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Perdeuteration, large crystal growth and neutron data collection of Leishmania mexicana triose-phosphate isomerase E65Q variant

Kelpšas, Vinardas LU ; Lafumat, Bénédicte LU ; Blakeley, Matthew P.; Coquelle, Nicolas; Oksanen, Esko LU and Von Wachenfeldt, Claes LU (2019) In Acta Crystallographica Section F: Structural Biology Communications 75(4). p.260-269
Abstract

Triose-phosphate isomerase (TIM) catalyses the interconversion of dihydroxyacetone phosphate and glyceraldehyde 3-phosphate. Two catalytic mechanisms have been proposed based on two reaction-intermediate analogues, 2-phosphoglycolate (2PG) and phosphoglycolohydroxamate (PGH), that have been used as mimics of the cis-enediol(ate) intermediate in several studies of TIM. The protonation states that are critical for the mechanistic interpretation of these structures are generally not visible in the X-ray structures. To resolve these questions, it is necessary to determine the hydrogen positions using neutron crystallography. Neutron crystallography requires large crystals and benefits from replacing all hydrogens with deuterium. Leishmania... (More)

Triose-phosphate isomerase (TIM) catalyses the interconversion of dihydroxyacetone phosphate and glyceraldehyde 3-phosphate. Two catalytic mechanisms have been proposed based on two reaction-intermediate analogues, 2-phosphoglycolate (2PG) and phosphoglycolohydroxamate (PGH), that have been used as mimics of the cis-enediol(ate) intermediate in several studies of TIM. The protonation states that are critical for the mechanistic interpretation of these structures are generally not visible in the X-ray structures. To resolve these questions, it is necessary to determine the hydrogen positions using neutron crystallography. Neutron crystallography requires large crystals and benefits from replacing all hydrogens with deuterium. Leishmania mexicana triose-phosphate isomerase was therefore perdeuterated and large crystals with 2PG and PGH were produced. Neutron diffraction data collected from two crystals with different volumes highlighted the importance of crystal volume, as smaller crystals required longer exposures and resulted in overall worse statistics.

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author
organization
publishing date
type
Contribution to journal
publication status
published
subject
keywords
large crystals, Laue method, neutron diffraction, perdeuteration, triose-phosphate isomerase
in
Acta Crystallographica Section F: Structural Biology Communications
volume
75
issue
4
pages
10 pages
publisher
Wiley-Blackwell
external identifiers
  • scopus:85063956842
ISSN
2053-230X
DOI
10.1107/S2053230X19001882
language
English
LU publication?
yes
id
6bb03569-4607-467f-96f4-e1a36e26c96f
date added to LUP
2019-04-26 09:01:53
date last changed
2019-05-14 04:54:17
@article{6bb03569-4607-467f-96f4-e1a36e26c96f,
  abstract     = {<p>Triose-phosphate isomerase (TIM) catalyses the interconversion of dihydroxyacetone phosphate and glyceraldehyde 3-phosphate. Two catalytic mechanisms have been proposed based on two reaction-intermediate analogues, 2-phosphoglycolate (2PG) and phosphoglycolohydroxamate (PGH), that have been used as mimics of the cis-enediol(ate) intermediate in several studies of TIM. The protonation states that are critical for the mechanistic interpretation of these structures are generally not visible in the X-ray structures. To resolve these questions, it is necessary to determine the hydrogen positions using neutron crystallography. Neutron crystallography requires large crystals and benefits from replacing all hydrogens with deuterium. Leishmania mexicana triose-phosphate isomerase was therefore perdeuterated and large crystals with 2PG and PGH were produced. Neutron diffraction data collected from two crystals with different volumes highlighted the importance of crystal volume, as smaller crystals required longer exposures and resulted in overall worse statistics.</p>},
  author       = {Kelpšas, Vinardas and Lafumat, Bénédicte and Blakeley, Matthew P. and Coquelle, Nicolas and Oksanen, Esko and Von Wachenfeldt, Claes},
  issn         = {2053-230X},
  keyword      = {large crystals,Laue method,neutron diffraction,perdeuteration,triose-phosphate isomerase},
  language     = {eng},
  number       = {4},
  pages        = {260--269},
  publisher    = {Wiley-Blackwell},
  series       = {Acta Crystallographica Section F: Structural Biology Communications},
  title        = {Perdeuteration, large crystal growth and neutron data collection of Leishmania mexicana triose-phosphate isomerase E65Q variant},
  url          = {http://dx.doi.org/10.1107/S2053230X19001882},
  volume       = {75},
  year         = {2019},
}