Carbonic Acid

So far, the determined residues consist solely of C $\alpha$ atoms. A complete protein backbone also consists of carbon, nitrogen, and oxygen atoms. Previous research has introduced various methods for reconstruction of a protein backbone from a reduced representation, such as one that contains only C $\alpha$ atoms (28 (link)). Instead of employing these theoretical methods, we chose to implement our own backbone reconstruction method to make use of the information captured from the 3D cryo-EM maps. This section presents our all-atom backbone reconstruction method. This is necessary for the next step in the pipeline, resolving the side-chain atoms.
In addition to C $\alpha$ prediction, the U-Net also provides information about carbon and nitrogen atoms in the confidence map predicted by the U-Net. We can use this information in combination with the previously determined C $\alpha$ atom positions to place the carbon and nitrogen atoms. Between the C $\alpha$ atoms of two connected amino acids, there is always a nitrogen and a carbon atom. Therefore, we can guess the initial position of these atoms by calculating the vector from one C $\alpha$ atom to the other and then placing the nitrogen and carbon atoms at one-third and two-thirds of the distance of this vector. To refine these initial positions, we calculated the center of mass around them in the carbon and nitrogen confidence maps. In Fig. 7A, we can see an example for the initial and refined placement of the carbon and nitrogen atoms.
After the initial refinement, we can further refine the positions of the carbon and nitrogen atoms by applying well-known molecular mechanics of a peptide chain. We made several assumptions about the positions of carbon, nitrogen, and oxygen atoms relative to the C $\alpha$ atoms, as seen in Fig. 7B. First, we assumed the planar peptide geometry in which the C $\alpha$ atom and carbon atom in the carbonyl group of an amino acid are in the same plane as the next amino acid’s nitrogen and C $\alpha$ atom (29 (link)). Second, we constructed a virtual bond between the neighboring C $\alpha$ atoms. The angles between this bond and the C ${\alpha }_{\left(i\right)}$ – ${\mathrm{C}}_{\left(i\right)}$ bond ( ${\theta }_2$ ) and between this bond and the C ${\alpha }_{\left(i+1\right)}$ – ${\mathrm{N}}_{\left(i+1\right)}$ bond ( ${\varphi }_2$ ) are 20. $\mathrm{9}°$ and 14. $\mathrm{9}°$ , respectively (29 (link)). Third, the peptide bonds in a protein are in the stable trans configuration (30 ).
To refine the position of the carbon atoms, we relied on the previous refinement. Let us call the unit vector pointing from C ${\alpha }_{\left(i\right)}$ to ${\mathrm{C}}_{\left(i\right)refined}\hspace{0.17em}{v}_1$ , the unit vector pointing from C ${\alpha }_{\left(i\right)}$ to ${\mathrm{C}}_{\left(i\right)}\hspace{0.17em}{v}_2$ , and the unit vector pointing from C ${\alpha }_{\left(i\right)}$ to C ${\alpha }_{\left(i+1\right)}\hspace{0.17em}{v}_3$ . $\begin{array}{l}\hfill v_1\hspace{0.17em}=\hspace{0.17em}<a_1,a_2,a_3>\\ \hfill v_2\hspace{0.17em}=\hspace{0.17em}<b_1,b_2,b_3>\\ \hfill v_3\hspace{0.17em}=\hspace{0.17em}<c_1,c_2,c_3>.\end{array}$ The goal is to solve for the components of ${\mathrm{v}}_1$ . Due to the planar peptide geometry, $v_1$ , $v_2$ , and $v_3$ exist in the same plane. Thus, their triple product equals zero. $v_1\times \left(v_2\cdot v_3\right)=0$ $\begin{array}{l}\hfill or\\ \hfill a_1\left(b_2{c}_3-b_3{c}_2\right)-a_2\left(b_1{c}_3-b_3{c}_1\right)+a_3\left(b_1{c}_2-b_2{c}_1\right)=0.\end{array}$ From this relation and the cross-product of $v_1$ and $v_2$ , and that of $v_2$ , $v_3$ , we can construct a system of equations, $\begin{array}{l}\hfill \left\{\begin{array}{c}{a}_1{b}_1+a_2{b}_2+a_3{b}_3=cos\left({\theta }_2-{\theta }_1\right)\hspace{1em}\hfill \\ a_1{c}_1+a_2{c}_2+a_3{c}_3=cos\left({\theta }_2\right)\hspace{1em}\hfill \\ a_1\left(b_2{c}_3-b_3{c}_2\right)-a_2\left(b_1{c}_3-b_3{c}_1\right)+a_3\left(b_1{c}_2-b_2{c}_1\right)=0\hspace{1em}\hfill \end{array}\right..\\ \hfill \end{array}$ Solving this system of equations yields $a_1$ , $a_2$ and $a_3$ . Next, the vector $v_1$ is scaled appropriately to resolve the new position of the carbon atom. The position of the nitrogen atom is refined in a similar manner.
To determine the location of the oxygen atom in the carbonyl group, we assumed the coplanar relationship between the oxygen, C $\alpha$ , carbon, and nitrogen atom (29 (link)), and that the angles ${\mathrm{A}}_{\alpha CO}$ and ${\mathrm{A}}_{OCN}$ (Fig. 7C) are approximately identical. We then derived a unit vector pointing in the direction of the C–O bond and scaled it with the C–O bond length to get the position of the oxygen atom.

The properties of the bottom sediment, which have been analysed, are as follows: particle size fractions, pH and redox potential. The analytical methods, as well as the above parameters, have been described in our previous studies (Tarnawski and Baran 2018 (link)). The content of total organic carbon (TOC) in sediments was determined using a CNS analyser (Vario EL Cube, Elementar Analysensysteme 2013). The content of humus compounds was extracted from bottom sediments using a mixture of 0.1 mol dm⁻³ Na₄P₂O₇ solution and 0.1 mol dm⁻³ NaOH (Mierzwa-Hersztek et al. 2018 ). The carbon of humic acids (Cha) was isolated in the extract of sodium pyrophosphate and a sodium base, whereas the carbon of fulvic acids (Ckf) was calculated from the difference between the amount of carbon (C ext) as well as the amount of humic acid carbon (Cha) in the extract. The extraction residue—non-hydrolysing carbon (Cnh)—was computed from the difference between the total organic carbon content (TOC) and the amount of carbon in the extract. In the prepared solutions of humic acids, light absorbance was measured at the 465 and 665 nm wavelength and the colour ratio (E₄/E₆) was computed (Mierzwa-Hersztek et al. 2018 ). In order to determine the dissolved organic carbon (DOC), the sediment samples were extracted in sediment: water ratio 1:10 v/v, by shaking on a rotary shaker for 24 h. Next, the samples were centrifuged in 50-ml tubes at 3000×g for 10 min, and filtered through a 0.45 μm membrane filter (Akkanen et al. 2005 (link)). The DOC content was measured using TOC analyser 1200 (Thermo Elektron).

AEs and SEs were first screened using GC fitted with a flame ionization detector (FID) for quantification and general screening of preservation. An Agilent 7890A Series gas chromatograph was fitted with a DB1-high temperature (HT) column (15 m × 0.32 mm × 0.1 µm). One microlitre of the extract was injected via a splitless injector maintained at a temperature of 300°C. The temperature of the column was kept at 100°C for 2 min and then increased by 20°C every minute until a final temperature of 325°C was reached. A temperature of 325°C was then held for 2 min. Helium was used as the carrier gas at constant flow. The detector was kept at 300°C with hydrogen flow of 30 ml min⁻¹. For SEs, the temperature of the column was kept at 50°C for 2 min and then increased by 10°C every minute until a final temperature of 375°C was reached. A temperature of 375°C was then held for 10 min.
To identify molecular profiles, all extracts were analysed using GC-MS. For analysis of AEs and ABEs, the GC component was an Agilent 7890A series attached to an MS Agilent 5975 Inert XL mass selective detector with a quadrupole mass analyser (Agilent Technologies, Cheadle, UK). A DB-5MS (5%-phenyl)-methylpolysiloxane column (30 m × 0.250 mm × 0.25 µm; J&W Scientific, Folsom, CA, USA) was used. The GC column was inserted directly into the ion source of the mass spectrometer. One microlitre of sample was injected via a splitless injector maintained at a temperature of 300°C. Helium at constant flow was used as the carrier gas. The ionization energy of the spectrometer was 70 eV and spectra were obtained by scanning between m/z 50 and 800. The temperature of the column was kept at 50°C for 2 min and then increased by 10°C every minute until a final temperature of 325°C was reached. A temperature of 325°C was then held for 15 min. For SEs, a HT column and programme were used to detect the presence of tri-, di and mono-acylglycerols (TAGs, DAGs and MAGs) and wax esters. A DB5-HT column (30 m × 0.25 mm × 0.1 µm) was used, and the temperature of the column was kept at 50°C for 2 min and then increased by 10°C every minute until a final temperature of 375°C was reached. To target ions specific to alkylresorcinols SEs were analysed using the same chromatographic conditions with the mass spectrometer in SIM mode. The ions m/z 73, 268, 464, 492, 520, 548, 576, 604 and 632, corresponding to alkylresorcinols with cyclic carbon chain lengths C₁₇ to C₂₅, were monitored.
AEs were also analysed using an Agilent 7890A series chromatograph attached to an MS Agilent 5975 Inert XL mass selective detector with a quadrupole mass analyser (Agilent Technologies, Cheadle, UK) equipped with a DB-23 (50%-Cyanopropyl)-methylpolysiloxane column (60 m × 0.250 mm × 0.25 µm; J&W Scientific, Folsom, CA, USA). The temperature of the column was kept at 50°C for 2 min and then increased by 10°C every minute until 100°C. The temperature increased then until 140°C by 4°C every minute, then until 160°C by 0.5°C every minute and finally until 250°C by 20°C every minute. A SIM mode was used to target different groups of ions. These groups were: m/z 74, 105, 262, 290, 318 and 346 for the detection of ω-(o-alkyl phenyl)alkanoic acids of carbon lengths C₁₆ to C₂₂ (APAA_16–22), m/z 74, 87, 213, 270 for TMTD, m/z 74, 88, 101, 312 for pristanic acid, m/z 74, 101, 171, 326 for phytanic acid and m/z 74, 105, 262, 290, 318, 346 for the detection of ω-(o-alkyl phenyl)alkanoic acids of carbon lengths C₁₆ to C₂₂ (APAA_16–22).