2ip1: Difference between revisions

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==Overview==

Saccharomyces cerevisiae is an ideal host from which to obtain high levels, of posttranslationally modified eukaryotic proteins for x-ray, crystallography. However, extensive replacement of methionine by, selenomethionine for anomalous dispersion phasing has proven intractable, in yeast. We report a general method to incorporate selenomethionine into, proteins expressed in yeast based on manipulation of the appropriate, metabolic pathways. sam1(-) sam2(-) mutants, in which the conversion of, methionine to S-adenosylmethionine is blocked, exhibit reduced, selenomethionine toxicity compared with wild-type yeast, increased, production of protein during growth in selenomethionine, and efficient, replacement of methionine by selenomethionine, based on quantitative mass, spectrometry and x-ray crystallography. The structure of yeast, tryptophanyl-tRNA synthetase was solved to 1.8 A by using multiwavelength, anomalous dispersion phasing with protein that was expressed and purified, from the sam1(-) sam2(-) strain grown in selenomethionine. Six of eight, selenium residues were located in the structure.

Saccharomyces cerevisiae is an ideal host from which to obtain high levels of posttranslationally modified eukaryotic proteins for x-ray crystallography. However, extensive replacement of methionine by selenomethionine for anomalous dispersion phasing has proven intractable in yeast. We report a general method to incorporate selenomethionine into proteins expressed in yeast based on manipulation of the appropriate metabolic pathways. sam1(-) sam2(-) mutants, in which the conversion of methionine to S-adenosylmethionine is blocked, exhibit reduced selenomethionine toxicity compared with wild-type yeast, increased production of protein during growth in selenomethionine, and efficient replacement of methionine by selenomethionine, based on quantitative mass spectrometry and x-ray crystallography. The structure of yeast tryptophanyl-tRNA synthetase was solved to 1.8 A by using multiwavelength anomalous dispersion phasing with protein that was expressed and purified from the sam1(-) sam2(-) strain grown in selenomethionine. Six of eight selenium residues were located in the structure.

==About this Structure==

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[[Category: Single protein]]

[[Category: Tryptophan--tRNA ligase]]

[[Category: CHTSB, Center.for.High-Throughput.Structural.Biology.]]

[[Category: CHTSB, Center for High-Throughput Structural Biology.]]

[[Category: Malkowski, M.G.]]

[[Category: Malkowski, M G.]]

[[Category: PG4]]

[[Category: center for high-throughput structural biology]]

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[[Category: structural genomics]]

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@@ Line 4: / Line 4: @@
 ==Overview==
-Saccharomyces cerevisiae is an ideal host from which to obtain high levels, of posttranslationally modified eukaryotic proteins for x-ray, crystallography. However, extensive replacement of methionine by, selenomethionine for anomalous dispersion phasing has proven intractable, in yeast. We report a general method to incorporate selenomethionine into, proteins expressed in yeast based on manipulation of the appropriate, metabolic pathways. sam1(-) sam2(-) mutants, in which the conversion of, methionine to S-adenosylmethionine is blocked, exhibit reduced, selenomethionine toxicity compared with wild-type yeast, increased, production of protein during growth in selenomethionine, and efficient, replacement of methionine by selenomethionine, based on quantitative mass, spectrometry and x-ray crystallography. The structure of yeast, tryptophanyl-tRNA synthetase was solved to 1.8 A by using multiwavelength, anomalous dispersion phasing with protein that was expressed and purified, from the sam1(-) sam2(-) strain grown in selenomethionine. Six of eight, selenium residues were located in the structure.
+Saccharomyces cerevisiae is an ideal host from which to obtain high levels of posttranslationally modified eukaryotic proteins for x-ray crystallography. However, extensive replacement of methionine by selenomethionine for anomalous dispersion phasing has proven intractable in yeast. We report a general method to incorporate selenomethionine into proteins expressed in yeast based on manipulation of the appropriate metabolic pathways. sam1(-) sam2(-) mutants, in which the conversion of methionine to S-adenosylmethionine is blocked, exhibit reduced selenomethionine toxicity compared with wild-type yeast, increased production of protein during growth in selenomethionine, and efficient replacement of methionine by selenomethionine, based on quantitative mass spectrometry and x-ray crystallography. The structure of yeast tryptophanyl-tRNA synthetase was solved to 1.8 A by using multiwavelength anomalous dispersion phasing with protein that was expressed and purified from the sam1(-) sam2(-) strain grown in selenomethionine. Six of eight selenium residues were located in the structure.
 ==About this Structure==
@@ Line 14: / Line 14: @@
 [[Category: Single protein]]
 [[Category: Tryptophan--tRNA ligase]]
-[[Category: CHTSB, Center.for.High-Throughput.Structural.Biology.]]
+[[Category: CHTSB, Center for High-Throughput Structural Biology.]]
-[[Category: Malkowski, M.G.]]
+[[Category: Malkowski, M G.]]
 [[Category: PG4]]
 [[Category: center for high-throughput structural biology]]
@@ Line 24: / Line 24: @@
 [[Category: structural genomics]]
-''Page seeded by [http://oca.weizmann.ac.il/oca OCA ] on Wed Jan 23 14:45:15 2008''
+''Page seeded by [http://oca.weizmann.ac.il/oca OCA ] on Thu Feb 21 17:54:46 2008''