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G protein-coupled receptors (GPCRs) constitute the largest human membrane protein superfamily with over 800 members. They reside in plasma membranes mediating cell signaling and regulating the majority of physiological processes, making them successful targets of about 30–40% of current drugs. Detailed understanding of the mechanism of GPCR action requires high-resolution structural information for many representative members of the family captured in different conformational states, but progress has been limited by the difficulty of preparing large amounts of homogenous and stable samples and by the need for sufficiently large crystals. In this chapter we summarize the work that led to the development and successful application of serial femtosecond crystallography (SFX) to micron-sized GPCR crystals grown and delivered to an X-ray free electron laser (XFEL) beam in a lipid membrane mimetic mesophase, known as lipidic cubic phase (LCP). The LCP-SFX approach obviates the need for growing larger crystals, while also minimizing radiation damage effects on room-temperature samples, and so could substantially shorten the time to success, with concomitant significant reduction in cost and effort. It therefore has the potential to revolutionize structural studies on challenging membrane proteins and their complexes with soluble partners.

The Protein Data Bank (PDB) at Brookhaven National Laboratory is a database with approximately 10 000 entries containing information on experimentally determined three-dimensional structures of proteins, nucleic acids and other biological macromolecules. The PDB has a 27-year history of service to a global community of researchers, educators and students in a wide variety of scientific disciplines. Data are easily submitted via the PDB’s web-based tool AutoDep in either PDB or mmCIF format and are most conveniently examined via the PDB’s web-based tool 3DB Browser. Collaborative centres have been, and continue to be, established worldwide to assist in data deposition, archiving and distribution. As of 1 July 1999 the operation of the PDB in the USA is being transferred from Brookhaven National Laboratory to the Research Collaboratory for Structural Bioinformatics (RCSB). Keywords: databases; deposition of data; Protein Data Bank

The protein data bank (PDB), at Brookhaven National Laboratory, is a database containing information on experimentally determined three-dimensional structures of proteins, nucleic acids, and other biological macromolecules, with approximately 9000 entries. The PDB has a 27-year history of service to a global community of researchers, educators, and students in a wide variety of scientific disciplines. Data are easily submitted via PDB's WWW-based tool AutoDep, in either PDB or mmCIF format, and are most conveniently examined via PDB's WWW-based tool 3DB Browser. Collaborative centers have been, and continue to be, established worldwide to assist in data deposition, archiving, and distribution.

The covalently linked hybrid of two human lambda-type light chains (Mcg and Weir) crystallizes as trigonal bipyramids in ammonium sulfate [Ely et al., Molec. Immun. 22, 85-92 (1985)]. While markedly different in appearance from the barrel-shaped crystals of the parental Mcg dimer, the bipyramids of the hybrid have the same space group: trigonal P3(1)21. Moreover, the unit cell dimensions are practically identical: a = 72.3 A in both proteins; c = 188.1 A in the hybrid and 185.9 A in the Mcg dimer. These observations imply that the crystal packing and the main features of the three-dimensional structures are closely similar in the Mcg X Weir hybrid and the Mcg dimer. The "constant" domains of the Mcg and Weir proteins belong to the same genetic subclass and were expected to interact in comparable ways in hybrids and parental dimers. However, the overall similarities in the "variable" domain pairs in the hybrid and Mcg dimer were completely unpredicted, since the amino acid sequences of the heterologous variable domains differ by 36 residues. By difference Fourier analysis the Weir light chain has been tentatively identified as monomer 1 (heavy-chain analogue) and the Mcg protein as monomer 2 (light-chain analogue) in the hybrid dimer. Substitutions in key positions in the hypervariable loops explain the differences in binding activity of the Mcg and Weir dimers. In the Mcg dimer bis(dinitrophenyl)lysine spans two relatively spacious subsites (A and B), with primary contacts involving tyrosines 34 and 38 of monomer 2. The Weir dimer, which does not bind dinitrophenyl ligands, has serine and phenylalanine in homologous positions. Moreover, the bilateral replacement of valine 48 and serine 91 in Mcg by leucine and methionine in the Weir dimer should effectively block access to subsite B. In the hybrid binding activity for bis(dinitrophenyl)lysine is restored because the Mcg light chain is present as the monomer 2 subunit.

A Fortran minicomputer program for the collection of X-ray diffraction data from protein crystals is described. Data collection efficiency has been achieved by means of: ω step scans for reflection intensity, radial step scan along 2θ to estimate background intensity and preselected reflection files. The program includes a standard reflection monitor, a means for slight misalignment compensation, empirical absorption correction data, Bijvoet pair collection, and the ability to collect data for the crystal lifetime without operator intervention.

Immunoglobulin light chains are examples of single polypeptide chains synthesized under the control of two genes. The three-dimensional structure of a human (Mcg) lambda-type chain (Bence-Jones) dimer supports the hypothesis of a common primordial gene for the amino ("variable" or V) and carboxyl ("constant" or C) halves of each monomer. However, sequence homologies have been obscurred by divergent evolution of the V and C regions ("domains"). The types of evolutionary changes that have occurred in the domain can be surmised by a comparison of the sequences, using the three-dimensional structures as a basis for alignment. Despite substantial differences in sequences, the hydrophobic character of key internal sites has been maintained in each domain. Regions present in only one domain are situated in position appropriate for their functions, but not deleterious to the general structural integrity of a common fold. The divergence of the V and C domains can be interpreted in terms of rotational allomerism. The cylinders of beta-pleated sheets have rotated in such a way that homologous regions in the two domains perform different functions in their interactions with a second molecule of light or heavy chain. These regions include complementarity-determining sites for antigen binding in the V domains and crossover sites stabilizing dimer formation in the C domains. Differences in surface properties between the V1-V2 and C1-C2 dimeric modules may partially explain why the V regions have been implicated in the formation of amyloid fibrils and in the characteristic thermal behavior of Bence-Jones proteins.

The three-dimensional structure of an IgG1(lambda) immunoglobulin from a patient (Mcg) with amyloidosis was determined at 6.5-A resolution with X-ray diffraction techniques. The protein crystallized from water in the space group C2221, with a = 87.8, b = 111.3 and c = 186.3 A; the crystallographic asymmetric unit was a half-molecule consisting of one light and one heavy chain. The structure was solved by the multiple isomorphous replacement method with five heavy-atom derivatives. Electron density maps were interpreted with the aid of a protein modeling system used in conjunction with an Evans and Sutherland Picture System II graphics station. IgG1 molecules were tightly packed in the crystal lattice, with numerous intermolecular contacts. The two-fold axis relating identical halves of each molecule was found to be parallel to the y crystallographic axis. Electron density modules collectively representing one molecule were identified as three lobes representing the two antigen-binding (Fab) arms and the Fc region. An interchain disulfide bond connecting the two CL domains was located on the molecular diad and used as a landmark in the interpretation of the electron density map. A computer graphics method was developed to produce a solid image model of the IgG1 molecule in any prescribed orientation.

Crystals were prepared from samples of nonjovalent and covalently linked dimers previously isolated as intermediates and end products in the interconversion of conformational isomers of immunoglobulin light chains from the patient Mcg. In ammonium sulfate these dimers crystallized in the trigonal form characteristic of the native Bence-Jones protein and in an abnormal needle form associated with conformational changes in the vicinity of the interchain disulfide bond. Trigonal forms were compared with the native covalent dimer by difference Fourier analysis at 3.5-A resolution. The packing of molecules in the trigonal crystal lattice was examined in detail to determine the steric limitations governing chemical modifications, chemical modifications within crystals or in solutions slated for crystallization. When the modifications involved only the cleavage and alkylation of the interchain disulfide bond, the difference Fourier maps indicated only local conformational changes mainly in the COOH-terminal pentapeptide segment of monomer 2 of the dimer. When light chains were dissociated from heavy chains and reassembled into a dimer, there were changes in segments which interact to stabilize the V domain dimer. These changes, as well as the more extensive changes in the needle forms, could be reversed by dissolving the crystals, cleaving the interchain disulfide bond, andmore » allowing it to reoxidize. The resulting proteins crystallized as trigonal forms indistinguishable from those of the native dimer. After the binding of two molecules of bis(dinitrophenyl)lysine and subsequent removal of the ligands by dialysis, the dimer crystallized only as needles.« less

The application of crystallographic techniques to Fab, Fc, and light-chain components of immunoglobulins has provided a three-dimensional framework for interpreting chemical and biological observations like those summarized by Metzger in the next chapter (p. 119) (Poljak et al.,1974, 1976; Segal et al.,1974; Padian et al.,1974; Davies et al., 1975; Deisenhofer et al., 1976; Epp et al., 1974; Fehlhammer et al.,1975; Wang et al., 1975; Schiffer et al., 1973; Edmundson et al., 1975). These high-resolution analyses have been supplemented by low-resolution studies of intact IgG1 and IgG2 immunoglobulins (Sarma et al., 1971; Silverton et al.,1977; Colman et al., 1976; Edmundson et al.,1970; Ely et al.,1978a; Colman et al.,unpublished data). The structures of immunoglobulin components are well described in the above articles and they will not be discussed in detail in the present report. Instead, we shall concentrate on recent crystallographic work relating to the capacities of immunoglobulins to assume different conformations.