Figure 1.
Proteome fractionation and purification flow chart.
Approximately 500 g of E. coli cells were lysed at pH 7 using a microfluidizer and the cell debris pelleted. The supernatant was applied to a tangential flow column with a nominal molecular weight cut off of 500 kDa, generating 2 fractions (retentate and flow through). The fraction above 500 kDa (retentate) was further purified via sucrose gradients, size exclusion, and ion exchange chromatography prior to crystallization trials. The fraction less than 500 kDa was applied to multiple affinity and ion exchange columns followed by phenyl sepharose, ion exchange, and size exclusion prior to crystallization trials in microfluidic chips.
Figure 2.
E. coli proteome predicted and experimental characterization.
(A) Predicted size distribution of all ORFs in the E. coli proteome. (B) Size exclusion chromatograph of crude E. coli lysate with the largest peak at approximately 100 kDa. (C) Final step ion exchange (MonoQ) purification in a typical fractionation experiment. Peaks marked with a star were sent for downstream crystallization trials.
Figure 3.
Crystallization of native source E. coli proteins.
(A) Capillary electrophoresis of purified protein fractions. White stars indicate samples successfully crystallized and black stars represent solved structures. (B) Crystals of 5-keto-4-deoxyuronate isomerase crystallized from fractions of varying purity. Crystal quality was not always correlated with sample purity. (C) Resolution of the data collected versus percent purity of the starting sample based on quantification of protein concentrations by capillary gel electrophoresis with the Caliper system. Sample purity did not correlate with higher resolution data.
Table 1.
Crystallization conditions and data collection statistics for previously deposited structures.
Figure 4.
Structures of previously deposited proteins solved during the pipeline.
All proteins were oligomers as shown above. Proteins are from top left - inorganic pyrophosphatase (1IPW), 5-keto-4-deoxyuronate isomerase (1XRU), Hsp31 (1N57), pyruvate kinase (1PKY), phosphoserine aminotransferase (1BJN), Citrate synthase (1NXG), ycaC gene product (1YAC), Cystathione gamma-synthase (1CS1), Dihydrodipicolinate synthase (1DHP), Arginosuccinate lyase (1TJ7), Nicotinamide nucleotide transhydrogenase domain I (1×12), MoaB (molybdopterin biosynthesis protein B) (1R2K), Catalase HPII (1GG9), Lysyl-tRNA synthetase (constitutive) (1BBW), Glutamate decarboxylase (1PMO), Glucosamine 6-phosphate deaminase (IHOT), Malate dehydrogenase (2PWZ), Adenylosuccinate synthetase (1CG1), catalase HPII truncated (1YE9).
Figure 5.
YghZ tetramer and active site.
Left, the YghZ tetramer viewed along the four-fold axis. Putative active site residues are depicted as ball-and-stick and colored by atom with the active site of one monomer outlined by a gray box. Right, close -up view of the active site with putative active site residues colored by atom and labeled.
Figure 6.
PGI dimer and putative active site.
Left, the pGI dimer. Right, close -up view of the active site with putative active site residues colored by atom and labeled. The active site is formed at the dimer interface and has contributions from both monomers.
Figure 7.
BglA dimer and putative active site.
Left, BglA dimer with the putative active site outlined in a gray box. Right, close up of the active site with glucose-6-phosphate modeled based of the position of the sulfate ion from crystallization. Active site residues are depicted as ball-and-stick. Putative hydrogen bonds to the substrate are drawn as dashed lines.
Figure 8.
The protein forms a hexamer (dimer of trimers). Left, view of the GDH hexamer along the two-fold axis. Right, view of the GDH hexamer along the three-fold axis.
Table 2.
Data collection and refinement statistics for methylglyoxal reductase (YghZ).
Table 3.
Data collection and refinement statistics for Glucose-6-phosphate isomerase (pGI).
Table 4.
Data collection and refinement statistics for 6-phospho-beta-glucosidase (BglA).
Table 5.
Data collection and refinement statistics for glutamate dehydrogenase (GDH).