1. 3D class averages & cryo-EM
Preliminary electron microscopy studies of bacterial phosphoglucosamine mutase Andrew Muenks1, Tommi A. White1&2, and Lesa J. Beamer1 Department of Biochemistry1 and Electron Microscopy Core2, University of Missouri, Columbia, MO
Abstract
Sample preparation
3D class averages & cryo-EM
Phosphoglucosamine mutase (PNGM) is an essential bacterial enzyme in the cytoplasmic steps of peptidoglycan biosynthesis. Peptidoglycan is used to synthesize the cell wall, and thus this pathway has been a target for design of antimicrobial agents. The crystal structure of PNGM from Bacillus anthracis has been solved and reveals an apparent dimer, along with significant conformational mobility of its C-terminal domain. To date, however, crystallization of an enzyme-ligand complex has proven elusive, a major setback in inhibitor design and for understanding the role of conformational change in enzyme function. In order to obtain further structural information on PNGM, including the possibility of enzyme-ligand complexes, we have initiated electron microscopy (EM) studies on the enzyme from B. anthracis at the University of Missouri’s Electron Microscopy Core. Preliminary efforts at negative staining with nano-W (methyl amine tungsten) and uranyl formate show well-distributed particles. Micrographs from these stains have been collected and are being processed for 2D classification and averaging. Upcoming studies include preparation of enzyme samples in vitrified ice for cryo-EM to conduct single particle reconstruction. Using this method, we hope to capture novel conformational states of the PNGM dimer that give clues regarding changes in protein structure from ligand binding.
Negative staining was performed using 2% Nano-W and 0.75% uranyl formate. Micrographs of negative stains were collected on the JEOL JEM-1400 TEM.
2D and 3D classifications from the micrographs were generated using Relion
Samples for cryo-EM were frozen in liquid ethane using the FEI Mark IV Vitribot. Micrographs were collected using the FEI Tecnai F30 G2 Twin TEM.
A low resolution 3D class average from the negative staining reveals an envelope (top) similar to the PNGM crystal structure (middle). As a comparison, the crystal structure of B. anthracis PNGM filtered to 50 Å is provided (bottom).
JEOL JEM-1400 TEM at University of Missouri’s EM Core
Negative staining & image processing
Background
Negatively stained images of PNGM from B. anthracis at concentrations of 2 μg/mL (top) and 0.2 mg/mL (bottom). PNGM can be seen as white specks (see arrows).
PNGM catalyzes the phosphoryl transfer between glucosamine-6-P and glucosamine-1-P. Glucosamine-1-P is eventually converted to UDP-N-acetylglucosamine, a building unit of peptidoglycan used in bacterial cell walls.
Micrographs for protein samples in vitrified ice at 0.2 mg/mL (left) and 20 μg/mL (right) taken at 39000x on a FEI Technai F30 G2 Twin TEM at 200 kV. Protein specimens (black specks, see arrows) were either too concentrated or too dilute for further processing, and will need to be optimized.
GlmM
(PNGM)
The crystal structure of PNGM from Bacillus anthracis is known. The protein has 456 amino acids arranged in a four-domain architecture with a centrally located active site cleft. An apparent dimer (molecular mass 93.1 kDa) observed in the crystal was confirmed in solution. Comparisons of the two protomers within the dimer showed significant conformational mobility (30° rotation) the C-terminal domain.
Negative staining done with nano-W (top) and uranyl formate (bottom) of PNGM. Images were taken at 25000x (top) and 15000x (bottom) on a JEOL JEM-1400 TEM at 120 kV on a Gatan Ultrascan 1000 CCD camera.
Future work
Upcoming studies on PNGM include:
Optimizing protein dilutions for plunge freezing to enable cryo-EM.
Data collection for single particle reconstruction at the Midwest High Resolution Cryo-electron Microscopy Consortium (Purdue University).
Use of the FEI Volta Phase Plate to enhance contrast
Preparation PNGM-ligand complexes to observe conformational change
Expansion to different PNGMs from Francisella tularenis and Pyrococcus horikoshii. A 4 B
Micrographs from the uranyl formate negative staining were processed using Relion. 2D class averages were calculated from 1028 hand picked particles, representing different orientations of the protein. These class averages autopicked 8183 particles to create reference free 2D class averages. Reference free class averages generated 3D class averages.
(A) The crystal structure of PNGM (PDB ID: 3PDK) colored by domain. (B) Aligned structures of the opened (magenta) and closed (light blue) protomers, showing the 30° change in conformation of the C-terminal domain (domain 4). (C) The dimer of PNGM.
References 1
Mehra-Chaudhary R, Mick J, Beamer LJ. Crystal structure of Bacillus anthracis phosphoglucosamine mutase, an enzyme in the peptidoglycan biosynthetic pathway. J Bacteriol. 2011; 193: 4081–4087. doi: /JB
Mehra-Chaudhary R, Mick J, Tanner JJ, Beamer LJ. Quaternary structure, conformational variability and global motions of phosphoglucosamine mutase. FEBS J. 2011; 278: 3298–3307.
Scheres SH. RELION: implementation of a Bayesian approach to cryo-EM structure determination. J of Struct Bio. 2012; 180: 2 3 C
2D class averages from uranyl formate negatively stained images. Classes outlined in red were used for autopicking.
Acknowledgements
A.G.M. was supported by the Life Sciences Undergraduate Research Opportunity Program and an Electron Microscopy Core Fellowship. Part of this work was supported by an Excellence in Electron Microscopy Award from the MU EM Core and Office of Research.