Structural genomics projects are providing large quantities of new 3D structural data for proteins. To monitor the quality of these data, we have developed the protein structure validation software suite (PSVS), for assessment of protein structures generated by NMR or X-ray crystallographic methods. PSVS is broadly applicable for structure quality assessment in structural biology projects. The software integrates under a single interface analyses from several widely-used structure quality evaluation tools, including PROCHECK (Laskowski et al., J Appl Crystallog 1993;26:283-291), MolProbity (Lovell et al., Proteins 2003;50:437-450), Verify3D (Luthy et al., Nature 1992;356:83-85), ProsaII (Sippl, Proteins 1993;17: 355-362), the PDB validation software, and various structure-validation tools developed in our own laboratory. PSVS provides standard constraint analyses, statistics on goodness-of-fit between structures and experimental data, and knowledge-based structure quality scores in standardized format suitable for database integration. The analysis provides both global and site-specific measures of protein structure quality. Global quality measures are reported as Z scores, based on calibration with a set of high-resolution X-ray crystal structures. PSVS is particularly useful in assessing protein structures determined by NMR methods, but is also valuable for assessing X-ray crystal structures or homology models. Using these tools, we assessed protein structures generated by the Northeast Structural Genomics Consortium and other international structural genomics projects, over a 5-year period. Protein structures produced from structural genomics projects exhibit quality score distributions similar to those of structures produced in traditional structural biology projects during the same time period. However, while some NMR structures have structure quality scores similar to those seen in higher-resolution X-ray crystal structures, the majority of NMR structures have lower scores. Potential reasons for this "structure quality score gap" between NMR and X-ray crystal structures are discussed.

Sequence-specific 'H and '-N resonance asignments have been determined for the major cold shock protein (CspA) from Escherichia coli with recently developed three-dimensional triple-resonance NMR experiments. By use of these asgments, five antp lel 1-strands were identified from analysis of NMR data. Strands 1-4 have a clsical 3-2-1-4 Greek key -sheet topology and there are two 1-bulges, at positions Lys'0-Trp" and Gly"5-Asn". Threedimensional structures of CspA were generated from NMR data by using smulated annling with molecular dynamics. The overall chain fold of CspA Is a 1-barrel structure, with a tightly packed hydrophobic core. Two-dimensional isotopeedited pulsed-field gradient 15N-1H heteronuclear singlequantum coherence spectroscopy was used to chracerie the "N-1H fingerprint spectrum with and without a 24-base oligodeoxyribonucleotide, 5'-AACGGTTTGACGTACAGAC-CATTA-3'. Protein-DNA complex formation perturbs a subset of the amide resonances that are located mostly on one face of the CspA molecule. This portion of the CspA molecular surface includes two putative RNA-binding sequence motifs which contribute to an unusual cluster of eight surface aromatic side chais: Trpol Phel", Phe", P , Phe3, His33, Phe3, and Tyr42. These surface aromatic groups, and also residues Lys"6, Ser", and Lys"' located on this same face of CspA, are higly conserved in the family of CspA homologues. These isotopeedited pulsed-field gradient NMR data provide a low-resolution mapping of a DNA-binding epitope on CspA.eukaryotic Y-box family of transcription-regulating proteins (6)(7)(8) (4,18), and these also contain RNP1 and RNP2 sequence motifs. The three-dimensional structure of one of these, the major cold shock protein CspB from Bacillus subtilis, has been determined recently by solution NMR (19) and x-ray crystallography (16). CspB has structural homology with several nucleic acid-binding proteins and forms complexes with ssDNA oligomers containing a CCAAT site in gel retardation experiments (16,19). In this paper we describe the overall chain fold of E. coli CspA determined in solution by NMR spectroscopy and demonstrate that CspA forms a complex with a 24-base oligodeoxyribonucleotide corresponding in sequence to the 5' leader region of cspA mRNA. 11 Many bacteria respond to low temperature stress by production ofcold shock proteins (1-5). In Escherichia coli this cold shock response involves enhanced production of at least 14 proteins (1, 2). One of these, the major cold shock protein (CspA), is not detectable when cells are grown at 370C but is produced at levels of 10-15% of total protein synthesis when cells are shifted to 10-15TC (2), with expression peaking 60-120 min following cold shock. The gene for CspA (cspA) has been cloned and sequenced (2), and primer extension studies indicate that levels of cspA transcripts also increase and then decrease in the cold shock response, paralleling the production kinetics of CspA protein. The transient nature of cold shock-induced cspA expression indicates a regulator...

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