Isomorphous Replacement Method Research Articles

A pipeline for structure determination of in vivo-grown crystals using in cellulo diffraction.

While structure determination from micrometre-sized crystals used to represent a challenge, serial X-ray crystallography on microfocus beamlines at synchrotron and free-electron laser facilities greatly facilitates this process today for microcrystals and nanocrystals. In addition to typical microcrystals of purified recombinant protein, these advances have enabled the analysis of microcrystals produced inside living cells. Here, a pipeline where crystals are grown in insect cells, sorted by flow cytometry and directly analysed by X-ray diffraction is presented and applied to in vivo-grown crystals of the recombinant CPV1 polyhedrin. When compared with the analysis of purified crystals, in cellulo diffraction produces data of better quality and a gain of ∼0.35 Å in resolution for comparable beamtime usage. Importantly, crystals within cells are readily derivatized with gold and iodine compounds through the cellular membrane. Using the multiple isomorphous replacement method, a near-complete model was autobuilt from 2.7 Å resolution data. Thus, in favourable cases, an in cellulo pipeline can replace the complete workflow of structure determination without compromising the quality of the resulting model. In addition to its efficiency, this approach maintains the protein in a cellular context throughout the analysis, which reduces the risk of disrupting transient or labile interactions in protein-protein or protein-ligand complexes.

The first protein Structure Determinations

Max Perutz had been studying the structure of haemoglobin for 21 years using highly original and creative techniques, when I arrived in Cambridge in the summer of 1958. Most recently he had explored the use of isomorphous replacement by substituting specific amino acids with heavy metal compounds. Together with Ann Cullis, he had collected three dimensional diffraction data on native haemoglobin crystals and on three heavy atom derivatives. But now Max needed help in analyzing this data. I had had experience in determining three small aromatic hydrocarbon structures as a graduate student with J.M. Robertson at the University of Glasgow using only mechanical hand calculators, a slide rule and Beevers-Lipson strips. At the University of Minnesota, as a post doc with Bill Lipscomb, I had learned how to program one of the earliest commercial computers and solved a plant terpenoid structure in three dimensions. Thus, when I joined Max's lab, I was soon busy programming the bran new "home build" EDSAC2 computer to determine and refine the heavy atom parameters for phase determination by isomorphous replacement. When we calculated the 5.5Å resolution horse oxy haemoglobin electron density map in the summer of 1959 we were amazed to see that the two polypeptide chains of haemoglobin each had the same fold as that of myoglobin, another oxygen carrying protein determined by John Kendrew at 6Å resolution two years earlier at the Cavendish. John and his group were extending the structure of myoglobin to 2Å resolution. The high resolution map of myglobin was calculated using my Fourier program the week after the low resolution haemoglobin map. We then saw the atomic structure of α-helices as had been predicted by Linus Pauling. Seeing evolution for the first time at a molecular level has inspired my thinking and choice of research topics ever since. In the following few years, stimulated by these discoveries , David Blow (who had returned from the US later that summer after two years of post-doc studies) and I developed the single isomorphous replacement method, the use of anomalous dispersion for phase determination, phase combination procedures and, most importantly, molecular replacement.

Exploiting subtle structural differences in heavy-atom derivatives for experimental phasing.

Structure determination using the single isomorphous replacement (SIR) or single-wavelength anomalous diffraction (SAD) methods with weak derivatives remains very challenging. In a recent structure determination of glycoprotein E2 from bovine viral diarrhea virus, three isomorphous uranium-derivative data sets were merged to obtain partially interpretable initial experimental maps. Small differences between them were then exploited by treating them as three independent SAD data sets plus three circular pairwise SIR data sets to improve the experimental maps. Here, how such subtle structural differences were exploited for experimental phasing is described in detail. The basis for why this approach works is also provided: the effective resolution of isomorphous signals between highly isomorphous derivatives is often much higher than the effective resolution of the anomalous signals of individual derivative data sets. Hence, the new phasing approaches outlined here will be generally applicable to structure determinations involving weak derivatives.

Expression, purification and preliminary crystallographic studies of the C-terminal SH3 domain of human Tks4.

The Src homology 3 (SH3) domain is a small, noncatalytic domain with a conserved sequence of about 60 amino-acid residues that interacts with proline-rich peptides to form a protein complex. In this study, the C-terminal SH3 domain of human Tks4 (residues 853-911) was expressed, purified and crystallized. X-ray diffraction data were collected to 2.3 Å resolution. The crystal belonged to the trigonal space group P3121 (or P3221), with unit-cell parameters a = b = 83.87, c = 108.44 Å, α = β = 90, γ = 120°. Calculating the self-rotation and the native Patterson function did not lead to the detection of any noncrystallographic translational symmetry. Six, seven or eight protein molecules are likely to be present in the asymmetric unit, resulting in a Matthews coefficient and approximate solvent content of 2.71 Å(3) Da(-1) and 55%, 2.32 Å(3) Da(-1) and 47%, and 2.03 Å(3) Da(-1) and 39%, respectively. To solve the crystal structure of the C-terminal SH3 domain of human Tks4, the isomorphous replacement method is presently being utilized.

Addressing the Inverse Problem of Far-Field Imaging: A Noniterative Exact Solution for Phase in Imaging

This talk describes an approach to a solution of inverse problems in far-field imaging with application to optical, electron/ion beam, x-ray and other imaging modalities. Two aspects of the inverse problem in optical imaging are phase and resolution. Whereas techniques such as STED, PALM, STORM etc address the latter, they lack phase information as does near-field optics. Furthermore, all of these techniques are serial. We introduce an approach based on a controllable nanoscopic far-field optical point source integrated into parallel imaging. In the first demonstration of this development we focus on the inverse problem of the phase of an image which has never been solved exactly and can only be approached through iterative methods with all their problems of nonconvergence, slow convergence, convergence to local minima, and stagnation. We now show that it is possible to obtain, with such a method, an exact solution to the inverse problem of phase both experimentally and theoretically. Our method is based on the breakthrough that crystallography experienced in phase retrieval for large molecular entities by Max Perutz's introduction of “heavy atoms” using the method of isomorphous replacement. Scanning probe microscopy and its full integration with optical microscopy allows us to apply these X-ray concepts to implement “heavy atom” restoration of phase in optical phase retrieval. In analogy to the heavy atom method, we acquire Fourier intensities in place of an X-ray diffraction pattern, and in place of the heavy atom, we utilize a nanometrically translatable point source of light, coherently related to the far-field illumination this leads to 3D optical imaging. The methodology has super-resolution potential, and thus, heavy atom restoration of phase with super-resolution (HARPS) has the potential to provide super-resolution 3D images in real time.

Noniterative Exact Solution to the Phase Problem in Optical Imaging Implemented with Scanning Probe Microscopy

An object is characterized by its amplitude and phase. However, when acquiring optical data about such an object, using a recording medium such as a camera, phase information is lost. Crystallography experienced a breakthrough in phase retrieval for large molecular entities by Max Perutz's introduction of "heavy atoms" using the method of isomorphous replacement. The availability of scanning probe microscopy and its full integration with optical microscopy allows us to apply these X-ray concepts to implement "heavy atom" restoration of phase in optical phase retrieval. In analogy to the heavy atom method, we acquire Fourier intensities in place of an X-ray diffraction pattern, and in place of the heavy atom, we utilize a nanometrically translatable point source of light based on the propagating field of a cantilevered near-field scanning optical microscopic (NSOM) probe controlled by an atomic force microscope (AFM). This integration of NSOM/AFM technology with far-field imaging achieves robust phase retrieval independent of external parameters, leading to 3D optical imaging. The methodology has super-resolution potential, and thus, heavy atom restoration of phase with super-resolution (HARPS) shows the potential of transparently integrated scanning probe microscopy with optical and other imaging modalities such as electron or ion optical imaging.

Features of the secondary structure of a protein molecule from powder diffraction data

Protein powder diffraction is shown to be suitable for obtaining de novo solutions to the phase problem at low resolution via phasing methods such as the isomorphous replacement method. Two heavy-atom derivatives (a gadolinium derivative and a holmium derivative) of the tetragonal form of hen egg-white lysozyme were crystallized at room temperature. Using synchrotron radiation, high-quality powder patterns were collected in which pH-induced anisotropic lattice-parameter changes were exploited in order to reduce the challenging and powder-specific problem of overlapping reflections. The phasing power of two heavy-atom derivatives in a multiple isomorphous replacement analysis enabled molecular structural information to be obtained up to approximately 5.3 A resolution. At such a resolution, features of the secondary structure of the lysozyme molecule can be accurately located using programs dedicated to that effect. In addition, the quoted resolution is sufficient to determine the correct hand of the heavy-atom substructure which leads to an electron-density map representing the protein molecule of proper chirality.

Structural Basis for the Kexin-like Serine Protease from Aeromonas sobria as Sepsis-causing Factor

The anaerobic bacterium Aeromonas sobria is known to cause potentially lethal septic shock. We recently proposed that A. sobria serine protease (ASP) is a sepsis-related factor that induces vascular leakage, reductions in blood pressure via kinin release, and clotting via activation of prothrombin. ASP preferentially cleaves peptide bonds that follow dibasic amino acid residues, as do Kex2 (Saccharomyces cerevisiae serine protease) and furin, which are representative kexin family proteases. Here, we revealed the crystal structure of ASP at 1.65 A resolution using the multiple isomorphous replacement method with anomalous scattering. Although the overall structure of ASP resembles that of Kex2, it has a unique extra occluding region close to its active site. Moreover, we found that a nicked ASP variant is cleaved within the occluding region. Nicked ASP shows a greater ability to cleave small peptide substrates than the native enzyme. On the other hand, the cleavage pattern for prekallikrein differs from that of ASP, suggesting the occluding region is important for substrate recognition. The extra occluding region of ASP is unique and could serve as a useful target to facilitate development of novel antisepsis drugs.

Dipeptidyl Aminopeptidase IV fromStenotrophomonas maltophiliaExhibits Activity against a Substrate Containing a 4-Hydroxyproline Residue

The crystal structure of dipeptidyl aminopeptidase IV from Stenotrophomonas maltophilia was determined at 2.8-A resolution by the multiple isomorphous replacement method, using platinum and selenomethionine derivatives. The crystals belong to space group P4(3)2(1)2, with unit cell parameters a = b = 105.9 A and c = 161.9 A. Dipeptidyl aminopeptidase IV is a homodimer, and the subunit structure is composed of two domains, namely, N-terminal beta-propeller and C-terminal catalytic domains. At the active site, a hydrophobic pocket to accommodate a proline residue of the substrate is conserved as well as those of mammalian enzymes. Stenotrophomonas dipeptidyl aminopeptidase IV exhibited activity toward a substrate containing a 4-hydroxyproline residue at the second position from the N terminus. In the Stenotrophomonas enzyme, one of the residues composing the hydrophobic pocket at the active site is changed to Asn611 from the corresponding residue of Tyr631 in the porcine enzyme, which showed very low activity against the substrate containing 4-hydroxyproline. The N611Y mutant enzyme was generated by site-directed mutagenesis. The activity of this mutant enzyme toward a substrate containing 4-hydroxyproline decreased to 30.6% of that of the wild-type enzyme. Accordingly, it was considered that Asn611 would be one of the major factors involved in the recognition of substrates containing 4-hydroxyproline.

Crystallization and preliminary X-ray analysis of the inducible lysine decarboxylase from Escherichia coli.

The decameric inducible lysine decarboxylase (LdcI) from Escherichia coli has been crystallized in space groups C2 and C222(1); the Ta6Br12(2+) cluster was used to derivatize the C2 crystals. The method of single isomorphous replacement with anomalous scattering (SIRAS) as implemented in SHELXD was used to solve the Ta6Br12(2+)-derivatized structure to 5 A resolution. Many of the Ta6Br12(2+)-binding sites had twofold and fivefold noncrystallographic symmetry. Taking advantage of this feature, phase modification was performed in DM. The electron-density map of LdcI displays many features in agreement with the low-resolution negative-stain electron-density map [Snider et al. (2006), J. Biol. Chem. 281, 1532-1546].

Crystal twinning of human MD-2 recognizing endotoxin cores of lipopolysaccharide.

Twinning of crystals causes overlapping of two or more reciprocal lattice points, and hence structure amplitudes for a single crystalline domain are hardly obtained from X-ray diffraction intensities. MD-2 protein forms a stable complex with Toll-like receptor 4 and recognizes bacterial lipopolysaccharide (LPS). Excessive immune responses activated by LPS cause septic shocks. Saccharide-trimmed human MD-2 crystallizes in the tetragonal form with apparent Laue symmetry of 4/mmm, and diffraction intensities from these crystals indicate crystal twinning. The crystal consists of two different domains, A and B. The c(A) axis of domain A coincides with the c(B) axis of domain B with a smaller lattice, and the a(A) axis corresponds to the (a(B) + b(B)) axis. This twinning severely imposes difficulty in structure determination. Through optimization of cryoprotectant, domain A was thoroughly transformed into domain B. The crystal containing only domain B is in space group P4(1)2(1)2 with one MD-2 molecule in the asymmetric unit. The structure of this form of MD-2 as well as its complex with antiendotoxic lipid IVa was successfully determined using the multiple isomorphous replacement method.

Crystal structure of the λ repressor and a model for pairwise cooperative operator binding

Bacteriophage lambda has for many years been a model system for understanding mechanisms of gene regulation. A 'genetic switch' enables the phage to transition from lysogenic growth to lytic development when triggered by specific environmental conditions. The key component of the switch is the cI repressor, which binds to two sets of three operator sites on the lambda chromosome that are separated by about 2,400 base pairs (bp). A hallmark of the lambda system is the pairwise cooperativity of repressor binding. In the absence of detailed structural information, it has been difficult to understand fully how repressor molecules establish the cooperativity complex. Here we present the X-ray crystal structure of the intact lambda cI repressor dimer bound to a DNA operator site. The structure of the repressor, determined by multiple isomorphous replacement methods, reveals an unusual overall architecture that allows it to adopt a conformation that appears to facilitate pairwise cooperative binding to adjacent operator sites.

Molecular envelopes derived from protein powder diffraction data

The preparation of single crystals suitable for X-ray analysis is frequently the most difficult step in structural studies of proteins. With the aid of two examples, it is shown thatde novosolution of the crystallographic phase problem can be achieved at low resolution using microcrystalline powder samplesviathe single isomorphous replacement method. With synchrotron radiation and optimized instrumentation, high-quality powder patterns have been recorded, from which it was possible to generate phase information for structure factors up to 6 Å resolution. pH- and radiation-induced anisotropic lattice changes were exploited to reduce the problem of overlapping reflections, which is a major challenge in protein powder diffraction. The resulting data were of sufficient quality to compute molecular envelopes of the protein molecule and to map out the solvent channels in the crystals. The results show that protein powder diffraction can yield low-resolution data that are potentially useful for the characterization of microcrystalline proteins as novel micro- and mesoporous materials as well as for structural studies of biologically important macromolecules.

Zinc Induces Dimerization of the Class II Major Histocompatibility Complex Molecule That Leads to Cooperative Binding to a Superantigen

Dimerization of class II major histocompatibility complex (MHC) plays an important role in the MHC biological function. Mycoplasma arthritidis-derived mitogen (MAM) is a superantigen that can activate large fractions of T cells bearing specific T cell receptor Vbeta elements. Here we have used structural, sedimentation, and surface plasmon resonance detection approaches to investigate the molecular interactions between MAM and the class II MHC molecule HLA-DR1 in the context of a hemagglutinin peptide-(306-318) (HA). Our results revealed that zinc ion can efficiently induce the dimerization of the HLA-DR1/HA complex. Because the crystal structure of the MAM/HLA-DR1/hemagglutinin complex in the presence of EDTA is nearly identical to the structure of the complex crystallized in the presence of zinc ion, Zn(2+) is evidently not directly involved in the binding between MAM and HLA-DR1. Sedimentation and surface plasmon resonance studies further revealed that MAM binds the HLA-DR1/HA complex with high affinity in a 1:1 stoichiometry, in the absence of Zn(2+). However, in the presence of Zn(2+), a dimerized MAM/HLA-DR1/HA complex can arise through the Zn(2+)-induced DR1 dimer. In the presence of Zn(2+), cooperative binding of MAM to the DR1 dimer was also observed.

Localization of dihalogenated phenols in vesicle systems determined by contrast variation X-ray scattering

Localization of 2,4-dichloro- and 2,4-dibromophenol in multilamellar vesicles in a 1/1 dihalogenated phenol/lipid molar ratio was investigated by classical contrast variation X-ray scattering using the isomorphous replacement method. The results were compared with those obtained by anomalous small-angle X-ray scattering from a vesicle system doped with 2,4-dibromophenol. Dissimilarities in the results of the two methods are discussed, taking into account the advantages and disadvantages of both techniques in studying multilamellar systems.

Crystallization and Preliminary X-Ray Analysis of the Catalytic Domain of Chitinase D from Bacillus circulans WL-12

We report here on crystallization and preliminary X-ray analysis of the catalytic domain of chitinase D from Bacillus circulans WL-12. The native crystals of this domain were found to belong to the orthorhombic space group P2(1)2(1)2(1). To elucidate the structure of the catalytic domain by the multiple isomorphous replacement method, 30 kinds of derivatized crystals were prepared by soaking the native crystals into a mother liquor containing salts of heavy metal atoms. Difference Patterson maps calculated for four derivatives showed strong peaks in the Harker sections.

Cloning, expression, purification, crystallization and initial crystallographic analysis of the preprotein translocation ATPase SecA from Thermus thermophilus.

The Thermus thermophilus gene encoding the preprotein translocation ATPase SecA was cloned and expressed and the purified protein was crystallized by the hanging-drop vapour-diffusion technique in two different space groups P3(1(2))21 (a = b = 168.6, c = 149.8 A) and P6(1(5))22 (a = b = 130.9, c = 564.6 A). The crystals, improved by macroseeding, diffracted to beyond 2.8 and 3.5 A resolution for the trigonal and hexagonal crystal forms, respectively. Structure determination using the multiple isomorphous replacement method is in progress.

Present at the Flood: A First Person Guided Tour through the Early Literature in Structural Biology

Present at the Flood: A First Person Guided Tour through the Early Literature in Structural Biology

タンパク質結晶構造解析入門―ブラックボックスの中身―（３）モデリングの裏側をのぞいてみよう 位相改良による電子密度図のリファイン

Before starting model building, the first set of protein phase, which is obtained with the isomorphous replacement method, the multiple wavelength anomalous dispersion method, and/or the molecular replacement method, should be improved by the phase improvement and density modification techniques. An insufficient quality of the electron density map might be a snag for model building. Methods of phase improvement and density modification techniques must usually be performed.

Crystal structure and some properties of a major house dust mite allergen, Derf 2

Pyroglyphid house dust mites are a major source of allergens in house dust. Mite allergens sensitize and induce asthma, rhinitis, and eczema in a large portion of patients with allergic diseases. Here, the crystal structure of a major mite allergen, Derf 2, derived from Dermatophagoides farinae was solved by single isomorphous replacement method with anomalous scattering (SIRAS) at 2.1 Å resolution. The present study also demonstrated that the conformation of the allergen was critical in the determination of Th1/Th2 shift based on physicochemical and immunological analyses. This indicates that rigidly folded and singly dispersed structure is essentially required for the generation of Th2 type cells by the allergen, while conformational variant protein leads to Th1 skewing, irrespective of the same amino acid sequence. This structure/function relationship may allow us to develop a novel strategy for hyposensitization therapy in patients with allergic diseases triggered by house dust mite allergens.