| Summary: | 博士 === 國立臺灣大學 === 生化科學研究所 === 107 === Nowadays, structural biology has become a reliable subject for providing biophysical interaction evidence in near atomic scale. Decades ago, X-ray crystallography was the most common way to get high resolution protein structural information. However, with the technologies improve, more and more non-crystallographic methods have been developed to breakthrough sample preparation bottleneck, even more, speed-up data process. In this research, we compared two modern strategies used for measuring small virus capsid protein structural information: cryo-electron microscopy (cryo-EM) single particle reconstruction, and X-ray free-electron laser coherent diffractive imaging (XFEL-CDI), not only for benchmark, but also for dig-out any biological significance among these virus sample targets.
In the first part, we purified HeDNV virus from mosquito C6/36 cell line and successfully solved mosquito Haemagogus equinus densovirus (HeDNV) at 2.7 Å near-atomic resolution by cryo-EM single particle reconstruction. Mosquito densovirus infection suggests markedly reduced severity of Dengue virus infection in mosquito cell. The main purpose of this project is to provide high-resolution structural information from a native mosquito densovirus by cryo-EM for fighting mosquito-borne diseases. HeDNV belongs to Brevidensovirus in virus taxonomy, contains small, non-enveloped, T=1 icosahedra (60 subunits), 20-22nm in diameter capsid structure, and a linear, negative-sensed single-stranded DNA of 4–4.2 kb T-shaped hairpin genome. Since the resolution is high enough to interpret amino acid side chain backbone, the results of this research has demonstrate the power of cryo-EM single particle reconstruction. The topology and geometry of HeDNV asymmetric unit shared a classical “jelly roll” core which is highly conserved in all densoviruses. Besides, the electron map distribution of ssDNA genome and capsid protein represents three groups of ordered ssDNA bases interactions with specific amino acids, which is the most abundant of nucleotide density solved so far in densovirus. Furthermore, this is the first report of detailed protein / DNA / metal ion interaction networks contribute to subunit-subunit interaction in densovirus. By comparing of available invertebrate DNV structures, we hope can provide a structural based analysis to classify the sub-classes of densoviruses.
Second part, we are interested in another light resource: X-ray free electron laser (XFEL). It provides femto-second pulse and high coherence. With the combination of coherent diffractive imaging (CDI) method, it may suggest to realize “solving structure without crystal.” In order to investigate “low resolution structural order” of XFEL-CDI technique revealing 30nm biological sample and validate by cryo-EM. In this research, we are offered to use a liquid-enclosure chamber for sample loading in order to mimic the native state of biological sample, and we tend to use icosahedral Human Hepatitis B virus (HBV) capsid, a smaller and structure-known virus particle as a benchmark for our XFEL setup, with the help of cryo-EM. The results showed that there are three more diffraction rings appear in high concentrated HBV capsid samples, which represented the “structure factor” of HBV capsid. To validate from cryo-EM by increasing low frequency signal, we used “defocus” and “phase plate” method, compared with CDI results in reciprocal space. The power spectrum of XFEL-CDI, CEM, and CEM with VPP fits well in low frequency represents the structure factor of HBV capsid. The limited power spectrum of was collected in high concentrated HBV capsid solution, brings out the insufficiency of single particle XFEL-CDI in 30nm Virus-like sample so far. Furthermore, phase plate contributes significant contrast intensity in low frequency area, which becomes a useful tool to get macro-molecule shape information.
Taken together, I hope this research can provide a clear answer to researchers choosing suitable light source for their structural biology research purpose.
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