopes has been installed at the cryo-EM facility BECM at VIB-VUB, Brussels, where the microscope is used for continuous data collection on multiple projects. Here, the suitability and performance of the microscope is assessed for high-throughput single-particle data collection. In particular, the properties of the illumination system, the stage stability and ice contamination rates are reported. The microscope was benchmarked using mouse heavy-chain apoferritin which was reconstructed to a resolution of 1.9 Å. Finally, uptime and throughput statistics of the instrument accumulated during the first six months of the facility operation in user access mode are reported.
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http://dx.doi.org/10.1107/S2052252520006065DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7340256PMC
July 2020

Cryo-EM shows stages of initial codon selection on the ribosome by aa-tRNA in ternary complex with GTP and the GTPase-deficient EF-TuH84A.

Nucleic Acids Res 2018 06;46(11):5861-5874

Department of Biochemistry and Molecular Biophysics, Columbia University, New York, NY, USA.

The GTPase EF-Tu in ternary complex with GTP and aminoacyl-tRNA (aa-tRNA) promotes rapid and accurate delivery of cognate aa-tRNAs to the ribosomal A site. Here we used cryo-EM to study the molecular origins of the accuracy of ribosome-aided recognition of a cognate ternary complex and the accuracy-amplifying role of the monitoring bases A1492, A1493 and G530 of the 16S rRNA. We used the GTPase-deficient EF-Tu variant H84A with native GTP, rather than non-cleavable GTP analogues, to trap a near-cognate ternary complex in high-resolution ribosomal complexes of varying codon-recognition accuracy. We found that ribosome complexes trapped by GTPase-deficicent ternary complex due to the presence of EF-TuH84A or non-cleavable GTP analogues have very similar structures. We further discuss speed and accuracy of initial aa-tRNA selection in terms of conformational changes of aa-tRNA and stepwise activation of the monitoring bases at the decoding center of the ribosome.
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http://dx.doi.org/10.1093/nar/gky346DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6009598PMC
June 2018

Invited review: MnmE, a GTPase that drives a complex tRNA modification reaction.

Biopolymers 2016 Aug;105(8):568-79

Structural Biology Brussels, Vrije Universiteit Brussel, Pleinlaan 2, Brussel, 1050, Belgium.

MnmE is a multi-domain GTPase that is conserved from bacteria to man. Together with its partner protein MnmG it is involved in the synthesis of a tRNA wobble uridine modification. The orthologues of these proteins in eukaryotes are targeted to mitochondria and mutations in the encoding genes are associated with severe mitochondrial diseases. While classical small GTP-binding proteins are regulated via auxiliary GEFs and GAPs, the GTPase activity of MnmE is activated via potassium-dependent homodimerization of its G domains. In this review we focus on the catalytic mechanism of GTP hydrolysis by MnmE and the large scale conformational changes that are triggered throughout the GTPase cycle. We also discuss how these conformational changes might be used to drive and tune the complex tRNA modification reaction. © 2016 Wiley Periodicals, Inc. Biopolymers 105: 568-579, 2016.
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http://dx.doi.org/10.1002/bip.22813DOI Listing
August 2016

SAXS analysis of the tRNA-modifying enzyme complex MnmE/MnmG reveals a novel interaction mode and GTP-induced oligomerization.

Nucleic Acids Res 2014 May 14;42(9):5978-92. Epub 2014 Mar 14.

Structural Biology Research Center, VIB, Pleinlaan 2, 1050 Brussel, Belgium Structural Biology Brussels, Vrije Universiteit Brussel, Pleinlaan 2, 1050 Brussel, Belgium

Transfer ribonucleic acid (tRNA) modifications, especially at the wobble position, are crucial for proper and efficient protein translation. MnmE and MnmG form a protein complex that is implicated in the carboxymethylaminomethyl modification of wobble uridine (cmnm(5)U34) of certain tRNAs. MnmE is a G protein activated by dimerization (GAD), and active guanosine-5'-triphosphate (GTP) hydrolysis is required for the tRNA modification to occur. Although crystal structures of MnmE and MnmG are available, the structure of the MnmE/MnmG complex (MnmEG) and the nature of the nucleotide-induced conformational changes and their relevance for the tRNA modification reaction remain unknown. In this study, we mainly used small-angle X-ray scattering to characterize these conformational changes in solution and to unravel the mode of interaction between MnmE, MnmG and tRNA. In the nucleotide-free state MnmE and MnmG form an unanticipated asymmetric α2β2 complex. Unexpectedly, GTP binding promotes further oligomerization of the MnmEG complex leading to an α4β2 complex. The transition from the α2β2 to the α4β2 complex is fast, reversible and coupled to GTP binding and hydrolysis. We propose a model in which the nucleotide-induced changes in conformation and oligomerization of MnmEG form an integral part of the tRNA modification reaction cycle.
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http://dx.doi.org/10.1093/nar/gku213DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4027165PMC
May 2014

Structure of an early native-like intermediate of β2-microglobulin amyloidogenesis.

Protein Sci 2013 Oct 20;22(10):1349-57. Epub 2013 Aug 20.

Structural Biology Research Centre, VIB, Pleinlaan 2, 1050, Brussel, Belgium; Structural Biology Brussels, Vrije Universiteit Brussel, Pleinlaan 2, 1050, Brussel, Belgium.

To investigate early intermediates of β2-microglobulin (β2m) amyloidogenesis, we solved the structure of β2m containing the amyloidogenic Pro32Gly mutation by X-ray crystallography. One nanobody (Nb24) that efficiently blocks fibril elongation was used as a chaperone to co-crystallize the Pro32Gly β2m monomer under physiological conditions. The complex of P32G β2m with Nb24 reveals a trans peptide bond at position 32 of this amyloidogenic variant, whereas Pro32 adopts the cis conformation in the wild-type monomer, indicating that the cis to trans isomerization at Pro32 plays a critical role in the early onset of β2m amyloid formation.
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http://dx.doi.org/10.1002/pro.2321DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3795493PMC
October 2013

NrdH-redoxin of Mycobacterium tuberculosis and Corynebacterium glutamicum dimerizes at high protein concentration and exclusively receives electrons from thioredoxin reductase.

J Biol Chem 2013 Mar 28;288(11):7942-7955. Epub 2013 Jan 28.

Department of Structural Biology, Vlaams Instituut voor Biotechnologie, 1050 Brussels, Belgium; Structural Biology Brussels, Vrije Universiteit Brussel, 1050 Brussels, Belgium; Brussels Center for Redox Biology, 1050 Brussels, Belgium. Electronic address:

NrdH-redoxins are small reductases with a high amino acid sequence similarity with glutaredoxins and mycoredoxins but with a thioredoxin-like activity. They function as the electron donor for class Ib ribonucleotide reductases, which convert ribonucleotides into deoxyribonucleotides. We solved the x-ray structure of oxidized NrdH-redoxin from Corynebacterium glutamicum (Cg) at 1.5 Å resolution. Based on this monomeric structure, we built a homology model of NrdH-redoxin from Mycobacterium tuberculosis (Mt). Both NrdH-redoxins have a typical thioredoxin fold with the active site CXXC motif located at the N terminus of the first α-helix. With size exclusion chromatography and small angle x-ray scattering, we show that Mt_NrdH-redoxin is a monomer in solution that has the tendency to form a non-swapped dimer at high protein concentration. Further, Cg_NrdH-redoxin and Mt_NrdH-redoxin catalytically reduce a disulfide with a specificity constant 1.9 × 10(6) and 5.6 × 10(6) M(-1) min(-1), respectively. They use a thiol-disulfide exchange mechanism with an N-terminal cysteine pKa lower than 6.5 for nucleophilic attack, whereas the pKa of the C-terminal cysteine is ~10. They exclusively receive electrons from thioredoxin reductase (TrxR) and not from mycothiol, the low molecular weight thiol of actinomycetes. This specificity is shown in the structural model of the complex between NrdH-redoxin and TrxR, where the two surface-exposed phenylalanines of TrxR perfectly fit into the conserved hydrophobic pocket of the NrdH-redoxin. Moreover, nrdh gene deletion and disruption experiments seem to indicate that NrdH-redoxin is essential in C. glutamicum.
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http://dx.doi.org/10.1074/jbc.M112.392688DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3597831PMC
March 2013

Crystal structures of the tRNA:m2G6 methyltransferase Trm14/TrmN from two domains of life.

Nucleic Acids Res 2012 Jun 22;40(11):5149-61. Epub 2012 Feb 22.

VIB Department of Structural Biology, Vrije Universiteit Brussel, Pleinlaan 2, 1050 Brussel, Belgium.

Methyltransferases (MTases) form a major class of tRNA-modifying enzymes needed for the proper functioning of tRNA. Recently, RNA MTases from the TrmN/Trm14 family that are present in Archaea, Bacteria and Eukaryota have been shown to specifically modify tRNA(Phe) at guanosine 6 in the tRNA acceptor stem. Here, we report the first X-ray crystal structures of the tRNA m(2)G6 (N(2)-methylguanosine) MTase (TTC)TrmN from Thermus thermophilus and its ortholog (Pf)Trm14 from Pyrococcus furiosus. Structures of (Pf)Trm14 were solved in complex with the methyl donor S-adenosyl-l-methionine (SAM or AdoMet), as well as the reaction product S-adenosyl-homocysteine (SAH or AdoHcy) and the inhibitor sinefungin. (TTC)TrmN and (Pf)Trm14 consist of an N-terminal THUMP domain fused to a catalytic Rossmann-fold MTase (RFM) domain. These results represent the first crystallographic structure analysis of proteins containing both THUMP and RFM domain, and hence provide further insight in the contribution of the THUMP domain in tRNA recognition and catalysis. Electrostatics and conservation calculations suggest a main tRNA binding surface in a groove between the THUMP domain and the MTase domain. This is further supported by a docking model of TrmN in complex with tRNA(Phe) of T. thermophilus and via site-directed mutagenesis.
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http://dx.doi.org/10.1093/nar/gks163DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3367198PMC
June 2012

The open reading frame TTC1157 of Thermus thermophilus HB27 encodes the methyltransferase forming N²-methylguanosine at position 6 in tRNA.

RNA 2012 Apr 15;18(4):815-24. Epub 2012 Feb 15.

Institut de Recherches Microbiologiques Jean-Marie Wiame, B-1070 Bruxelles, Belgium.

N(2)-methylguanosine (m(2)G) is found at position 6 in the acceptor stem of Thermus thermophilus tRNA(Phe). In this article, we describe the cloning, expression, and characterization of the T. thermophilus HB27 methyltransferase (MTase) encoded by the TTC1157 open reading frame that catalyzes the formation of this modified nucleoside. S-adenosyl-L-methionine is used as donor of the methyl group. The enzyme behaves as a monomer in solution. It contains an N-terminal THUMP domain predicted to bind RNA and contains a C-terminal Rossmann-fold methyltransferase (RFM) domain predicted to be responsible for catalysis. We propose to rename the TTC1157 gene trmN and the corresponding protein TrmN, according to the bacterial nomenclature of tRNA methyltransferases. Inactivation of the trmN gene in the T. thermophilus HB27 chromosome led to a total absence of m(2)G in tRNA but did not affect cell growth or the formation of other modified nucleosides in tRNA(Phe). Archaeal homologs of TrmN were identified and characterized. These proteins catalyze the same reaction as TrmN from T. thermophilus. Individual THUMP and RFM domains of PF1002 from Pyrococcus furiosus were produced. These separate domains were inactive and did not bind tRNA, reinforcing the idea that the THUMP domain acts in concert with the catalytic domain to target a particular position of the tRNA molecule.
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http://dx.doi.org/10.1261/rna.030411.111DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3312568PMC
April 2012

ELP3 controls active zone morphology by acetylating the ELKS family member Bruchpilot.

Neuron 2011 Dec;72(5):776-88

VIB, Department of Molecular and Developmental Genetics, B3000 Leuven, Belgium.

Elongator protein 3 (ELP3) acetylates histones in the nucleus but also plays a role in the cytoplasm. Here, we report that in Drosophila neurons, ELP3 is necessary and sufficient to acetylate the ELKS family member Bruchpilot, an integral component of the presynaptic density where neurotransmitters are released. We find that in elp3 mutants, presynaptic densities assemble normally, but they show morphological defects such that their cytoplasmic extensions cover a larger area, resulting in increased vesicle tethering as well as a more proficient neurotransmitter release. We propose a model where ELP3-dependent acetylation of Bruchpilot at synapses regulates the structure of individual presynaptic densities and neurotransmitter release efficiency.
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http://dx.doi.org/10.1016/j.neuron.2011.10.010DOI Listing
December 2011

Crystallization and preliminary X-ray crystallographic analysis of putative tRNA-modification enzymes from Pyrococcus furiosus and Thermus thermophilus.

Acta Crystallogr Sect F Struct Biol Cryst Commun 2011 Nov 27;67(Pt 11):1432-5. Epub 2011 Oct 27.

VIB Department of Structural Biology, Pleinlaan 2, 1050 Brussels, Belgium.

Methyltransferases form a major class of tRNA-modifying enzymes that are needed for the proper functioning of tRNA. Here, the expression, purification and crystallization of two related putative tRNA methyltransferases from two kingdoms of life are reported. The protein encoded by the gene pf1002 from the archaeon Pyrococcus furiosus was crystallized in the monoclinic space group P2(1). A complete data set was collected to 2.2 Å resolution. The protein encoded by the gene ttc1157 from the eubacterium Thermus thermophilus was crystallized in the trigonal space group P3(2)21. A complete data set was collected to 2.05 Å resolution.
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http://dx.doi.org/10.1107/S1744309111036347DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3212469PMC
November 2011