Visualizing the translation landscape in human cells at high resolution.

Obtaining comprehensive structural descriptions of macromolecules within their natural cellular context holds immense potential for understanding fundamental biology and improving health. Here, we present the landscape of protein synthesis inside human cells in unprecedented detail obtained using an approach which combines automated cryo-focused ion beam (FIB) milling and in situ single-particle cryo-electron microscopy (cryo-EM). With this in situ cryo-EM approach we resolved a 2.19 Å consensus structure of the human 80S ribosome and unveiled its 21 distinct functional states, nearly all higher than 3 Å resolution. In contrast to in vitro studies, we identified protein factors, including SERBP1, EDF1 and NAC/3, not enriched on purified ribosomes. Most strikingly, we observed that SERBP1 binds to the ribosome in almost all translating and non-translating states to bridge the 60S and 40S ribosomal subunits. These newly observed binding sites suggest that SERBP1 may serve an important regulatory role in translation. We also uncovered a detailed interface between adjacent translating ribosomes which can form the helical polysome structure. Finally, we resolved high-resolution structures from cells treated with homoharringtonine and cycloheximide, and identified numerous polyamines bound to the ribosome, including a spermidine that interacts with cycloheximide bound at the E site of the ribosome, underscoring the importance of high-resolution in situ studies in the complex native environment. Collectively, our work represents a significant advancement in detailed structural studies within cellular contexts.

Ice nucleation proteins self-assemble into large fibres to trigger freezing at near 0 °C.

In nature, frost can form at a few degrees below 0 °C. However, this process requires the assembly of tens of thousands of ice-like water molecules that align together to initiate freezing at these relatively high temperatures. Water ordering on this scale is mediated by the ice nucleation proteins (INPs) of common environmental bacteria like Pseudomonas syringae and Pseudomonas borealis. However, individually, these 100 kDa proteins are too small to organize enough water molecules for frost formation, and it is not known how giant, megadalton-sized multimers, which are crucial for ice nucleation at high sub-zero temperatures, form. The ability of multimers to self-assemble was suggested when the transfer of an INP gene into Escherichia coli led to efficient ice nucleation. Here, we demonstrate that a positively charged subdomain at the C-terminal end of the central ß-solenoid of the INP is crucial for multimerization. Truncation, relocation, or change of the charge of this subdomain caused a catastrophic loss of ice nucleation ability. Cryo-electron tomography of the recombinant E. coli showed that the INP multimers form fibres that are ~5 nm across and up to 200 nm long. A model of these fibres as an overlapping series of antiparallel dimers can account for all their known properties and suggests a route to making cell-free ice nucleators for biotechnological applications.

Ice nucleation proteins self-assemble into large fibres to trigger freezing at near 0 °C.

In nature, frost can form at a few degrees below 0 °C. However, this process requires the assembly of tens of thousands of ice-like water molecules that align together to initiate freezing at these relatively high temperatures. Water ordering on this scale is mediated by the ice nucleation proteins of common environmental bacteria like Pseudomonas syringae and P. borealis. However, individually, these 100-kDa proteins are too small to organize enough water molecules for frost formation, and it is not known how giant, megadalton-sized multimers, which are crucial for ice nucleation at high sub-zero temperatures, form. The ability of multimers to self-assemble was suggested when the transfer of an ice nucleation protein gene into Escherichia coli led to efficient ice nucleation. Here we demonstrate that a positively-charged sub-domain at the C-terminal end of the central beta-solenoid of the ice nucleation protein is crucial for multimerization. Truncation, relocation, or change of the charge of this subdomain caused a catastrophic loss of ice nucleation ability. Cryo-electron tomography of the recombinant E. coli showed that the ice nucleation protein multimers form fibres that are ~ 5 nm across and up to 200 nm long. A model of these fibres as an overlapping series of antiparallel dimers can account for all their known properties and suggests a route to making cell-free ice nucleators for biotechnological applications.

Anti-σ28 Factor FlgM Regulates Flagellin Gene Expression and Flagellar Polarity of Treponema denticola.

FlgM, an antagonist of FliA (also known as σ28), inhibits transcription of bacterial class 3 flagellar genes. It does so primarily through binding to free σ28 to prevent it from forming a complex with core RNA polymerase. We recently identified an FliA homolog (FliATd) in the oral spirochete Treponema denticola; however, its antagonist FlgM remained uncharacterized. Herein, we provide several lines of evidence that TDE0201 functions as an antagonist of FliATd. TDE0201 is structurally similar to FlgM proteins, although its sequence is not conserved. Heterologous expression of TDE0201 in Escherichia coli inhibits its flagellin gene expression and motility. Biochemical and mutational analyses demonstrate that TDE0201 binds to FliATd and prevents it from binding to the σ28-dependent promoter. Deletions of flgM genes typically enhance bacterial class 3 flagellar gene expression; however, deletion of TDE0201 has an opposite effect (e.g., the mutant has a reduced level of flagellins). Follow-up studies revealed that deletion of TDE0201 leads to FliATd turnover, which in turn impairs the expression of flagellin genes. Swimming plate, cell tracking, and cryo-electron tomography analyses further disclosed that deletion of TDE0201 impairs spirochete motility and alters flagellar number and polarity: i.e., instead of having bipolar flagella, the mutant has flagella only at one end of cells. Collectively, these results indicate that TDE0201 is a FlgM homolog but acts differently from its counterparts in other bacteria. IMPORTANCE Spirochetes are a group of bacteria that cause several human diseases. A unique aspect of spirochetes is that they have bipolar periplasmic flagella (PFs), which bestow on the spirochetes a unique spiral shape and distinct swimming behaviors. While the structure and function of PFs have been extensively studied in spirochetes, the molecular mechanism that regulates the PFs' morphogenesis and assembly is poorly understood. In this report, FlgM, an anti-σ28 factor, is identified and functionally characterized in the oral spirochete Treponema denticola. Our results show that FlgM regulates the number and polarity of PFs via a unique mechanism. Identification of FliA and FlgM in T. denticola sets a benchmark to investigate their roles in other spirochetes.

Three-dimensional analysis of membrane structures associated with tomato spotted wilt virus infection.

To study viral infection, the direct structural visualization of the viral life cycle consisting of virus attachment, entry, replication, assembly and transport is essential. Although conventional electron microscopy (EM) has been extremely helpful in the investigation of virus-host cell interactions, three-dimensional (3D) EM not only provides important information at the nanometer resolution, but can also create 3D maps of large volumes, even entire virus-infected cells. Here, we determined the ultrastructural details of tomato spotted wilt virus (TSWV)-infected plant cells using focused ion beam scanning EM (FIB-SEM). The viral morphogenesis and dynamic transformation of paired parallel membranes (PPMs) were analyzed. The endoplasmic reticulum (ER) membrane network consisting of tubules and sheets was related to viral intracellular trafficking and virion storage. Abundant lipid-like bodies, clustering mitochondria, cell membrane tubules, and myelin-like bodies were likely associated with viral infection. Additionally, connecting structures between neighboring cells were found only in infected plant tissues and showed the characteristics of tubular structure. These novel connections that formed continuously in the cell wall or were wrapped by the cell membranes of neighboring cells appeared frequently in the large-scale 3D model, suggesting additional strategies for viral trafficking that were difficult to distinguish using conventional EM.

FIB-SEM analysis on three-dimensional structures of growing organelles in wild Chlorella pyrenoidosa cells.

To clarify dynamic changes of organelle microstructures in Chlorella pyrenoidosa cells during photosynthetic growth with CO2 fixation, three-dimensional (3D) organelle microstructures in three growth periods of meristem, elongation, and maturity were quantitatively determined and comprehensively reconstructed with focused ion beam scanning electron microscopy (FIB-SEM). The single round-pancake mitochondria in each cell split into a dumbbell and then into a circular ring, while the barycenter distance of mitochondria to chloroplast and nucleus was reduced to 45.5% and 88.3% to strengthen energy transfer, respectively. The single pyrenoid consisting of a large part and another small part in each chloroplast gradually developed to a mature state in which the two parts were nearly equal in size. The nucleolus progressively became larger with euchromatin replication. The number of starch grains gradually increased, but the mean grain volume remained nearly unchanged.

Life-cycle assessment of flue gas CO2 fixation from coal-fired power plant and coal chemical plant by microalgae.

The technology of flue gas CO2 fixation by microalgae is highly attractive in the era of CO2 neutrality. However, CO2 emission along the whole process has yet to be sufficiently evaluated. Here, a life-cycle assessment was performed to evaluate the energy conversion characteristics and environmental impacts of flue gas CO2 fixation from coal-fired power plant (Case 1) and coal chemical plant (Case 2) by microalgae. The results show that total energy consumption and CO2 gas emissions for Case 1 are 27.5-38.0 MJ/kg microalgae power (MP) and 5.7-7.7 kg CO2 equiv/kg MP, respectively, which are lower than that for Case 2 (122.5-181.3 MJ/kg MP and 32.7-48.6 kg CO2 equiv/kg MP). The CO2 gas aeration rate and microalgae growth rate are the two most sensitive parameters for the energy conversion and net CO2 emission. Therefore, increasing the CO2 aeration efficiency and microalgae growth rate are key to advance the technology of flue gas CO2 fixation by microalgae which will contribute to carbon naturality.

Developing staggered woven mesh aerator with three variable-micropore layers in recycling water pipeline to enhance CO2 conversion for improving Arthrospira growth.

A staggered woven mesh (SWM) aerator equipped with three variable-micropore layers was developed to enhance the CO2 conversion into HCO3- in a recycling water pipeline for promoting CO2 utilization efficiency and Arthrospira growth in large-scale raceway ponds. The input CO2 gas was broken into smaller bubbles (0.78- 2.43 mm) through the first-stage shear with axial rectangles, second-stage shear with radial rectangles (equivalent pore diameter = 150 µm), and third-stage shear with uniform micropores. A high-speed camera (MotionXtra HG-100K CMOS) and an Image J image processing software were employed to capture the bubble pictures. Compared to the traditional steel pipe (TSP) aerator, the bubble generation diameter and time in the SWM aerator reduced by 72.3% and 48.6%, respectively. The optimized structure (ε = 14, pore = 23 µm) of the SWM aerator promoted the carbonization efficiency and HCO3- conversion efficiency into biomass by 78.6% and 64.6% than the TSP aerator. Further, the chlorophyll fluorescence and biomass measurements showed an increase in the actual photochemical efficiency (analyzed by Hansatech FMS1 chlorophyll fluorescence instrument) and biomass yield by 1.8 times and 80.1%.

Orange light spectra filtered through transparent colored polyvinyl chloride sheet enhanced pigment content and growth of Arthrospira cells.

Microalgae are significantly affected by the spectra composition with various wavelengths. The development of light harvesting pigments can be controlled with specific wavelength of filtered light received by microalgae. Coverage of open raceway pond using transparent colored polyvinyl chloride sheets (PVCS) to filter light spectra, was assessed for the capacity to enhance biomass growth rate. Results showed that orange PVCS filtered light spectra at wavelengths from 480 to 665 nm, increased biomass dry weight (3.3 g/L) by 61% compared with control condition (white PVCS = 350-750 nm). Light spectra filtered through orange PVCS were more easily absorbed by the light harvesting pigment protein complex (phycobilisome) of Arthrospira platensis cells and subsequently transferred to intracellular photosynthesis reaction centers. Therefore, A. platensis cells cultivated with light spectra filtered through orange PVCS contained 62.7 mg/L chlorophyll-a and 23.5 mg/L carotenoid, which were 40% and 29% higher than control condition (with white PVCS).

The flagellar motor of Vibrio alginolyticus undergoes major structural remodeling during rotational switching.

The bacterial flagellar motor switches rotational direction between counterclockwise (CCW) and clockwise (CW) to direct the migration of the cell. The cytoplasmic ring (C-ring) of the motor, which is composed of FliG, FliM, and FliN, is known for controlling the rotational sense of the flagellum. However, the mechanism underlying rotational switching remains elusive. Here, we deployed cryo-electron tomography to visualize the C-ring in two rotational biased mutants in Vibrio alginolyticus. We determined the C-ring molecular architectures, providing novel insights into the mechanism of rotational switching. We report that the C-ring maintained 34-fold symmetry in both rotational senses, and the protein composition remained constant. The two structures show FliG conformational changes elicit a large conformational rearrangement of the rotor complex that coincides with rotational switching of the flagellum. FliM and FliN form a stable spiral-shaped base of the C-ring, likely stabilizing the C-ring during the conformational remodeling.

Development of a single helical baffle to increase CO2 gas and microalgal solution mixing and Chlorella PY-ZU1 biomass yield.

A single helical baffle (SHB), consisting of twisted turns, was developed to convert straight flow into spiral flow in a Chlorella PY-ZU1 open raceway pond (ORWP) bubbled with 15% CO2. Microalgal solution flowing through the SHB alternative helical interspaces generated whirling flow both vertically and horizontally, which decreased mixing and increased mass transfer rates. The optimized SHB had a pitch length to total SHB length ratio of 0.13 and SHB diameter to ORWP single channel width ratio of 0.30, which decreased mixing times and increased mass transfer coefficients by 41.1% and 38.4% respectively. SHB moved Chlorella PY-ZU1 from the ORWP bottom to the top, increasing light exposure for photosynthesis. Cellular electron transfer rates and photochemical efficiency (φPSII) increased by 18%, chlorophyll a content increased by 16% and variable to maximum fluorescence ratio increased by 13%. The microalgal biomass of SHB ORWP was 23% higher than that of conventional ORWP.

Improving light distribution and light/dark cycle of 900 L tangential spiral-flow column photobioreactors to promote CO2 fixation with Arthrospira sp. cells.

The light distribution and light/dark cycle were improved in 900 L tangential spiral-flow column photobioreactors (TSCP) to promote CO2 fixation with Arthrospira sp. cells. Solar irradiation model was employed in CFD simulation to investigate light distribution and light/dark cycle in flow field composed of culture medium, CO2 bubbles and Arthrospira sp. cells under actual sunlight irradiation considering geolocation and time. An accurate way to divide light/dark zone based on saturate light intensity and light intensity field was adopted for the first time. When Arthrospira sp. cell concentration increased from 0.1 to 0.9 g/L, light/dark cycle frequency of cells firstly increased from 0.650 Hz to 0.868 Hz and then decreased to 0.117 Hz. Intracellular chlorophyll a content and carotenoids content of Arthrospira sp. cells in TSCP were 6% and 41% higher than those in conventional bubble column photobioreactor. This promoted cellular photosynthesis and stress resistance, which contributed to increase CO2 fixation rate of Arthrospira sp. cells by 59%. When CO2 aeration rate, CO2 volume concentration, and circulating pump power were 0.210 L/min, 15%, and 30 W, chlorophyll a content, helix pitch, and CO2 fixation rate of Arthrospira sp. cells all reached peak values of 8.769 mg/L, 78.26 µm and 0.358 g/L/d, respectively.

A novel porous nickel-foam filled CO2 absorptive photobioreactor system to promote CO2 conversion by microalgal biomass.

In order to solve problems associated with a short residence time and low conversion efficiency when CO2 gas is aerated directly into raceway ponds, a novel porous nickel-foam filled CO2 absorptive photobioreactor system was developed to promote CO2 conversion to NaHCO3 in a short time to improve photosynthesis of microalgal cells. Numerical simulation showed that the porous nickel-foam promoted the Na2CO3 solution radial velocity and CO2 volume fraction in the CO2 absorption reactor, which enhanced the reaction rate of CO2 gas and soluble Na2CO3. The conversion efficiency of CO2 gas to soluble NaHCO3 gradually increased with an increasing nickel-foam pore diameter and a decreasing CO2 gas outflow rate, while it first increased and then decreased with an increasing relative nickel-foam height in the CO2 absorption reactor. The conversion efficiency from soluble NaHCO3 to microalgal biomass first increased and then decreased with an increasing nickel-foam pore diameter (peaking at 2 mm) and relative height (peaking at 0.24); and CO2 gas outflow rate (peaking at 2 L/min). The chlorophyll fluorescence measurements showed that a sufficient HCO3- supply promoted the quantum ratio used for electron transfer (from 0.19 to 0.23) and the maximum photochemical efficiency (from 0.48 to 0.52), resulting in an increased biomass growth rate (by 1.1 times) when the nickel-foam pore diameter increased from 0.1 to 2 mm.

Conversion of NaHCO3 to Na2 CO3 with a growth of Arthrospira platensis cells in 660 m2 raceway ponds with a CO2 bicarbonation absorber.

The weight ratio of Na2 CO3 /NaHCO3 was investigated in order to improve microalgal productivity in large-scale industrial operations by converting NaHCO3 to Na2 CO3 with a growth of Arthrospira platensis cells in 660 m2 raceway ponds. Two microalgal cultivation systems with a NaHCO3 by-product (SPBP) and a CO2 bicarbonation absorber (CBAP) were firstly thoroughly introduced. There was a 13.3% decrease in the initial weight ratio of Na2 CO3 /NaHCO3 resulting in a 25.3% increase in the biomass growth rate with CBAP, compared to that of SPBP. Increased sunlight intensity, solution temperature and pH all resulted in both a higher HCO 3 - absorbance and CO 3 2 - release, thereby increasing the weight ratio of Na2 CO3 /NaHCO3 during the growth of A. platensis. The biomass growth rate was peaked at 39.9 g m-2  day-1 when the weight ratio of Na2 CO3 /NaHCO3 was 3.7. Correspondingly, the cell pigments (chlorophyll a and carotenoid) and trichome size (helix pitch and trichome length) reached to a maximum state of 8.47 mg l-1 , 762 µg l-1 , 57 and 613 µm under the CBAP system.

Three-dimensional numerical simulation of light penetration in an optimized flow field composed of microalgae cells, carbon dioxide bubbles and culture medium.

In order to evaluate light penetration and its influence on microalgae growth in a raceway pond with alternatively permutated conic baffles (RWP-APCB), 3D numerical simulation of light penetration was performed using computational fluid dynamics in an optimized flow field composed of microalgae cells, CO2 bubbles and culture medium. Results showed that light intensity in the culture medium attenuated faster in accordance with solution depth, with increased microalgae cell concentration, increased bubble volume fraction and decreased CO2 bubble diameter. Light zone fraction (i.e. ratio of light zone length to solution depth) increased with promoted incident irradiation. It was found that around 75% of microalgae cells were distributed in light zone and non-photochemical quenching coefficient of microalgae decreased by 32% in RWP-APCB. This resulted in a 16% increase of the Chlorella pyrenoidosa biomass growth rate, to 0.36 g/L/d.

A novel jet-aerated tangential swirling-flow plate photobioreactor generates microbubbles that enhance mass transfer and improve microalgal growth.

To reduce bubble diameter and enhance mass transfer, a novel jet-aerated tangential swirling-flow plate photobioreactor was developed that improves the growth rate of microalgae. In this system, the circulating microalgal solution enters a jet aerator that takes up 15% CO2 by vacuum suction and then injects into a plate photobioreactor through four centrally symmetric nozzles. Each jetflow is tangent to a tangential circle, driving vertical vortex movement of the surrounding microalgal solution, which markedly reduced the bubble diameter and enhanced mass transfer. The mass transfer coefficient was enhanced by decreasing the nozzle number (n) and increasing the ratio of tangential circle diameter to plate photobioreactor equivalent diameter (d/D). The average bubble diameter decreased by 80.2% to 0.37 mm and the mass transfer coefficient increased 4.6 times to 48.9 h-1 when n was 4 and d/D was 0.34. Finally, the optimized system increased the biomass dry weight of microalgae by 49.4%.

Self-rotary propellers with clockwise/counterclockwise blades create spiral flow fields to improve mass transfer and promote microalgae growth.

In this work, self-rotary propellers (SRPs) with clockwise/counterclockwise blades were investigated to create spiral flow fields without external power to strengthen gas-liquid mixing and promote microalgal growth in an open raceway pond. The rotational flow around the propellers and spiral flow between the propellers generated extensive wall shear stress in three dimensions. Four-clockwise blades on the propellers exerted better mixing than three-counterclockwise blades. The bubble generation diameter was reduced by 69% and the mass transfer coefficient increased by 49% when the propeller diameter was increased from 32 to 60 mm. The photochemical efficiency (φPSII) of Arthrospira platensis cells was enhanced by 25%, while the helix pitch and trichome lengths were enlarged by 7-16%. Self-rotary propellers (60 mm diameter) with four-clockwise blades enhanced the growth rate of A. platensis biomass by 35% compared to that in an unmodified raceway pond without propellers.

Developing microporous fibrous-diaphragm aerator to decrease bubble generation diameter for improving microalgal growth with CO2 fixation in a raceway pond.

A novel microporous fibrous-diaphragm aerator (FDA) was proposed to generate pressurized gas with check valve to penetrate through millions of micropores (6-126 µm) of expanded fibrous diaphragm on internal support, thus decreasing bubble generation diameter and increasing bubble residence time for improving microalgal growth with CO2 fixation in a raceway pond. When installation angle of FDA internal support increased from 0° to 45°, bubble generation time and diameter first decreased (to valley bottoms of 4 ms and 0.45 mm at 22°) and then increased. Compared to traditional strip aerator, bubble generation time and diameter decreased by 50% and 60% through FDA with support installation angle of 22° and average pore diameter of 28 µm, while gas-solution mixing time decreased by 22% and mass transfer coefficient increased by 40%, leading to increased actual photochemical efficiency (by 80%) and increased biomass yield (by 38.5%) of Arthrospira cells with pure CO2 aeration through FDA.

Developing a CO2 bicarbonation absorber for promoting microalgal growth rates with an improved photosynthesis pathway.

In order to solve the problems of the short residence time and low utilization efficiency of carbon dioxide (CO2) gas added directly to a raceway pond, a CO2 bicarbonation absorber (CBA) was proposed to efficiently convert CO2 gas and sodium carbonate (Na2CO3) solution to sodium bicarbonate (NaHCO3), which was dissolved easily in the culture medium and left to promote the microalgal growth rate. The CO2 gas reacted with the Na2CO3 solution (initial concentration = 200 mM L-1 and volume ratio in CBA = 60%) for 90 min at 0.3 MPa to give the optimized molar proportion (92%) of NaHCO3 product in total inorganic carbon and increase the microalgal growth rate by 5.0 times. Quantitative label-free protein analysis showed that the expression levels of the photosystem II (PSII) reaction centre protein (PsbH) and PSII cytochrome (PsbV2) in the photosynthesis pathway increased by 4.8 and 3.4 times, respectively, while that of the RuBisCO enzyme (rbcL) in the carbon fixation pathway increased by 3.5 times in Arthrospira platensis cells cultivated with the NaHCO3 product in the CBA at 0.3 MPa.