Carboxylate Shifts in the Active Site of the Hydroxylase Component of Soluble Methane Monooxygenase from Methylococcus capsulatus (Bath)

1996 ◽  
pp. 141-149 ◽  
Author(s):  
Amy C. Rosenzweig ◽  
Christin A. Frederick ◽  
Stephen J. Lippard
Keyword(s):  
Active Site ◽  
Methane Monooxygenase ◽  
Methylococcus Capsulatus ◽  
Soluble Methane Monooxygenase

scholarly journals Batch Production of High-Quality Graphene Grids for Cryo-EM: Cryo-EM Structure of Methylococcus capsulatus Soluble Methane Monooxygenase Hydroxylase

2021 ◽  
Author(s):  
Eungjin Ahn ◽  
byungchul Kim ◽  
uhn-soo Cho
Keyword(s):  
Protein Denaturation ◽  
Methane Monooxygenase ◽  
Atomic Layer ◽  
Water Interface ◽  
Methylococcus Capsulatus ◽  
Force Microscopy ◽  
Voltage Electron ◽  
Soluble Methane Monooxygenase ◽  
Air Water Interface ◽  
Scanning Em

Cryogenic electron microscopy (cryo-EM) has become a widely used tool for determining protein structure. Despite recent advances in instruments and algorithms, sample preparation remains a major bottleneck for several reasons, including protein denaturation at the air/water interface and the presence of preferred orientations and nonuniform ice layers. Graphene, a two-dimensional allotrope of carbon consisting of a single atomic layer, has recently attracted attention as a near-ideal support film for cryo-EM that can overcome these challenges because of its superior properties, including mechanical strength and electrical conductivity. Graphene minimizes background noise and provides a stable platform for specimens under a high-voltage electron beam and cryogenic conditions. Here, we introduce a reliable, easily implemented, and reproducible method of producing 36 graphene-coated grids at once within 1.5 days. The quality of the graphene grids was assessed using various tools such as scanning EM, Raman spectroscopy, and atomic force microscopy. To demonstrate their practical application, we determined the cryo-EM structure of Methylococcus capsulatus soluble methane monooxygenase hydroxylase (sMMOH) at resolutions of 2.9 and 2.4 angstrom using Quantifoil and graphene-coated grids, respectively. We found that the graphene-coated grid has several advantages; for example, it requires less protein, enables easy control of the ice thickness, and prevents pro-tein denaturation at the air/water interface. By comparing the cryo-EM structure of sMMOH with its crystal structure, we revealed subtle yet significant geometrical differences at the non-heme di-iron center, which may better indicate the active site configuration of sMMOH in the resting/oxidized state.

scholarly journals Mutational and structural analyses of the regulatory protein B of soluble methane monooxygenase from Methylococcus capsulatus (Bath)

1999 ◽  
Vol 6 (7) ◽  
pp. 441-449 ◽  
Author(s):  
Hans Brandstetter ◽  
Douglas A Whittington ◽  
Stephen J Lippard ◽  
Christin A Frederick
Keyword(s):  
Methane Monooxygenase ◽  
Regulatory Protein ◽  
Methylococcus Capsulatus ◽  
Soluble Methane Monooxygenase ◽  
Protein B

Sequencing and analysis of the Methylococcus capsulatus (Bath) soluble methane monooxygenase genes

2000 ◽  
Vol 267 (8) ◽  
pp. 2174-2185 ◽  
Author(s):  
David E. Coufal ◽  
Jessica L. Blazyk ◽  
Douglas A. Whittington ◽  
Wayne W. Wu ◽  
Amy C. Rosenzweig ◽  
...  
Keyword(s):  
Methane Monooxygenase ◽  
Methylococcus Capsulatus ◽  
Soluble Methane Monooxygenase
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