To the editor:Obtaining diffraction-quality crystals is a major bottleneck in protein X-ray crystallography. For example, the current success rate for protein structure solution at the Midwest Center for Structural Genomics (starting from purified protein) is ~10%. Protein crystallization is influenced by many factors, and many methods have been developed to enhance crystallization. In particular, reductive methylation of proteins has been successfully applied to obtain high-quality crystals 1-4 . Several studies 3,5,6 have indicated that methylating the solvent-exposed ε-amino group of lysines changes protein properties (pI, solubility and hydropathy) 7,8 , which may promote crystallization via improving crystal packing. Reductive methylation of proteins is a simple, generic method; it is fast, specific and requires few steps under relatively mild buffer and chemical conditions and can be executed for several proteins in parallel. Native and methylated proteins have very similar structures, and, in most cases, methylated proteins maintain their biochemical function 2,5,9 . Some proteins can only be crystallized after methylation 3,10 , and crystals of modified proteins often diffract to higher resolution 3,9 . The efficacy of the method has been previously tested on 10 proteins, with a 30% success rate 3 .Here we investigated the application of reductive methylation on a large scale. We applied a previously described reductive methylation protocol 2,11 (Supplementary Methods online) to 370 sequence-diverse proteins selected from protein families that had no structural homologs with >30% sequence identity. We expressed 370 recombinant proteins and purified them using standard methods 12 and screened them using standard crystal screening methods (Supplementary Methods). Of the 370 proteins, 269 proteins had not previously yielded crystals suitable for structure determination (crystals were too small, poorly ordered, twinned, highly mosaic or multiple), 85 proteins had previously failed to crystallize and 16 proteins were a reference set (not previously screened for crystallization; Table 1 and Supplementary Tables 1 and 2 online). After reductive methylation, we obtained diffraction-quality crystals for 40 of the 370 proteins, and so far we solved 26 crystal structures ( NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author ManuscriptWe also determined the structures of 4 proteins in their native as well as their methylated states (Supplementary Methods). By comparing these structures, we obtained insight into how methylation affects protein crystallization. We observed a decrease in the isotropic B factor (Fig. 1), which is likely a result of more ordered crystal packing and which leads to better diffraction limits. Indeed, the resolution of the methylated structures (average, 2.07 Å) was better than that of their native counterparts (average, 3.05 Å; Supplementary Table 1). The methylated lysines were engaged in various intra-and intermolecular interactions with protein and solvent (carboxylates and ...

In structural biology, the most critical issue is the availability of high-quality samples. “Structural-biology-grade” proteins must be generated in a quantity and quality suitable for structure determination using X-ray crystallography or nuclear magnetic resonance. The additional challenge for structural genomics is the need for high numbers of proteins at low cost where protein targets quite often have low sequence similarities, unknown properties and are poorly characterized. The purification procedures must reproducibly yield homogeneous proteins or their derivatives containing marker atom(s) in milligram quantities. The choice of protein purification and handling procedures plays a critical role in obtaining high-quality protein samples. Where the ultimate goal of structural biology is the same—to understand the structural basis of proteins in cellular processes, the structural genomics approach is different in that the functional aspects of individual protein or family are not ignored, however, emphasis here is on the number of unique structures, covering most of the protein folding space and developing new technologies with high efficiency. At the Mid-west Center Structural Genomics (MCSG), we have developed semiautomated protocols for high-throughput parallel protein purification. In brief, a protein, expressed as a fusion with a cleavable affinity tag, is purified in two immobilized metal affinity chromatography (IMAC) steps: (i) first IMAC coupled with buffer-exchange step, and after tag cleavage using TEV protease, (ii) second IMAC and buffer exchange to clean up cleaved tags and tagged TEV protease. Size exclusion chromatography is also applied as needed. These protocols have been implemented on multidimensional chromatography workstations AKTAexplorer and AKTAxpress (GE Healthcare). All methods and protocols used for purification, some developed in MCSG, others adopted and integrated into the MCSG purification pipeline and more recently the Center for Structural Genomics of Infectious Disease (CSGID) purification pipeline, are discussed in this chapter.

Many bacteria express phosphoenolpyruvate-dependent phosphotransferase systems (PTS). Phosphoenolpyruvate-dependent phosphotransferase systems (PTS)2 primarily consist of a transmembrane transporter and enzymes responsible for phosphoryl group transfer from phosphoenolpyruvate to a transporter-bound sugar acceptor (1, 2). The PTS are generally substrate-defined, and their components may vary across species (2, 3). D-Mannitol, or 1,2,3,4,5,6-hexanehexol, is a polyol formed by reduction of mannose or fructose. It is one of the hexitols involved in bacteria catabolism pathways, where it serves as a source of fermentable sugar (4, 5). The D-mannitol-specific PTS was first discovered and sequenced in Escherichia coli (6 -8). Taking into consideration that the system was regulated like other hexitol PTS systems, an open reading frame was sought and identified within the mannitol operon (9). Experiments indicated that the loss of the gene (mtlR) led to the constitutive expression of the operon, and its gene product MtlR was proposed to be a transcription factor: mannitol operon repressor (MtlR, COG2213) (9). Two putative DNA operator palindromes that might serve as MltR binding sites were identified within the operator-promoter region.Besides MtlR, a typical mannitol operon also encodes a mannitol-specific PTS system ABC transporter II component (MtlA, COG2213) and a mannitol-1-phosphate 5-dehydrogenase (MtlD, COG0246). The mannitol operon is conserved and has been cloned from many Gram-negative bacterial families, such as Shigella (10), Salmonella (11), Yersinia (12), Klebsiella (13, 14), and Vibrio (15). For example, in the genome sequence of Vibrio parahemeolyticus RIMD 2210633 (15), a typical mannitol operon, including MtlR, MtlA, and MtlD, has been identified on chromosome I. A paralogue gene yggD annotated as putative transcriptional regulator was found clustered with cmtA and cmtB on chromosome II of V. parahemeolyticus. The cmtA and cmtB genes seem to encode the equivalents of MtlA components according to a recent study (16).In genomes of many other Gram-negative bacteria, a gene named yggD, encoding a sequence homolog of MtlR, has also been identified. The gene yggD is not clustered with mtlA and mtlD as mtlR in a typical mannitol operon. Its gene neighbors vary considerably among organisms, even among strains, and its position provides little insight into its function. In the complete genome sequence of Shigella flexneri serotype 2a strain 2457T (10), for example, the yggD gene is located about 1 megabase pair away from the mannitol operon on the same strand of DNA (10). Its neighboring genes include uncharacterized yggC and yggF. The latter may encode a fructose-1,6-bisphosphatase II-like protein (10). The function of YggD has never been described. However, based on its sequence similarity to MtlR, it has been assigned to the same protein family, Pfam05068 (mannitol operon repressor) (17).