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The successful structure solution of the integral membrane diacylglycerol kinase and the CRISPR-associated endonuclease RNA–DNA complex by native SAD phasing is demonstrated. The structures were solved with a combined low-dose multi-orientation, multi-crystal data-collection strategy., Recent improvements in data-collection strategies have pushed the limits of native SAD (single-wavelength anomalous diffraction) phasing, a method that uses the weak anomalous signal of light elements naturally present in macromolecules. These involve the merging of multiple data sets from either multiple crystals or from a single crystal collected in multiple orientations at a low X-ray dose. Both approaches yield data of high multiplicity while minimizing radiation damage and systematic error, thus ensuring accurate measurements of the anomalous differences. Here, the combined use of these two strategies is described to solve cases of native SAD phasing that were particular challenges: the integral membrane diacylglycerol kinase (DgkA) with a low Bijvoet ratio of 1% and the large 200 kDa complex of the CRISPR-associated endonuclease (Cas9) bound to guide RNA and target DNA crystallized in the low-symmetry space group C2. The optimal native SAD data-collection strategy based on systematic measurements performed on the 266 kDa multiprotein/multiligand tubulin complex is discussed.

Sulfur SAD phasing facilitates the structure determination of diverse native proteins using femtosecond X-rays from free-electron lasers via serial femtosecond crystallography., Serial femtosecond crystallography (SFX) allows structures to be determined with minimal radiation damage. However, phasing native crystals in SFX is not very common. Here, the structure determination of native lysozyme from single-wavelength anomalous diffraction (SAD) by utilizing the anomalous signal of sulfur and chlorine at a wavelength of 1.77 Å is successfully demonstrated. This sulfur SAD method can be applied to a wide range of proteins, which will improve the determination of native crystal structures.

We provide a review about the current and previous use of anomalous diffraction of x-rays in the analysis of compound semiconductors. Among the large number of available techniques, those that have been used in successful experiments on this class of compounds are identified. An exhaustive overview of the compound semiconductor systems that have been studied successfully is provided and the kind of results derived from experiments is discussed.

A case study has been made on the treatment of the SIRAS (single isomorphous replacement with anomalous scattering) data of the originally unknown protein LegC3N. An alternative treatment has been proposed which led to improved results in this particular test case. The treatment involves iterative direct-method SAD (single-wavelength anomalous diffraction) phasing and direct-method-aided model completion, both of which are implanted in theIPCAS(Iterative Protein Crystal-structure Automatic Solution) pipeline. Apart from the experimental data, a simulated SIRAS data set for LegC3N with the derivative data truncated to 5.0 Å resolution has also been tested. SAD phasing and phase/model extension inPHENIXwithout direct methods failed to solve the structure using these simulated SIRAS data. However, the procedure proposed here involving direct methods in both SAD phasing and phase/model extension led to a nearly complete structure model. This shows the potential ability of treating SIRAS data with a derivative diffracting to lower resolution.

Another iron in the fire: X-ray anomalous diffraction studies on the nitrogenase MoFe protein show the presence of a mononuclear iron site, designated as Fe16, which was previously identified as either Ca^(2+) or Mg^(2+). The position of the absorption edge indicates that this site is in the oxidation state +2. The high sequence conservation of the residues coordinated to Fe16 emphasizes the potential importance of the site in nitrogenase.

A tetrahydrofolate-dependentO-demethylase, LigM, fromSphingobiumsp. SYK-6 was crystallized by the hanging-drop vapour-diffusion method. However, the obtainedP3121 orP3221 crystals, which diffracted to 2.5–3.3 Å resolution, were hemihedrally twinned. To overcome the twinning problem, microseeding usingP3121/P3221 crystals as microseeds was performed with optimization of the reservoir conditions. As a result, another crystal form was obtained. The newly obtained crystal diffracted to 2.5–3.0 Å resolution and belonged to space groupP21212, with unit-cell parametersa= 102.0,b= 117.3,c= 128.1 Å. TheP21212 crystals diffracted to better than 2.0 Å resolution after optimizing the cryoconditions. Phasing using the single anomalous diffraction method was successful at 3.0 Å resolution with a Pt-derivative crystal. This experience suggested that microseeding is an effective method to overcome the twinning problem, even when twinned crystals are utilized as microseeds.

The structure of a β-lactamase-like protein from B. melitensis was solved independently using two data sets with anomalous signal. Anomalous Fourier maps could confirm the identity of two metal ions in the active site. AMP-bound and GMP-bound structures provide hints to the possible function of the protein., The crystal structure of a β-lactamase-like protein from Brucella melitensis was initially solved by SAD phasing from an in-house data set collected on a crystal soaked with iodide. A high-resolution data set was collected at a synchroton at the Se edge wavelength, which also provided an independent source of phasing using a small anomalous signal from metal ions in the active site. Comparisons of anomalous peak heights at various wavelengths allowed the identification of the active-site metal ions as manganese. In the native data set a partially occupied GMP could be identified. When co-crystallized with AMPPNP or GMPPNP, clear density for the hydrolyzed analogs was observed, providing hints to the function of the protein.

We have determined the structure of a PNA-DNA duplex to 1.7 A resolution by multiple-wavelength anomalous diffraction phasing method on a zinc derivative. This structure represents the first high-resolution 3D view of a hybrid duplex containing a contiguous chiral PNA strand with complete gamma-backbone modification ("gammaPNA"). Unlike the achiral counterpart, which adopts a random-fold, this particular gammaPNA is already preorganized into a right-handed helix as a single strand. The new structure illustrates the unique characteristics of this modified PNA, possessing conformational flexibility while maintaining sufficient structural integrity to ultimately adopt the preferred P-helical conformation upon hybridization with DNA. The unusual structural adaptability found in the gammaPNA strand is crucial for enabling the accommodation of backbone modifications while constraining conformational states. In conjunction with NMR analysis characterizing the structures and substructures of the individual building blocks, these results provide unprecedented insights into how this new class of chiral gammaPNA is preorganized and stabilized, before and after hybridization with a cDNA strand. Such knowledge is crucial for the future design and development of PNA for applications in biology, biotechnology, and medicine.

The exploitation of anomalous signals for biological structural solution is maturing. Single-wavelength anomalous diffraction (SAD) is dominant in de novo structure analysis. Nevertheless, for challenging structures where the resolution is low (dmin ≥ 3.5 Å) or where only lighter atoms (Z ≤ 20) are present, as for native macromolecules, solved SAD structures are still scarce. With the recent rapid development in crystal handling, beamline instrumentation, optimization of data collection strategies, use of multiple crystals and structure determination technologies, the weak anomalous diffraction signals are now robustly measured and should be used for routine SAD structure determination. The review covers these recent advances on weak anomalous signals measurement, analysis and utilization.

For years, three groups worked meticulously to generate hCG crystals for X-ray crystallography and multi-wavelength anomalous diffraction methods in hopes of determining the fine three-dimensional (3D) structure of hCG. Unfortunately, the heterogeneity of the eight carbohydrate side chains on hCG prevented any crystallization. Even desialylization, or removal of the principal charged carbohydrate component, did not allow fine crystal formation. Using an anhydrous hydrofluoric acid treatment, they carefully removed the entire N-linked oligosaccharides from hCG, leaving the deglycosylated protein intact. To remove the four O-linked oligosaccharides, they cleaved the 34-amino-acid C-terminal peptide at β111. It was assumed that the C-terminal peptide was a random, nonfolded component, similar to a semipolymer of proline and serine.

The mouse {beta}PIX-SH3 domain, residues 8-63 of P21-activated kinase interacting exchange factor, has been characterized by X-ray diffraction. Crystals belonging to space group P3{sub 2}21 diffracted to 2.0 A and the structure was phased by the single-wavelength anomalous diffraction method. The domain is a compact {beta}-barrel with an overall conformation similar to the general SH3 structure. The X-ray structure shows mouse {beta}PIX-SH3 domain binding the way in which the {beta}PIX characteristic amino acids do so for an unconventional ligand binding surface. This arrangement provides a rationale for the unusual ligand recognition motif exhibited by mouse {beta}PIX-SH3 domain. Comparison with another SH3/peptide complex shows that the recognition mode of the mouse {beta}PIX-SH3 domain should be very similar to the RXXK ligand binding mode. The unique large and planar hydrophobic pocket may contribute to the promiscuity of {beta}PIX-SH3 domain resulting in its multiple biological functions.

FIP is a French Collaborating Research Group (CRG) beamline at the European Synchrotron Radiation Facility (ESRF) dedicated exclusively to crystallography of biological macromolecules, with a special emphasis on multiwavelength anomalous diffraction data collection in the 0.7-1.81 A wavelength range. The optics, consisting of long cylindrical grazing-angle mirrors associated with a cryocooled double-crystal monochromator, delivers an optimal beam in the corresponding energy range. The high level of automation, which includes automated crystal centring, automated data-collection management and data processing, makes the use of this beamline very easy. This is illustrated by the large number of challenging structures that have been solved since 1999.

Acetophenone reductase (APRD) fromGeotrichum candidiumNBRC 4597 was crystallized by the hanging-drop vapour-diffusion method using PEG 3350 as a precipitant. The crystal belonged to space groupP6522, with unit-cell parametersa=b= 104.5,c= 273.7 Å, and diffracted to 2.6 Å resolution. Phasing using the single-wavelength anomalous diffraction method was successful. Model building and crystallographic refinement are in progress.

International audience; The phage-proximal part of the long tail fibres of bacteriophage T4 consists of a trimer of the 1289 amino-acid gene product 34 (gp34). Different carboxy-terminal parts of gp34 have been produced and crystallized. Crystals of gp34(726-1289) diffracting X-rays to 2.9 Å resolution, crystals of gp34(781-1289) diffracting to 1.9 Å resolution and crystals of gp34(894-1289) diffracting to 3.0 and 2.0 Å resolution and belonging to different crystal forms were obtained. Native data were collected for gp34(726-1289) and gp34(894-1289), while single-wavelength anomalous diffraction data were collected for selenomethionine-containing gp34(781-1289) and gp34(894-1289). For the latter, high-quality anomalous signal was obtained.

Anomalous diffraction signals from typical native macromolecules are very weak, frustrating their use inde novostructure determination. Here, native SAD procedures are described to enhance signal to noise in anomalous diffraction by using multiple crystals in combination with synchrotron X-rays at 6 keV. Increased anomalous signals were obtained at 6 keV compared with 7 keV X-ray energy, which was used for previous native SAD analyses. A feasibility test of multi-crystal-based native SAD phasing was performed at 3.2 Å resolution for a known tyrosine protein kinase domain, and real-life applications were made to two novel membrane proteins at about 3.0 Å resolution. The three applications collectively serve to validate the robust feasibility of native SAD phasing at lower energy.

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.

The EMBL Hamburg Outstation currently operates two tunable protein crystallography beamlines suitable for single and multiple anomalous diffraction (SAD/MAD) experiments. The first beamline, designated X31, is located on a bending magnet of the DORIS III storage ring whereas the second beamline, BW7A, is positioned at a multipole wiggler at the same storage ring. X31 is equipped with an energy stabilization device to ensure constant wavelength during longer data-collection periods. The in-house built crystallographic end-station is now equipped with a Mar345 imaging-plate scanner as a detector. The wiggler beamline BW7A features a novel sagitally focusing monochromator. The end-station used here has also been developed and built in-house. The beamline is currently operated with a Mar 165 CCD detector. In this paper the hardware and software developments of the last years will be summarized and the outlook for substantial upgrades will be given. The future plans include the design and construction of a third tunable beamline, designated X12, for protein crystallography. The development of automated beamlines for protein crystallography is of particular importance with respect to structural genomics initiatives. The analysis of the projects of the last years shows the wide range of anomalous scatterer used on the tunable beamlines thus demonstrating the need of a wide range of accessible energies and fast and reliable energy changes.

The effect of Co addition on the atomic ordering of bcc FeCo-phase in partially nanocrystalline Fe81-xCoxNb7B12 alloys (x= 20.25, 27, 40.5, 54, 60.75) was investigated. In the partially nanocrystalline state of the alloy, the anomalous diffraction measurements provides unambiguous evidence of atomically ordered α′-FeCo phase (CsCl type) as the nanocrystalline ferromagnetic phase by the presence of (100) super lattice reflection in all the alloy compositions with x≥ 40.5, this reflection was found absent in specimens with x ≤ 27. The Bragg-Williams long range order parameter (S) possess high value for the alloys close to equi-atomic composition (x=40.5 and 54), however in the specimen with x=60.75 the value of S decreases drastically by almost 60 %, which signifies less degree of ordering in the same.The effect of Co addition on the atomic ordering of bcc FeCo-phase in partially nanocrystalline Fe81-xCoxNb7B12 alloys (x= 20.25, 27, 40.5, 54, 60.75) was investigated. In the partially nanocrystalline state of the alloy, the anomalous diffraction measurements provides unambiguous evidence of atomically ordered α′-FeCo phase (CsCl type) as the nanocrystalline ferromagnetic phase by the presence of (100) super lattice reflection in all the alloy compositions with x≥ 40.5, this reflection was found absent in specimens with x ≤ 27. The Bragg-Williams long range order parameter (S) possess high value for the alloys close to equi-atomic composition (x=40.5 and 54), however in the specimen with x=60.75 the value of S decreases drastically by almost 60 %, which signifies less degree of ordering in the same.

The widespread application of multiwavelength anomalous diffraction (MAD) for phase evaluation has been hampered in the past by the small selection of anomalous scattering centres that could be introduced into macromolecules. Recently, the use of chemical modification, protein engineering or biosynthetic labelling has provided suitable tools to overcome the previous limitations, thereby making most structural analyses amenable to a MAD approach.

Background The integrin family of cell-surface receptors mediates a wide variety of cell–cell and cell–extracellular matrix interactions. Integrin–ligand interactions are invariably dependent on the presence of divalent cations, and a subset of integrins contain a ∼200 amino acid inserted (I) domain that is important for ligand binding activity and contains a single divalent cation binding site. Many integrins are believed to respond to stimuli by undergoing a conformational change that increases their affinity for ligand, and there is a clear difference between two crystal structures of the CD11b I domain with different divalent cations (magnesium and manganese) bound. In addition to the different bound cation, a ‘ligand mimetic’ crystal lattice interaction in the CD11b I domain structure with bound magnesium has led to the interpretation that the different CD11b I domain structures represent different affinity states of I domains. The influence of the bound cation on I domain structure and function remains incompletely understood, however. The crystal structure of the CD11a I domain bound to manganese is known. We therefore set out to determine whether this structure changes when the metal ion is altered or removed.Results We report here the crystal structures of the CD11a I domain determined in the absence of bound metal ion and with bound magnesium ion. No major structural rearrangements are observed in the metal-binding site of the CD11a I domain in the absence or presence of bound manganese ion. The structures of the CD11a I domain with magnesium or manganese bound are extremely similar.Conclusions The conformation of the CD11a I domain is not altered by changes in metal ion binding. The cation-dependence of ligand binding thus indicates that the metal ion is either involved in direct interaction with ligand or required to promote a favorable quaternary arrangement of the integrin.

The program ANODE determines anomalous (or heavy-atom) densities by reversing the usual procedure for experimental phase determination. Instead of adding a phase shift to the heavy-atom phases to obtain a starting value for the native protein phase, this phase shift is subtracted from the native phase to obtain the heavy-atom substructure phase., The new program ANODE estimates anomalous or heavy-atom density by reversing the usual procedure for experimental phase determination by methods such as single- and multiple-wavelength anomalous diffraction and single isomorphous replacement anomalous scattering. Instead of adding a phase shift to the heavy-atom phases to obtain a starting value for the native protein phase, this phase shift is subtracted from the native phase to obtain the heavy-atom substructure phase. The required native phase is calculated from the information in a Protein Data Bank file of the structure. The resulting density enables even very weak anomalous scatterers such as sulfur to be located. Potential applications include the identification of unknown atoms and the validation of molecular replacement solutions.

Two new experiments (single isomorphous replacement including anomalous-scattering effects and radiation damage-induced phasing) have been designed to complement the five experiments (sulfur single-wavelength anomalous diffraction, multiple-wavelength anomalous diffraction, molecular replacement, ion binding and ligand binding) of the first edition of the previously described tutorial for learning and teaching macromolecular crystallography [Faust, Panjikar, Mueller, Parthasarathy, Schmidt, Lamzin & Weiss (2008). J. Appl. Cryst. 41, 1161–1172]. Furthermore, the tutorial has been re-organized and in part re-written to reflect the comments and suggestions of users. The most significant overhaul was applied to the data-processing part of the tutorial. Nevertheless, the convenient features that all of the utilized proteins used are commercially available and that they can be easily and reproducibly crystallized and mounted for diffraction data collection have been retained. Also, for all of the seven experiments the raw diffraction images and the processed data are provided for illustrating and teaching the steps of data processing and structure determination.

New opportunities for direct determination of three-dimensional structures of biological macromolecules are offered by multiwavelength anomalous diffraction. Recent successes of the method have demonstrated its potential, and the number of problems to which it is being applied is growing rapidly. Experimental considerations are somewhat different for this method than for more familiar crystallographic methods.

IscU is a key component of the ISC machinery and is involved in the biogenesis of iron-sulfur (Fe-S) proteins. IscU serves as a scaffold for assembly of a nascent Fe-S cluster prior to its delivery to an apo protein. Here, we report the first crystal structure of IscU with a bound [2Fe-2S] cluster from the hyperthermophilic bacterium Aquifex aeolicus, determined at a resolution of 2.3 angstrom, using multiwavelength anomalous diffraction of the cluster. The holo IscU formed a novel asymmetric trimer that harbored only one [2Fe-2S] cluster. One iron atom of the cluster was coordinated by the S-gamma atom of Cys36 and the N-epsilon atom of Hs106, and the other was coordinated by the S-gamma atoms of Cys63 and Cys107 on the surface of just one of the protomers. However, the cluster was buried inside the trimer between the neighboring protomers. The three protomers were conformationally distinct from one another and associated around a noncrystallographic pseudo-3-fold axis. The three flexible loop regions carrying the ligand-binding residues (Cys36, Cys63,His106 and Cys107) and the N-terminal alpha 1 helices were positioned at the interfaces and underwent substantial conformational rearrangement, which stabilized the association of the asymmetric trimer. This unique trimeric A. aeolicus holo-IscU architecture was clearly distinct from other known monomeric apo-IscU/SufU structures, indicating that asymmetric trimer organization, as well as its association/dissociation, would be involved in the scaffolding function of IscU. (C) 2008 Elsevier Ltd. All rights reserved.

The hybrid cluster protein (HCP) from the sulfate-reducing bacterium Desulfovibrio vulgaris strain Hildenborough has been isolated and crystallized anaerobically. The phase problem was solved for a P2(1)2(1)2(1) crystal form using multiple-wavelength anomalous diffraction data collected in the vicinity of the Fe K absorption edge. Although the overall protein structure is essentially the same as that previously obtained, it shows that the nature of the hybrid cluster has particular differences when isolated and crystallized in the absence of oxygen and this provides insight into the structural features associated with changes in the oxidation state. A comparison between HCPs and carbon monoxide dehydrogenases (CoDs) shows that they possess a similar fold and that the dehydrogenases have a related cluster at the equivalent HCP hybrid cluster position. This helps to understand the nature of the hybrid cluster and to predict a dimeric structure for class 3 HCPs, which lack the N-terminal region.

The gene product of structural genomics target Lmaj006129 from Leishmania major codes for a 164-residue protein of unknown function. When SeMet expression of the full-length gene product failed, several truncation variants were created with the aid of Ginzu, a domain-prediction method. 11 truncations were selected for expression, purification and crystallization based upon secondary-structure elements and disorder. The structure of one of these variants, Lmaj006129AAH, was solved by multiple-wavelength anomalous diffraction (MAD) using ELVES, an automatic protein crystal structure-determination system. This model was then successfully used as a molecular-replacement probe for the parent full-length target, Lmaj006129AAA. The final structure of Lmaj006129AAA was refined to an R value of 0.185 (R free = 0.229) at 1.60 A resolution. Structure and sequence comparisons based on Lmaj006129AAA suggest that proteins belonging to Pfam sequence families PF04543 and PF01878 may share a common ligand-binding motif.

A Bayesian treatment for phase calculation in the multiwavelength anomalous diffraction (MAD) technique is presented. This approach explicitly treats effects of errors correlated among measurements at different wavelengths and between Bijvoet pairs. The resulting method, which is called Bayesian correlated MAD phasing, gives proper statistical consideration to all data and does not give special treatment to data from a particular wavelength. Results obtained using Bayesian correlated MAD phasing and two other strategies on both a model test case and on data obtained in two actual MAD experiments are compared. Although all procedures performed well when the completeness of the data was high, it is shown that Bayesian correlated MAD phasing is more robust with respect to incompleteness of data than the other methods are. At 60% completeness the improvement over other methods for the examples given was nearly 50% in the correlation coefficients, and made a substantial difference in the interpretability of an electron-density map.

We describe a 2.9 Å X-ray structure of a complex between the aminocyclitol antibiotic streptomycin and an in vitro selected RNA aptamer, solved using the anomalous diffraction properties of Ba cations. The RNA aptamer, which contains two asymmetric internal loops, adopts a distinct cation-stabilized fold involving a series of S-shaped backbone turns anchored by canonical and noncanonical pairs and triples. The streptomycin streptose ring is encapsulated by stacked arrays of bases from both loops at the elbow of the L-shaped RNA architecture. Specificity is defined by direct hydrogen bonds between all streptose functional groups and base edges that line the inner walls of the cylindrical binding pocket. By contrast, the majority of intermolecular interactions involve contacts to backbone phosphates in the published structure of streptomycin bound to the 16S rRNA.

An alternative formulation of direct methods for phase refinement using the sign probability originally introduced for breaking the phase ambiguity intrinsic in single isomorphous replacement or single-wavelength anomalous diffraction data has been proposed. This formulation can incorporate known phase information through constrained phase refinement. This formulation was tested by application to the phase-extension problem using experimental data. The results showed that this formulation was valid and promising for designing better methods for phase extension.

Publisher Summary This chapter discusses the structure and adsorption properties of 5A zeolites. To correlate the adsorption and diffusion properties to the cation distribution in the structure of CaNaA zeolites, temperature-programmed desorption, anomalous X-ray powder diffraction, and quasi elastic neutron scattering experiments are performed. Water adsorption and diffusion behavior differs with the calcium content. Fourier maps obtained from anomalous diffraction experiments near the calcium (Ca) K-edge allow identifying the calcium crystallographic sites unambiguously.

The phase determination is one of the central problems for the determination of the electron density map during the X-ray crystallographic analysis of biomolecules. Thus several methods have been developed, which include isomorphous replacement [1], molecular replacement [2] and multi-wavelength anomalous diffraction (MAD) [3,4]. The last method uses the preparation of seleno-methionine derivatives of the protein structure. While molecular biology techniques unspecifically insert the selenomethionine in the protein structure, chemistry allows in general the rapid position-selective chemical synthesis. We report here a solid phase synthesis strategy for seleno-methionine derivatives of synthetic peptide and protein structures, which is tightly fitted into the overall Fmoc/tBu or tBoc/Bzl protection scheme.

In only a few years, multiple wavelength anomalous diffraction (MAD) phasing has advanced from an esoteric technique used in only a few favorable cases to the method of choice for solving new macromolecular structures. Before 1994, MAD phasing had been used for fewer than a dozen new structure determinations. In 1999 alone, well over 100 new structures were determined by MAD phasing. The meteoric rise in MAD applications resulted from the availability of new synchrotron beamlines, equipped with low bandpass optics, fast readout detectors, cryogenic cooling and user-friendly interfaces. The power of MAD phasing has been amplified by the availability of new computer programs for locating the positions of the anomalous scattering atoms and for calculating phases from the experimental data. Phasing by anomalous scattering techniques has been applied to structures as large as 640 kDa and 120 selenium atoms in the asymmetric unit. The practical size limitation for application of MAD phasing techniques has not yet been encountered.

Since it was first reported, the multiwavelength anomalous diffraction (MAD) technique for the determination of protein structures has become widely accepted and increasingly popular. Here, it is demonstrated that the anomalous signal from selenomethione (SeMet) substituted proteins can be significantly enhanced by oxidation.

Recent advances in ab initio direct methods have enabled the solution of crystal structures of small proteins from native X-ray data alone, that is, without the use of fragments of known structure or the need to prepare heavy-atom or selenomethionine derivatives, provided that the data are available to atomic resolution. These methods are also proving to be useful for locating the selenium atoms or other anomalous scatterers in the multiple wavelength anomalous diffraction phasing of larger proteins at lower resolution. peerReviewed

Multiwavelength anomalous diffraction (MAD) phasing, which relies on synchrotron radiation, has grown from a novelty technique to a mainstream method that has been used to determine macromolecule structures of up to 200,000 Mr.

Multiwavelength anomalous diffraction (MAD) is the fastest growing method of structure determination in macromolecular crystallography. At least twenty-five new structures solved with MAD were published in the past year. Many factors contribute to the growth of MAD, and its future is extremely bright. The experience gained over the past several years is now being generalized to make MAD more accessible. This paper aims to present a practical overview of MAD. I first review the observational equation for MAD and describe the basis of the phasing signal and how it is estimated for specific problems. This is followed by a discussion of the design of a MAD experiment, schemes for data analysis and phasing, and considerations in solving the anomalous-scatterer partial structure. Finally, there is a discussion of selenomethionine as a phasing vehicle. More comprehensive reviews of MAD have been published by W. A. Hendrickson, who pioneered its development and application in macromolecular crystallography [1,2]. Several short papers covering various aspects of MAD are included in the proceedings of the 1997 CCP4 Study Weekend [Recent Advances in Phasing, K. Wilson, G. Davies, and S. Bailey, eds., SERC Daresbury Laboratory, Warrington, U.K, 1997, in press].