Native SAD Research Articles

Application of CRISPRi for prokaryotic metabolic engineering involving multiple genes, a case study: Controllable P(3HB-co-4HB) biosynthesis

Clustered regularly interspaced short palindromic repeats interference (CRISPRi) is used to edit eukaryotic genomes. Here, we show that CRISPRi can also be used for fine-tuning prokaryotic gene expression while simultaneously regulating multiple essential gene expression with less labor and time consumption. As a case study, CRISPRi was used to control polyhydroxyalkanoate (PHA) biosynthesis pathway flux and to adjust PHA composition. A pathway was constructed in Escherichia coli for the production of poly(3-hydroxybutyrate-co-4-hydroxybutyrate) [P(3HB-co-4HB)] from glucose. The native gene sad encoding E. coli succinate semi-aldehyde dehydrogenase was expressed under the control of CRISPRi using five specially designed single guide RNAs (sgRNAs) for regulating carbon flux to 4-hydroxybutyrate (4HB) biosynthesis. The system allowed formation of P(3HB-co-4HB) consisting of 1–9mol% 4HB. Additionally, succinate, generated by succinyl-coA synthetase and succinate dehydrogenase (respectively encoded by genes sucC, sucD and sdhA, sdhB) was channeled preferentially to the 4HB precursor by using selected sgRNAs such as sucC2, sucD2, sdhB2 and sdhA1 via CRISPRi. The resulting 4HB content in P(3HB-co-4HB) was found to range from 1.4 to 18.4mol% depending on the expression levels of down-regulated genes. The results show that CRISPRi is a feasible method to simultaneously manipulate multiple genes in E. coli.

Multi-crystal native SAD analysis at 6 keV.

Anomalous diffraction signals from typical native macromolecules are very weak, frustrating their use in de novo structure determination. Here, native SAD procedures are described to enhance signal to noise in anomalous diffraction by using multiple crystals in combination with synchrotron X-rays at 6 keV. Increased anomalous signals were obtained at 6 keV compared with 7 keV X-ray energy, which was used for previous native SAD analyses. A feasibility test of multi-crystal-based native SAD phasing was performed at 3.2 Å resolution for a known tyrosine protein kinase domain, and real-life applications were made to two novel membrane proteins at about 3.0 Å resolution. The three applications collectively serve to validate the robust feasibility of native SAD phasing at lower energy.

Native SAD Structure Solution from Merohedrally Twinned Data

Up until now, comparatively few structures were solved by native SAD. Recent advances in multi crystal averaging [1] have shown that native SAD can be applied to an increasing number of cases. Though theoretically possible [2], successful structure solutions from twinned data have not been reported yet. Here, we report the structure solution of the human Centromere protein M from a merohedrally twinned crystal with a twinning fraction of 0.45 in the space group P3. The data were collected at the bending magnet beamline X06DA at the Swiss Light Source, which is equipped with the in-house developed multi-axis goniometer PRIGo and the PILATUS 2M detector. A highly redundant 2.2 Å dataset was collected in a number of different crystal orientations. A substructure solution could only be obtained after 50000 SHELXD [3] tries. Automatic model building after phasing and density modification resulted in a model with the majority of residues built correctly. We will present this particularly difficult case together with other more routine cases, all solved with the same experimental setup and at the beamline X06DA.

Structures of Biological Macromolecules from Multi-crystal Native SAD Phasing

Native metalloproteins have been routine subjects for mutli- and single-wavelength anomalous diffraction (respectively MAD and SAD) analyses for some time; however, despite notable early successes, native molecules without heavy atoms (Z ≥ 25) have only recently become routinely accessible. Crystals of native proteins and nucleic acids have substantial contents of light elements (P, S, Cl, K, Ca) of potential for use in SAD phasing. Anomalous signals from such elements can be enhanced by using a lower than usual x-ray energy; nevertheless, typical Bijvoet differences usually still remain at a level comparable to noise. We have devised robust SAD procedures to study native, light-atom-only biological structures. We have so far used a modestly low energy (6 - 7 keV), but we further enhance the signal-to-noise in anomalous diffraction by combining data from multiple crystals chosen to be statistically equivalent. We have applied our multi-crystal native SAD approach in several structure determinations (1,2) at sizes up to 1200 ordered residues per asymmetric unit and at resolutions so far as low as 3.2 Å. Our tested practices can be replicated readily, and we plan further improvements in computing protocols and in instrumentation.

Multi-crystal native SAD analysis of macromolecular structure

Multi-crystal native SAD analysis of macromolecular structure

Robust structural analysis of native biological macromolecules from multi-crystal anomalous diffraction data.

Structure determinations for biological macromolecules that have no known structural antecedents typically involve the incorporation of heavier atoms than those found natively in biological molecules. Currently, selenomethionyl proteins analyzed using single- or multi-wavelength anomalous diffraction (SAD or MAD) data predominate for such de novo analyses. Naturally occurring metal ions such as zinc or iron often suffice in MAD or SAD experiments, and sulfur SAD has been an option since it was first demonstrated using crambin 30 years ago; however, SAD analyses of structures containing only light atoms (Zmax ≤ 20) have not been common. Here, robust procedures for enhancing the signal to noise in measurements of anomalous diffraction by combining data collected from several crystals at a lower than usual X-ray energy are described. This multi-crystal native SAD method was applied in five structure determinations, using between five and 13 crystals to determine substructures of between four and 52 anomalous scatterers (Z ≤ 20) and then the full structures ranging from 127 to 1200 ordered residues per asymmetric unit at resolutions from 2.3 to 2.8 Å. Tests were devised to assure that all of the crystals used were statistically equivalent. Elemental identities for Ca, Cl, S, P and Mg were proven by f'' scattering-factor refinements. The procedures are robust, indicating that truly routine structure determination of typical native macromolecules is realised. Synchrotron beamlines that are optimized for low-energy X-ray diffraction measurements will facilitate such direct structural analysis.

High-pressure cryocooling for capillary sample cryoprotection and diffraction phasing at long wavelengths

Crystal cryocooling is usually employed to reduce radiation damage during X-ray crystallography. Recently, a high-pressure cryocooling method has been developed which results in excellent diffraction-quality crystals without the use of penetrative cryoprotectants. Three new developments of the method are presented here: (i) Xe-He high-pressure cryocooling for Xe SAD phasing, (ii) native sulfur SAD phasing and (iii) successful cryopreservation of crystals in thick-walled capillaries without additional cryoprotectants other than the native mother liquor. These developments may be useful for structural solution of proteins without the need for selenomethionine incorporation and for high-throughput protein crystallography.

X-ray crystallographic native sulfur SAD structure determination of laminarinase Lam16A fromPhanerochaete chrysosporium

Laminarinase Lam16A from Phanerochaete chrysosporium was recombinantly expressed in Pichia pastoris, crystallized and the structure was solved at 1.34 A resolution using native sulfur SAD X-ray crystallography. It is the first structure of a non-specific 1,3(4)-beta-D-glucanase from glycoside hydrolase family 16 (GH16). P. chrysosporium is a wood-degrading basidiomycete fungus and Lam16A is the predominant extracellular protein expressed when laminarin is used as the sole carbon source. The protein folds into a curved beta-sandwich homologous to those of other known GH16 enzyme structures (especially kappa-carrageenase from Pseudoalteromonas carrageenovora and beta-agarase from Zobelia galactanivorans). A notable likeness is also evident with the related glycoside hydrolase family 7 (GH7) enzymes. A mammalian lectin, p58/ERGIC, as well as polysaccharide lyase (PL7) enzymes also showed significant similarity to Lam16A. The enzyme has two potential N-glycosylation sites. One such site, at Asn43, displayed a branched heptasaccharide sufficiently stabilized to be interpreted from the X-ray diffraction data. The other N-glycosylation motif was found close to the catalytic centre and is evidently not glycosylated.