User:Francielle Aguiar Gomes/Sandbox 1

Introduction

Rhodospirillum (Rsp.) rubrum is an anoxygenic phototrophic purple bacterium with a long history as a model for the study of bacterial photosynthesis and related metabolic processes. It is unique among purple bacteria by producing both rhodoquinone (RQ) and ubiquinone (UQ)1 as electron carriers and bacteriochlorophyll (BChl) a esterified at the propionic acid side chain by geranylgeraniol (abbreviated as BChlaG) rather than phytol. The light-harvesting complexes (LHC) of photosynthetic purple sulfur and non-sulfur bacteria are responsible for the highly efficient collection and transfer of light energy to the photosynthetic reaction centres. This results in an initial separation of charge in the reaction centre (RC) and ultimately conversion of the light energy into a chemically useful form ^[1]. Rsp. rubrum has a single pair of αβ-polypeptides in its core light-harvesting (LH1) complex and lacks both the peripheral light-harvesting (LH2) complex and reaction center (RC) cytochrome (Cyt) c subunit present in many purple bacteria; thus, Rsp. rubrum is one of the simplest phototrophic bacteria known, in terms of its photosynthetic light reactions. Because the entire Rsp. rubrum LH1 complex and a stable B820 LH1-subunit can be reconstituted using the αβ-polypeptides and pigment molecules,3−5 both complexes have been intensively studied as models of the bacterial antenna apparatus6 and as such have provided a wealth of information on mechanisms of light energy acquisition, pigment−protein interactions, and assembly of multicomponent complexes. ^[2]

Inicial Structures

Structures of both purified LH1 and the RC-associated core complex (LH1-RC) of Rsp. rubrum have not been obtained at high resolution, and no RC atomic structure was known. The 8.5 Å resolution projection of R. rubrum LHCl represents the first glimpse of the structural architecture of the fundamental building block of the photosynthetic membrane in purple BChla-containing bacteria. The crystals diffract beyond 8 Å and the projection map was calculated to 8.5 A. The projection map shows 16 subunits in a 116 Å diameter ring with a 68 Å hole in the center.

Fourier components measure up to 8.0 Å resolution for estimation. The size of the boxes indicates the [IQ][[1]] value, with IQ=1 being the largest. This image shows the equivalent projection calculated from the average amplitudes and phases of eight images with symmetry applied and amplitudes enhanced by a resolution-dependent scaling factor. The positive density rings can represent the protein. 16-fold strong and 32-fold weak components can be seen above the noise level, thus indicating a 16-fold non-crystalline rotational symmetry in projection density. The 16 times rotationally filtered map is shown below.

Each of the 16 subunits contains three distinct domains. Assuming that the ring in our projection represents a 3 Da cylinder with outside diameter 116 Å, inside diameter 68 Å and length 45 Å (one membrane layer thick), this would correspond to a volume of 311000 Å^3. Assuming an average protein density of 0.77 Da/A3, this would give the cylinder a total mass of 266 kDa, corresponding to ~17 αβ-subunits with their associated bacteriochlorophylls and carotenoids, in reasonable agreement with our observation of 16 subunits ^[3].

Cryo-EM

Cryogenic electron microscopy (cryo-EM) is a cryomicroscopy technique applied to samples cooled to cryogenic temperatures. For biological samples, structure is preserved by embedding in a glassy ice environment. An aqueous sample is applied to a mesh grid and frozen by immersion in liquid ethane or a mixture of liquid ethane and propane ^[4]. This technique has advanced dramatically to become a viable tool for high-resolution structural biology research. The ultimate outcome of a cryoEM study is an atomic model of a macromolecule or its complex with interacting partners. Recent advances in direct electron detectors as well as reconstruction single particle algorithms have led to the determination of the structure of macromolecular complexes ranging from 2 to 5 Å resolution. At these resolutions, also known as “near atomic” resolution, it is possible to infer all-atom structures de novo. The first step in cryoEM structure determination is de novo structure determination, where an initial model can be built, given only one sequence and a reconstruction, when no other limited structural information is known. In the second stage, the model is optimized, where a wide range of class of methods for improving the fit of a model to the data and improving the geometry of a model. Finally, tools for model validation are described, in attempt to quantify the overall accuracy of a model given a reconstruction. An overview of three steps of atomic model determination from near-atomic resolution data. (Left) De novo building methods take primary sequence and map, and automatically produce a backbone model with sequence registered, identifying which regions in the map correspond to particular sequences. (Center) Model optimization takes an initial model—either produced from de novo building, or from a highresolution homologue—and optimizes the coordinates to better agree with the map, as well as adopt more physically realistic geometry. (Right) Model validation aims to assess—both globally and locally—the accuracy of a model, given experimental data. Such tools are useful not only for assessing overall accuracy but also for tuning parameters of optimization^[5].

Structure of Photosynthetic LH1-RC Super-complex of Rhodospirillum rubrum

The cryo-EM structure of Rsp. rubrum LH1-RC was determined at 2.76 Å resolution. The LH1 complex forms a closed, slightly elliptical double ring composed of 16 pairs of α(inner)β(outer)-polypeptides, 32 BChls aG and 16 all-trans-spirilloxanthins, as we can se on the Fig. 4. The RC of Rsp. rubrum is surrounded by the LH1 complex with only a few close contacts on the periplasmic surface with residues near Ser34 in the LH1 α-polypeptides. There is not apparent strong interactions of Rsp. rubrum RC with your LH1 polypeptides (Figure 4 a-c) as occurs in Cyt c-bound LH1-RCs where the C-terminal domains of some LH1 α-polypeptides interact extensively with the Cyt c subunit. The BChl aG molecules in Rsp. rubrum LH1 form an elliptical, partially overlapping ring with average Mg−Mg distances of 9.3 Å within a dimer and 8.5 Å between dimers (Figure 4c).

Fig. 4. Structure overview of the Rsp. rubrum LH1-RC complex. (a) Side view of the LH1-RC parallel to the membrane plane. (b) Top view of the LH1-RC from the periplasmic side of the membrane. (c) Tilted view of the cofactor arrangement. (d) Superposition of Cα carbons of the LH1 αβpolypeptides between Rsp. rubrum and Tch. tepidum (gray, PDB: 5Y5S). Color scheme: LH1-α, green; LH1-β, slate-blue; L-subunit, magenta; Msubunit, blue; BChl aG in LH1 and special pair, red sticks; Accessory BChl aG, cyan sticks; BPhe aG, light-pink sticks; Spirilloxanthin, yellow sticks; UQ10, blue sticks; RQ-10, green sticks; Fe, magenta ball. Phospholipids and detergents are omitted for clarity

The geranylgeranyl side chains in the BChl aG associated with βpolypeptides form a tail-up conformation (as shown on the image below) with a much higher structural homogeneity compared with those of purple bacteria whose BChl a is esterified by a phytyl group.

This may be due to the multiple double bonds present in the geranylgeranyl group, which results in a more rigid conformation. This unique conformation allows the geranylgeranyl side chains of the β-associated BChl aG to interact with the bacteriochlorin ring of the α-associated BChl aG, where the methyl group at geranylgeranyl g11 points to the macrocycle of a neighboring BChl aG at a distance of ∼3.5 Å.

This is a sample scene created with SAT to by Group, and another to make of the protein. You can make your own scenes on SAT starting from scratch or loading and editing one of these sample scenes.

References

1. ↑ 10.1002/j.1460-2075.1995.tb07041.x
2. ↑ 10.1021/acs.biochem.1c00360
3. ↑ 10.1002/j.1460-2075.1995.tb07041.x
4. ↑ 10.1017/S1431927608080781
5. ↑ 10.1016/bs.mie.2016.06.003