Lets first talk about the laws straight, The two fundamental laws that have contributed to protein x-ray crystallography are Bragg’s law and Fourier Transform. Bragg’s law determines the resolution of the diffraction patterns. Fourier transform equation calculates the electron density of the molecules for the electron density map (Grüne, 2005)(Rhodes, 1999).

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Bragg’s law states that when the x-ray is incident onto a crystal surface its angle of incidence it will reflect back with the same angle of scattering, when the plane of separation is equal to the wavelength of the incident x-rays a constructive interference will occur (Drenth, 2007).

In x-ray crystallography crystals are distinguished by its surface faces and by its anisotropy (Drenth, 2007).  Successful crystallization of proteins into 3D crystals is a prerequisite to X-ray crystallography and is considered a major challenge as well. Membrane proteins in particular form three types of 3D crystals type 1, type 2 (Lundstrom,2006) and type 3, is the recently discovered type of crystal where the protein is introduced into a micro liposome with the help of membrane vesicles crystallizes to form a macro crystal lattice (Liu et. al, 2004) .The  orientation of the crystal lattice is  proportional to diffraction data acquired (Brunger,1997).

In 2004 Liu and team had carried out research using  x-ray crystallography at 2.72 Å resolution on the structure of light harvesting protein (LH-II) in spinach chloroplasts using non-bilayer forming lipid digalactosyl triacylglycerol (DGDG), the crystallization conditions caused LH-II to insert into DGDG proteoliposomes which further resulted crystallized structure shown below resembled the crystallized structure of icosahedral viruses (Lundstrom, 2006) and further classed as type 3 membrane protein crystals .

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A. Drawing of the LHC-II-DGDG proteoliposome.  B. Packaging diagram of TypeIII  membrane protein crystals,N-terminus is located near  the Stromal surface(Liuet.al,2004).

 

Few factors that have to be considered in x-ray crystallography the purity of the protein, the concentration of the precipitants, size of detergent if the protein is insoluble (most transmembrane proteins) and the pH and temperature of the  environment the crystals are grown in (Drenth, 2007).

In crystallization the most common technique carried out in known as vapour diffusion.  Vapour diffusion can be carried out either hanging-drop or sitting-drop format, they are based on increasing the precipitation of the protein in solution using precipitants such as ammonium sulphate or polyethylene glycol (Drenth, 2007) leading to the crystallisation of the proteins (Rhodes, 1993).

A good example would be porins which are beta barrel transmembrane proteins(TMPs) were successfully crystallised by Hiroshi Nikaido using micro dialysis (Lundstrom, 2006).The X-ray crystallography of porins led to the study of substrate specific porins,  LamB porin also known as  maltoporin (Cowan et al.1992). Initially maltoporin was studied in reference to the outer membrane protein F (OmpF) refer to figures below. Maltoporin showed many similar folding properties to OmpF (16 stranded anti parallel beta strands) (Cowan et al.1992).

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By comparing the structures, maltoproin elucidated compact 18 beta barrel strands with monomer residues 80 residues long, beta hairpin turns  and  9 irregular cell surface  loops were observed .  Enclosed within the barrels are smaller channels 5-6 Å in diameter, linkage of the amino group valine (val1) to carboxyl group tryptophan (trp421) on the 18th strand indicated the C-terminus (Hofnug,1995). 3 inward folding loops determined the functional status that is to protect the constriction zone (entrance) of the pathway.

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Aromatic residues were found lining the channel called ‘greasy slide’ a pathway to the periplasmic outlet (Hofnug, 1995) (Schirmer et al.1995). Initially the functional status of maltoporin was limited to an E.coli receptor for phage lambda (Cowan et al.1992) until Schirmer et al. 1995 had elucidated on an alternative functional status of maltoporin. By  soaking the  crystals in  maltotriose  revealed the binding of  sugars to a hydrophobic track  which spread across the constriction zone  which further suggested  that maltose  and  other sugars( maltodextrins )are  translocated across the membrane .

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(1) Structure of  maltodextrin.   (2) The Channel is lined  with aromatic  residues that match the helical curvature of maltodextrin (Kukol,2014).

The next two mostly used crystallization methods are the cocrystallization and Lipid cubic phase. Co-crystallization is carried out when proteins consist of small extra membrane domains which are difficult to crystallize (Lacapère et al. 2007). This technique uses monoclonal antibody fragments which attach to the polar region of the membrane protein leading to the increase in the hydrophilic surface which facilitates crystallization. Co-crystallization was successfully used for the first time to determine the structure of cytochrome C oxidase at 2.7 Å resolutions from Paracoccus denitrificans in figure below (Ostermeier et al.1997)
5A study by Nobel Prize winner MacKinnon et al. in 1998 conducted an X-ray crystallography analysis of the potassium ion channels .The usually unsuitable Fab fragments, region on which the antibody binds to the antigen when proteolytically cleaved were successful in the structural determination of the KcsA K+channel from gram positive Streptomyces lividans. The potassium channel was used as a model channel to study potassium selectivity and permeation. It was studied that potassium leakage channel is 10,000 fold more selective for K ions than Na ions and transported 108 ions per second. The analysis determined a tetramer of 158 residue subunits which made the structure look like an inverted tepee  (Zhou et al. 2001). Each monomer in the channel was made up of two α helices, M1 being the helix that lies towards the outer lipid bi-layer and M2 lies around central pore (Williamson et al.2003).The channel showed three important sections the selectivity filter, high conductivity region and gating site .The size of the selectivity filter was 12 Å long and 3 Å wide lined with carbonyl groups which allows precise solvation of K ions into the water filled high conductivity cavity (Williamson et al.2003). In the cavity the helix dipole moment stabilizes of potassium ion leading to increased concentrations furthermore causing electrostatic repulsion of potassium ions into the gating site (MacKinnon et al.1998).

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7Sometimes 3D crystallization fail cause of short extra membrane loops prevent crystal growth, lipid cubic phase method overcame this drawback further proved successful in crystallization of bacteriorhodposin. In 1997 Landau and co-workers embedded membrane proteins into the 3D curved lipid bilayer matrix into a stabilizing medium where the protein remained in its native conformation, addition of precipitants to the mixture caused lateral diffusion along the bilayer eventually to nucleate and yield well-ordered crystals. Prior electron crystallography data already elucidated that bacteriorhodopsins captured light energy to move protons across the membrane and out of the cell. Crystals of bacteriorhodposin obtained in the lipid cubic phase were very small, between 25 and 75 µm and formed trimer in a hexagonal crystalline packing. The structure of bacteriorhodposin consists of seven transmembrane α-helices, linked by loops that formed two antiparallel β-strands on the extracellular surface (Landau et al .2000). Merohedral twinning at C-axis was detected by Luecke et al. in 1998. Landau, Pebay-Peyroula,Rummel and Rosenbusch (1998) appreciates the availability of a synchrotron as they were able to solve the  then first high resolution structure at 2.35 Å.

The structural determination of bacteriorhodposin lead to the study of its homologous protein Prokaryotic halorhodopsins sensory rhodopsin II with its transducer domain which in reference to lipid cubic phase crystallization lead to the discovery of the G- protein coupled receptors in eukaryotes (Landau et .al.2002).

8Ribbon structure of sensory rhodopsin II and transducing (heterotrimeric G protein) complex. Structural similarities with GPCRs are elucidated Rhodopsin is coloured in a rainbow with the N-terminus red and the C-terminus blue. Bound retinal chromophore inside the receptor. Transducin has the Gt-alpha subunit (red), beta (orange) and gamma (yellow). The Gt- a bound GDP is in yellow (Lambright et al, 1996) (Landau et .al.2002).

To complete the structural determination of proteins the obtained crystals have to undergo diffraction. Where beams of electromagnetic x-rays are transmitted through the crystals, the diffracted beam creates a diffraction pattern. These patterns are computationally recorded and mapped onto electron density map, a contour plot showing the distribution of electrons around the atoms of the molecule and finally to model building.

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Most studies have elucidated that synergising  x-ray crystallography with NMR spectroscopy has always come in handy, for example the cross beta structure in amyloid fibrils was solely discovered by x-ray diffraction and was further used as a reference in NMR spectroscopy. The combining of methods could help resolve complex structures like TMPs, understanding ligand-induced activation and inhibition of membrane proteins and also provide a base for designing membrane protein targeted drugs.

Which  will bring us to my upcoming write-up on NMR spectroscopy.

 

References you got to have them ! (follow the link below)

https://docs.google.com/document/d/1V_2fs-DnUCGSH5sPLwqbSMl0dhzOO6wYHl4qMZfwWe8/pub

 

 

 

 

 

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