Z.-Q. Fu

Characterization of a corrinoid protein involved in the C1 metabolism of strict anaerobic bacterium Moorella thermoacetica

The strict anaerobic, thermophilic bacterium Moorella thermoacetica metabolizes C1 compounds for example CO2/H2, CO, formate, and methanol into acetate via the Wood/Ljungdahl pathway. Some of the key steps in this pathway include the metabolism of the C1 compounds into the methyl group of methylenetetrahydrofolate (MTHF) and the transfer of the methyl group from MTHF to the methyl group of acetyl‐CoA catalyzed by methyltransferase, corrinoid protein and CO dehydrogenase/acetyl CoA synthase. Recently, we reported the crystallization of a 25 kDa methanol‐induced corrinoid protein from M. thermoacetica (Zhou et al., Acta Crystallogr F 2005; 61:537–540). In this study we analyzed the crystal structure of the 25 kDa protein and provide genetic and biochemical evidences supporting its role in the methanol metabolism of M. thermoacetia. The 25 kDa protein was encoded by orf1948 of contig 303 in the M. thermoacetica genome. It resembles similarity to MtaC the corrinoid protein of the methanol:CoM methyltransferase system of methane producing archaea. The latter enzyme system also contains two additional enzymes MtaA and MtaB. Homologs of MtaA and MtaB were found to be encoded by orf2632 of contig 303 and orf1949 of contig 309, respectively, in the M. thermoacetica genome. The orf1948 and orf1949 were co‐transcribed from a single polycistronic operon. Metal analysis and spectroscopic data confirmed the presence of cobalt and the corrinoid in the purified 25 kDa protein. High resolution X‐ray crystal structure of the purified 25 kDa protein revealed corrinoid as methylcobalamin with the imidazole of histidine as the α‐axial ligand replacing benziimidazole, suggesting base‐off configuration for the corrinoid. Methanol significantly activated the expression of the 25 kDa protein. Cyanide and nitrate inhibited methanol metabolism and suppressed the level of the 25 kDa protein. The results suggest a role of the 25 kDa protein in the methanol metabolism of M. thermoacetica. Proteins 2007.

A multi-dataset data-collection strategy produces better diffraction data

Theoretical analysis and experimental validation prove that a multi-dataset data-collection strategy produces better diffraction data. The readiness test is a simple and sensitive method for X-ray data-collection system evaluation and a benchmark.

SGXPro: a parallel workflow engine enabling optimization of program performance and automation of structure determination.

SGXPro consists of four components. (i) A parallel workflow engine that was designed to automatically manage communication between the different processes and build systematic searches of algorithm/program/parameter space to generate the best possible result for a given data set. This is performed by offering the user a palette of programs and techniques commonly used in X-ray structure determination in an environment that lets the user choose programs in a mix-and-match manner, without worrying about inter-program communication and file formats, during the structure-determination process. The current SGXPro program palette includes 3DSCALE, SHELXD, ISAS, SOLVE/RESOLVE, DM, SOLOMON, DMMULTI, BLAST, AMoRe, EPMR, XTALVIEW, ARP/wARP and MAID. (ii) A client/server architecture that allows the user to utilize the best computing facility available. (iii) Plug-in-and-play design, which allows easily integration of new programs into the system. (iv) User-friendly interface.

Monitoring the anomalous scattering signal and noise levels in X-ray diffraction of crystals

A statistical index R(as) is proposed in order to monitor the overall signal-to-noise ratio in an anomalous scattering data set. In this approach, symmetry-equivalent reflections are merged and grouped into centric and non-centric subsets. Reflections in the centric subset, which in theory should be equal, are used to estimate the noise level in the data. This approach differs from that used by most data-processing programs, in which the centric reflections are merged and averaged. By preserving the differences in centric reflections during data processing, an internal measure of the noise level can be estimated and used to analyze the quality of the anomalous signal in the data. An index R(as) is defined as the ratio of the average Bijvoet difference of merged acentric reflections to merged centric reflections. Test results on a variety of data show that R(as) has good correlation with the capability to determine the anomalous scattering substructure from the data. R(as) can also be useful in monitoring the quality of the data in terms of the data-collection strategy, instrument settings and data-processing software used. R(as) analysis has been implemented in the program 3DSCALE as part of a data-processing program suite that is under development in our laboratory.

Structure of the Archaeoglobus fulgidus orphan ORF AF1382 determined by sulfur SAD from a moderately diffracting crystal.

The crystal structure of the 11.14 kDa orphan ORF 1382 from Archaeoglobus fulgidus (AF1382) has been determined by sulfur SAD phasing using a moderately diffracting crystal and 1.9 Å wavelength synchrotron X-rays. AF1382 was selected as a structural genomics target by the Southeast Collaboratory for Structural Genomics (SECSG) since sequence analyses showed that it did not belong to the Pfam-A database and thus could represent a novel fold. The structure was determined by exploiting longer wavelength X-rays and data redundancy to increase the anomalous signal in the data. AF1382 is a 95-residue protein containing five S atoms associated with four methionine residues and a single cysteine residue that yields a calculated Bijvoet ratio (ΔF(anom)/F) of 1.39% for 1.9 Å wavelength X-rays. Coupled with an average Bijvoet redundancy of 25 (two 360° data sets), this produced an excellent electron-density map that allowed 69 of the 95 residues to be automatically fitted. The S-SAD model was then manually completed and refined (R = 23.2%, R(free) = 26.8%) to 2.3 Å resolution (PDB entry 3o3k). High-resolution data were subsequently collected from a better diffracting crystal using 0.97 Å wavelength synchrotron X-rays and the S-SAD model was refined (R = 17.9%, R(free) = 21.4%) to 1.85 Å resolution (PDB entry 3ov8). AF1382 has a winged-helix-turn-helix structure common to many DNA-binding proteins and most closely resembles the N-terminal domain (residues 1-82) of the Rio2 kinase from A. fulgidus, which has been shown to bind DNA, and a number of MarR-family transcriptional regulators, suggesting a similar DNA-binding function for AF1382. The analysis also points out the advantage gained from carrying out data reduction and structure determination on-site while the crystal is still available for further data collection.

A parallel program using SHELXD for quick heavy-atom partial structural solution on high-performance computers

A parallel algorithm has been designed for SHELXD to solve the heavy-atom partial structures of protein crystals quickly. Based on this algorithm, a program has been developed to run on high-performance multiple-CPU Linux PCs, workstations or clusters. Tests on the 32-CPU Linux cluster at SER-CAT, APS, Argonne National Laboratory, show that the parallelization dramatically speeds up the process by a factor of roughly the number of CPUs applied, leading to reliable and instant heavy-atom sites solution, which provides the practical opportunity to employ heavy-atom search as an alternative tool for anomalous scattering data quality evaluation during single/multiple-wavelength anomalous diffraction (SAD/MAD) data collection at synchrotron beamlines.

Three-dimensional model-free experimental error correction of protein crystal diffraction data with free-R test

Experimental error correction and scaling is the last step in X-ray diffraction data processing. It is also critical in obtaining good-quality data. In this study, an algorithm is proposed to more generally and efficiently correct experimental error in X-ray crystal diffraction data. With this algorithm, the experimental error is represented by a symbolic three-dimensional function C(xi, eta, t) in the detecting space. Here, (xi, eta) are the coordinates of the diffraction spots on the image and t represents the data-collection time. While the theoretical form of C(xi, eta, t) is not known, it will be determined from the data through computer-aided analysis. The free R(merge) is introduced to check the validity of the solution. The three-dimensional symbolic function does not carry any assumptions and thus can generally account for experimental errors in various X-ray crystal diffraction experiments that are normally too complicated to be described by any fixed formula. Tests will be given to compare the results from different algorithms.

Crystal structures of a subunit of the formylglycinamide ribonucleotide amidotransferase, PurS, from Thermus thermophilus, Sulfolobus tokodaii and Methanocaldococcus jannaschii.

The crystal structures of a subunit of the formylglycinamide ribonucleotide amidotransferase, PurS, from Thermus thermophilus, Sulfolobus tokodaii and Methanocaldococcus jannaschii were determined and their structural characteristics were analyzed. For PurS from T. thermophilus, two structures were determined using two crystals that were grown in different conditions. The four structures in the dimeric form were almost identical to one another despite their relatively low sequence identities. This is also true for all PurS structures determined to date. A few residues were conserved among PurSs and these are located at the interaction site with PurL and PurQ, the other subunits of the formylglycinamide ribonucleotide amidotransferase. Molecular-dynamics simulations of the PurS dimer as well as a model of the complex of the PurS dimer, PurL and PurQ suggest that PurS plays some role in the catalysis of the enzyme by its bending motion.

Data collection strategies for the Rayonix MX300HS 10 Hz CCD Detector

Investigating Data Collection Strategies for the Rayonix MX300HS 10 Hz CCD Detector Zhongmin Jin, John Chrzas, James Fait, Zheng-Qing Fu, Rod Salazar, John Gonczy, Unmesh Chinte, Palani Kandavelu, John P. Rose and Bi-Cheng Wang SERCAT, APS, Argonne National Lab, Argonne, IL 60439 and Department of Biochemistry & Molecular Biology, University of Georgia, Athens, GA 30602, USA. Earlier this year SER-CAT received its Rayonix MX300HS high-speed area detector purchased with funds awarded from an NIH grant. The detector is CCD based (4X4 taper/chip array) with a 1-2 millisecond readout speed and 8e-/pixel (78 micron/pixel) readout noise providing 10Hz data collection capabilities. In its slower readout mode, the readout noise can be further reduced to about 4e-/pixel, which is less than 1 ADU. The detector was installed in late January on beamline 22BM for integration with SER-CATs beamline control system and commissioning. This detector is the first of its kind and its fast readout time will allow us to collect data in the traditional shuttered mode with both normal and low noise readout; or in shutter-less mode using 0.1 second exposures with constant rotation of crystal. Importantly, the shutter-less mode may enhance system precision and improve the quality of the resulting data since it should eliminate mechanical errors associated with shutter jitter, shutter synchronization and goniometer backlash during data collection. Based on our preliminary tests, shutter-less mode data collection has confirmed the expectation of being capable for providing better quality data than that from shuttered mode. Shutter-less data collection also offers the ability to efficiently collect fine slice data, which will further improve the signal to noise in the data. We will present the results from systematic tests of the detector in terms of frame width (rotation angle per frame), rotation speed, exposure time, dynamic range etc. Work supported in part by NIH NCRR (S10RR028976), SER-CAT, The University of Georgia and the Advanced Photon Source.

Pushing the envelop of sulfur SAS structure determination at UGA/SER-CAT

1 small variability to cover Se and Br edges). The 24ID-E beamline is equipped with a MD2 microdiffractometer, which is used to provide stable and well collimated beam from 5 to 100 microns in diameter and capable of visualizing micron-sized crystals. These operational beamlines are currently open to general APS users. Installation of a bending magnet beamline is now in progress and expected to be completed by the end of this year. NE-CAT is a consortium of scientists organized to design, construct and operate a structural biology sector at the APS. This facility will be used to focus on NECAT research on structural studies involving technically challenging crystallographic projects. In order to meet these needs, an ALS robot for screening a large number of crystals is now being commissioned, a microfocus diffractometer MD-2 is installed on 24ID-E beamline and several novel hardware and software ideas will be implemented. Funding for NE-CAT is provided through P41 grant from the National Center for Research Resources and from the NE-CAT member institutions.