Coping with strong translational noncrystallographic symmetry and extreme anisotropy in molecular replacement with Phaser: human Rab27a

Data pathologies caused by effects such as diffraction anisotropy and translational noncrystallographic symmetry (tNCS) can dramatically complicate the solution of the crystal structures of macromolecules. Such problems were encountered in determining the structure of a mutant form of Rab27a, a member of the Rab GTPases. Mutant Rab27a constructs that crystallize in the free form were designed for use in the discovery of drugs to reduce primary tumour invasiveness and metastasis. One construct, hRab27aMut, crystallized within 24 h and diffracted to 2.82 Å resolution, with a unit cell possessing room for a large number of protein copies. Initial efforts to solve the structure using molecular replacement by Phaser were not successful. Analysis of the data set revealed that the crystals suffered from both extreme anisotropy and strong tNCS. As a result, large numbers of reflections had estimated standard deviations that were much larger than their measured intensities and their expected intensities, revealing problems with the use of such data at the time in Phaser. By eliminating extremely weak reflections with the largest combined effects of anisotropy and tNCS, these problems could be avoided, allowing a molecular-replacement solution to be found. The lessons that were learned in solving this structure have guided improvements in the numerical analysis used in Phaser, particularly in identifying diffraction measurements that convey very little information content. The calculation of information content could also be applied as an alternative to ellipsoidal truncation. The post-mortem analysis also revealed an oversight in accounting for measurement errors in the fast rotation function. While the crystal of mutant Rab27a is not amenable to drug screening, the structure can guide new modifications to obtain more suitable crystal forms

Versatile selective evolutionary pressure using synthetic defect in universal metabolism

Versatile selective evolutionary pressure using synthetic defect in universal metabolis

Engineering Nitrogenases for synthetic nitrogen fixation: From pathway engineering to directed evolution

Globally, agriculture depends on industrial nitrogen fertilizer to improve crop growth. Fertilizer production consumes fossil fuels and contributes to environmental nitrogen pollution. A potential solution would be to harness nitrogenases—enzymes capable of converting atmospheric nitrogen N2 to NH3 in ambient conditions. It is therefore a major goal of synthetic biology to engineer functional nitrogenases into crop plants, or bacteria that form symbiotic relationships with crops, to support growth and reduce dependence on industrially produced fertilizer. This review paper highlights recent work toward understanding the functional requirements for nitrogenase expression and manipulating nitrogenase gene expression in heterologous hosts to improve activity and oxygen tolerance and potentially to engineer synthetic symbiotic relationships with plants

Crystal structure of the [2Fe-2S] protein I (Shethna protein I) from Azotobacter vinelandii

Azotobacter vinelandii is a model diazotroph and is the source of most nitrogenase material for structural and biochemical work. Azotobacter can grow in above-atmospheric levels of oxygen, despite the sensitivity of nitrogenase activity to oxygen. Azotobacter has many iron–sulfur proteins in its genome, which were identified as far back as the 1960s and probably play roles in the complex redox chemistry that Azotobacter must maintain when fixing nitrogen. Here, the 2.1 Å resolution crystal structure of the [2Fe–2S] protein I (Shethna protein I) from A. vinelandii is presented, revealing a homodimer with the [2Fe–2S] cluster coordinated by the surrounding conserved cysteine residues. It is similar to the structure of the thioredoxin-like [2Fe–2S] protein from Aquifex aeolicus, including the positions of the [2Fe–2S] clusters and conserved cysteine residues. The structure of Shethna protein I will provide information for understanding its function in relation to nitrogen fixation and its evolutionary relationships to other ferredoxins

Crystal structure of CyanoQ from the thermophilic cyanobacterium Thermosynechococcus elongatus and detection in isolated photosystem II complexes.

The PsbQ-like protein, termed CyanoQ, found in the cyanobacterium Synechocystis sp. PCC 6803 is thought to bind to the lumenal surface of photosystem II (PSII), helping to shield the Mn(4)CaO(5) oxygen-evolving cluster. CyanoQ is, however, absent from the crystal structures of PSII isolated from thermophilic cyanobacteria raising the possibility that the association of CyanoQ with PSII might not be a conserved feature. Here, we show that CyanoQ (encoded by tll2057) is indeed expressed in the thermophilic cyanobacterium Thermosynechococcus elongatus and provide evidence in support of its assignment as a lipoprotein. Using an immunochemical approach, we show that CyanoQ co-purifies with PSII and is actually present in highly pure PSII samples used to generate PSII crystals. The absence of CyanoQ in the final crystal structure is possibly due to detachment of CyanoQ during crystallisation or its presence in sub-stoichiometric amounts. In contrast, the PsbP homologue, CyanoP, is severely depleted in isolated PSII complexes. We have also determined the crystal structure of CyanoQ from T. elongatus to a resolution of 1.6 Å. It lacks bound metal ions and contains a four-helix up-down bundle similar to the ones found in Synechocystis CyanoQ and spinach PsbQ. However, the N-terminal region and extensive lysine patch that are thought to be important for binding of PsbQ to PSII are not conserved in T. elongatus CyanoQ. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s11120-014-0010-z) contains supplementary material, which is available to authorized users

An integrated biostratigraphy and seismic stratigraphy for the late Neogene continental margin succession in northern Taranaki Basin, New Zealand

Our aim has been to develop an integrated biostratigraphy and seismic stratigraphy for the Pliocene and Pleistocene formations (Ariki, Mangaa, Giant Foresets) in northern Taranaki Basin to better understand the evolution of the modern continental margin offshore central-western North Island, New Zealand. Detailed mapping of seismic reflectors in part of the basin, when compared with correlations of late Neogene stage boundaries between 11 well sections, has highlighted crossover between the datasets. To help resolve this issue, the biostratigraphy of the Pliocene-Pleistocene parts of each of four well sections (Arawa-1, Ariki-1, Kora-1, and Wainui-1) has been re-examined using a dense suite of samples. In addition, the biostratigraphy of seven other well sections (Awatea-1, Kahawai-1, Mangaa-1, Taimana-1, Tangaroa-1, Te Kumi-1, and Turi-1) has been re-evaluated. The crossover is partly attributed to a combination of sampling resolution inherent in exploration well sections, the mixed nature of cuttings samples, and the general scarcity of age-diagnostic planktic foraminifera in the late Neogene formations. The achievement of seismic closure suggests that error in the mapping of the seismic reflectors is not a significant source of the uncertainty (crossover). We have developed a workable time-stratigraphic framework by qualitatively weighting the biostratigraphic data in each of the well sections, thereby identifying the parts of particular well sections with the highest resolution microfossil data and the optimal stratigraphic position of stage boundaries with respect to the mapped seismic horizons/seismic units. Hence, it is possible to assign the known numerical ages for these stage boundaries to reflection horizons/seismic units mapped within the basin. We have applied this information to produce a series of isopach maps for successive stage boundaries that help show the sedimentary evolution of the continental margin succession west of central North Island

Synthetic beta-solenoid proteins with the fragment-free computational design of a beta-hairpin extension

The ability to design and construct structures with atomic level precision is one of the key goals of nanotechnology. Proteins offer an attractive target for atomic design, as they can be synthesized chemically or biologically, and can self-assemble. However the generalized protein folding and design problem is unsolved. One approach to simplifying the problem is to use a repetitive protein as a scaffold. Repeat proteins are intrinsically modular, and their folding and structures are better understood than large globular domains. Here, we have developed a new class of synthetic repeat protein, based on the pentapeptide repeat family of beta-solenoid proteins. We have constructed length variants of the basic scaffold, and computationally designed de novo loops projecting from the scaffold core. The experimentally solved 3.56 ˚A resolution crystal structure of one designed loop matches closely the designed hairpin structure, showing the computational design of a backbone extension onto a synthetic protein core without the use of backbone fragments from known structures. Two other loop designs were not clearly resolved in the crystal structures and one loop appeared to be in an incorrect conformation. We have also shown that the repeat unit can accommodate whole domain insertions by inserting a domain into one of the designed loops

Structure of Psb29/Thf1 and its association with the FtsH protease complex involved in photosystem II repair in cyanobacteria

One strategy for enhancing photosynthesis in crop plants is to improve the ability to repair photosystem II (PSII) in response to irreversible damage by light. D espite the pivotal role of thylakoid embedded FtsH protease complexes in the selective degradation of PSII subunits during repair, little is known about the factors involved in regulating FtsH exp ression. Here we show using the cyanobacterium Synechocystis sp. PCC 6803 that the Psb29 subunit, originally identified as a minor component of His tagged PSII preparations, physically interacts with FtsH complexes in vivo and is required for normal accumulation of the FtsH2/FtsH3 hetero oligo meric complex involved in PSII repair. We show using X ray crystallography that Psb29 from Thermosynechococcus elongatus has a unique fold consisting of a helical bundle and an extended C terminal heli x and contains a highly conserved region that might be involved in binding to FtsH. A similar interaction is likely to occur in Arabidopsis chloroplasts between the Psb29 homologue, termed THF1, and the FTSH2/FTSH5 complex. The direct involvement of Psb29/THF1 in Ft sH accumulation helps explain why THF1 is a target during the hypersensitive response in plants induced by pathogen i nfection. Downregulating FtsH function and the PSII repair cycle via THF1 would cont ribute to the productio