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1.  PIM: Phase Integrated Method for Normal Mode Analysis of Biomolecules in Crystalline Environment 
Journal of molecular biology  2013;425(6):1082-1098.
In this study, a normal mode analysis, named phase integrated method (PIM), is developed for computing modes of biomolecules in crystalline environment. PIM can calculate low-frequency modes on one or a few asymmetric units and generate exact modes of a whole unit cell according to space group symmetry, while the translational symmetry between unit cells is maintained via periodic boundary condition. Therefore, the method can dramatically reduce computational cost in mode calculation in the presence of crystal symmetry. PIM also has an option to map modes onto a single asymmetric unit to form an orthonormalized mode set, which can be directly applied to normal-mode-based thermal parameter refinement in X-ray crystallography. The performance of PIM was tested on all 65 space groups available in protein crystals (one protein for each space group) and on another set of 83 ultra-high-resolution X-ray structures. The results showed that considering space group symmetry in mode calculation is crucial for accurately describing vibrational motion in crystalline environment. Moreover, the optimal inter-asymmetric-unit packing stiffness was found to be about 60% of that of intra-asymmetric-unit interactions (nonbonded interaction only).
doi:10.1016/j.jmb.2012.12.026
PMCID: PMC3594335  PMID: 23333742
Normal mode analysis; Crystal packing; Space group; Asymmetric unit; X-ray refinement; Phase shift; Phase integrated method; Inter- and Intra-asymmetric unit packing interactions
2.  A case of structure determination using pseudosymmetry 
When properly applied, pseudosymmetry can be used to improve crystallographic phases through averaging and to facilitate crystal structure determination.
Here, a case is presented of an unusual structure determination which was facilitated by the use of pseudosymmetry. Group A streptococcus uses cysteine protease Mac-1 (also known as IdeS) to evade the host immune system. Native Mac-1 was crystallized in the orthorhombic space group P21212. Surprisingly, crystals of the inactive C94A mutant of Mac-1 displayed monoclinic symmetry with space group P21, despite the use of native orthorhombic Mac-1 microcrystals for seeding. Attempts to solve the structure of the C94A mutant by MAD phasing in the monoclinic space group did not produce an interpretable map. The native Patterson map of the C94A mutant showed two strong peaks along the (1 0 1) diagonal, indicating possible translational pseudosymmetry in space group P21. Interestingly, one-third of the monoclinic reflections obeyed pseudo-orthorhombic P21212 symmetry similar to that of the wild-type crystals and could be indexed and processed in this space group. The pseudo-orthorhombic and monoclinic unit cells were related by the following vector operations: a m = b o − c o, b m = a o and c m = −2c o − b o. The pseudo-orthorhombic subset of data produced good SAD phases, leading to structure determination with one monomer in the asymmetric unit. Subsequently, the structure of the Mac-1 mutant in the monoclinic form was determined by molecular replacement, which showed six molecules forming three translationally related dimers aligned along the (1 0 1) diagonal. Knowing the geometric relationship between the pseudo-orthorhombic and the monoclinic unit cells, all six molecules can be generated in the monoclinic unit cell directly without the use of molecular replacement. The current case provides a successful example of the use of pseudosymmetry as a powerful phase-averaging method for structure determination by anomalous diffraction techniques. In particular, a structure can be solved in a higher pseudosymmetry subcell in which an NCS operator becomes a crystallographic operator. The geometrical relationships between the subcell and parental cell can be used to generate a complete molecular representation of the parental asymmetric unit for refinement.
doi:10.1107/S0907444909039912
PMCID: PMC2789005  PMID: 19966420
pseudosymmetry; structure determination; cysteine proteases; Mac-1
3.  Bis(1,3-dimethyl-1H-imidazolium) hexa­fluoro­silicate methanol 0.33-solvate 
The title compound, 6C5H9N2 +·3SiF6 2−·CH3OH, (I), was prepared by recrystallization of the crude salt from methanol along with solvent-free 2C5H9N2 +·SiF6 2− (II). Crystals of these solvatomorphs can be separated manually. The solvate (I) crystallizes in a rare hexa­gonal space group P6/mcc. Its asymmetric unit comprises one half of an imidazolium cation bis­ected by the crystallographic m-plane, one-sixth and one-twelfth of two crystallographically independent SiF6 2– dianions (Si atoms are located on the 3.2 and 6/m inversion centres), and one-twelfth of a methanol mol­ecule (C atoms are situated on the 622 inversion centres, other atoms are disordered between general positions). In (I), all F atoms of 3.2-located SiF6 2– dianions participate in the formation of symmetry-equivalent contacts to the H atoms of imidazolium fragments, thus forming rod-type ensembles positioned on the -6 axes. These ‘pillar’ rods are, in turn, F⋯H inter­linked through SiF6 2– dianions disordered around the 6/m centres. The twelvefold disordered methanol mol­ecules are appended to this array by O—H⋯F hydrogen bonds to the 6/m located SiF6 2– dianions. In terms of graph-set notation, the first and second level networks in (I) are N 1 = C 2 2(7)[3R 4 4(14)]D 2 2(4) and N 2 = D 2 2(5) (C—H⋯O hydrogen bonds are not considered). After locating all symmetrically independent atoms in the cation and anions, there remained a strong (> 3 e Å−3) residual electron density peak located at the 622 inversion centre. Treatment of this pre-refined model with the SQUEEZE procedure in PLATON [Spek (2009). Acta Cryst. D65, 148–155] revealed two voids per unit cell, indicative of the presence of the solvent methanol mol­ecule disordered about the 622 inversion centre.
doi:10.1107/S1600536813018230
PMCID: PMC3793724  PMID: 24109311
4.  Alternative models for two crystal structures of bovine rhodopsin 
Two crystal structures of rhodopsin that were originally described using trigonal symmetry can be interpreted in a hexagonal unit cell with a smaller asymmetric unit.
The space-group symmetry of two crystal forms of rhodopsin (PDB codes 1gzm and 2j4y; space group P31) can be re-interpreted as hexagonal (space group P64). Two molecules of the G protein-coupled receptor are present in the asymmetric unit in the trigonal models. However, the noncrystallographic twofold axes parallel to the c axis can be treated as crystallographic symmetry operations in the hexagonal space group. This halves the asymmetric unit and makes all of the protein molecules equivalent in these structures. Corrections for merohedral twinning were also applied in the refinement in the higher symmetry space group for one of the structures (2j4y).
doi:10.1107/S0907444908017162
PMCID: PMC2483493  PMID: 18645239
alternate space groups; rhodopsin; G protein-coupled receptors; integral membrane proteins
5.  Expression, purification, crystallization and preliminary crystallographic analysis of BipD, a component of the Burkholderia pseudomallei type III secretion system 
A construct consisting of residues 10–310 of mature BipD, a component of the B. pseudomallei type III secretion system, has been crystallized. Native BipD crystals and SeMet and K2PtCl4 derivative crystals have undergone preliminary crystallographic analysis.
A construct consisting of residues 10–310 of BipD, a component of the Burkholderia pseudomallei type III secretion system (T3SS), has been overexpressed as a GST fusion, cleaved from the GST tag and purified. Crystals were grown of native and selenomethionine-labelled BipD. The crystals grow in two different polymorphs from the same condition. The first polymorph belongs to space group C222, with unit-cell parameters a = 103.98, b = 122.79, c = 49.17 Å, a calculated Matthews coefficient of 2.4 Å3 Da−1 (47% solvent content) and one molecule per asymmetric unit. The second polymorph belongs to space group P21212, with unit-cell parameters a = 136.47, b = 89.84, c = 50.15 Å, and a calculated Matthews coefficient of 2.3 Å3 Da−1 (45% solvent content) for two molecules per asymmetric unit (analysis of the self-rotation function indicates the presence of a weak twofold non-crystallographic symmetry axis in this P21212 form). The native crystals of both forms give diffraction data to 2.7 Å resolution, while the SeMet-labelled P21212 crystals diffract to 3.3 Å resolution. A K2PtCl4 derivative of the P21212 form was also obtained and data were collected to 2.7 Å with radiation of wavelength λ = 0.933 Å. The Pt-derivative anomalous difference Patterson map revealed two self-peaks on the Harker sections.
doi:10.1107/S1744309106027035
PMCID: PMC1894745  PMID: 16946464
BipD; type III secretion system; Burkholderia pseudomallei
6.  On optimal placement of molecules in the unit cell 
Standard ways for the placement of molecules in the unit cell are proposed.
There are currently no rules for a unified, standard way of placing macromolecular structures in the crystal lattice. An analysis of all possible symmetry-equivalent representations of molecular structures in various space groups leads to the concept of the anti-Cheshire symmetry and suggests that the center of a unique structural motif can always be placed within the selected asymmetric unit of the anti-Cheshire cell. The placement of structures according to this suggestion will ensure uniformity of presentation of all structurally equivalent Protein Data Bank models and will therefore diminish the possibility of confusing less crystallographically knowledgeable users of the PDB. The anti-Cheshire cells and their asymmetric units are defined and tabulated for all 65 space groups relevant to macromolecular crystallography that exhibit only rotational symmetry operations.
doi:10.1107/S0907444913002722
PMCID: PMC4005360  PMID: 23633598
placement of molecules; Cheshire symmetry; anti-Cheshire symmetry
7.  A new specialized visual acuity chart for amblyopic children aged 3-5 years old: development and its clinical applications 
AIM
To introduce a new specialized visual acuity chart for amblyopic children aged 3-5 years old and its clinical applications.
METHODS
The new visual acuity chart and notations were designed based on Weber-Fechner law. The optotypes were red against a white background and were specially shaped four basic geometric symbols: circle, square, triangle, and cross. A regular geometric progression of the optotype sizes and distribution was employed to arrange in 14 lines. The progression rate of the optotype size between two lines was 1.2589 and the testing distance was 3m. Visual acuity score could be recorded as logMAR notation or decimal notation. Age-stratified diagnostic criteria for amblyopia established by consensus statement on diagnosis of amblyopia (2011) among members of the Strabismus and Pediatric Ophthalmology Group, Ophthalmology Society, Chinese Medical Association (SPOGOSCMA) were illustrated in the new visual acuity chart.
RESULTS
When assessing visual acuity in children aged 3-5 years old, this new visual acuity chart that consists of four symmetrical shapes (triangle, square, cross, and circle) overcame an inability to recognize the letters of the alphabet and difficulties in designating the direction of black abstract symbols such as the tumbling ‘E’ or Landolt ‘C’, which the subjects were prone to lose interest in. The visual acuity score may be recorded in different notations: decimal acuity and logMAR. These two notations can be easily converted each other in the new eye chart. The measurements of this new chart not only showed a significant correlation and a good consistency with the international standard logarithmic visual acuity chart (r=0.932, P<0.01), but also indicated a high test-retest reliability (89% of retest scores were within 0.1logMAR units of the initial test score).
CONCLUSION
The results of this study support the validity and reliability of distance visual acuity measurements using the new eye chart in children aged 3 to 5 years over a wide range of visual acuities, and the new eye chart is great for early detection of amblyopia. It can be applied in various clinical settings.
doi:10.3980/j.issn.2222-3959.2013.06.18
PMCID: PMC3874526  PMID: 24392335
amblyopic children; Weber-Fechner law; international standard logarithmic visual acuity chart
8.  Surprises and pitfalls arising from (pseudo)symmetry 
The presence of pseudosymmetry can cause problems in structure determination and refinement. The relevant background and representative examples are presented.
It is not uncommon for protein crystals to crystallize with more than a single molecule per asymmetric unit. When more than a single molecule is present in the asymmetric unit, various pathological situations such as twinning, modulated crystals and pseudo translational or rotational symmetry can arise. The presence of pseudosymmetry can lead to uncertainties about the correct space group, especially in the presence of twinning. The background to certain common pathologies is presented and a new notation for space groups in unusual settings is introduced. The main concepts are illustrated with several examples from the literature and the Protein Data Bank.
doi:10.1107/S090744490705531X
PMCID: PMC2394827  PMID: 18094473
pathology; twinning; pseudosymmetry
9.  Expression, Purification, Crystallization and Preliminary Crystallographic Analysis of BipD, a Component of the Burkholderia Pseudomallei Type III Secretion System 
A construct consisting of residues 10–310 of BipD, a component of the Burkholderia pseudomallei type III secretion system (T3SS), has been overexpressed as a GST fusion, cleaved from the GST tag and purified. Crystals were grown of native and selenomethionine-labelled BipD. The crystals grow in two different polymorphs from the same condition. The first polymorph belongs to space group C222, with unit-cell parameters a = 103.98, b = 122.79, c = 49.17 Å, a calculated Matthews coefficient of 2.4 Å3 Da−1 (47% solvent content) and one molecule per asymmetric unit. The second polymorph belongs to space group P21212, with unit-cell parameters a = 136.47, b = 89.84, c = 50.15 Å, and a calculated Matthews coefficient of 2.3 Å3 Da−1 (45% solvent content) for two molecules per asymmetric unit (analysis of the self-rotation function indicates the presence of a weak twofold non-crystallographic symmetry axis in this P21212 form). The native crystals of both forms give diffraction data to 2.7 Å resolution, while the SeMet-labelled P21212 crystals diffract to 3.3 Å resolution. A K2PtCl4 derivative of the P21212 form was also obtained and data were collected to 2.7 Å with radiation of wavelengt λ = 0.933 Å. The Pt-derivative anomalous difference Patterson map revealed two self-peaks on the Harker sections.
doi:10.1107/S1744309106027035
PMCID: PMC1894745  PMID: 16946464
10.  Crystallization of the nonameric small terminase subunit of bacteriophage P22 
The small terminase subunit of bacteriophage P22 was overexpressed in E. coli, purified under native conditions and crystallized as a nonamer. High-quality diffraction data were collected to 1.75 Å resolution using synchrotron radiation.
The packaging of viral genomes into preformed empty procapsids is powered by an ATP-dependent genome-translocating motor. This molecular machine is formed by a heterodimer consisting of large terminase (L-terminase) and small terminase (S-terminase) subunits, which is assembled into a complex of unknown stoichiometry, and a dodecameric portal protein. There is considerable confusion in the literature regarding the biologically relevant oligomeric state of terminases, which, like portal proteins, form ring-like structures. The number of subunits in a hollow oligomeric protein defines the internal diameter of the central channel and the ability to fit DNA inside. Thus, knowledge of the exact stoichiometry of terminases is critical to decipher the mechanisms of terminase-dependent DNA translocation. Here, the gene encoding bacteriophage P22 S-­terminase in Escherichia coli has been overexpressed and the protein purified under native conditions. In the absence of detergents and/or denaturants that may cause disassembly of the native oligomer and formation of aberrant rings, it was found that P22 S-terminase assembles into a concentration-independent nonamer of ∼168 kDa. Nonameric S-terminase was crystallized in two different crystal forms at neutral pH. Crystal form I belonged to space group P21212, with unit-cell parameters a = 144.2, b = 144.2, c = 145.3 Å, and diffracted to 3.0 Å resolution. Crystal form II belonged to space group P21, with unit-cell parameters a = 76.48, b = 100.9, c = 89.95 Å, β = 93.73°, and diffracted to 1.75 Å resolution. Preliminary crystallographic analysis of crystal form II confirms that the S-terminase crystals contain a nonamer in the asymmetric unit and are suitable for high-resolution structure determination.
doi:10.1107/S174430911004697X
PMCID: PMC3079985  PMID: 21206037
small terminase; large terminase; DNA packaging; bacteriophage P22; nonamers
11.  Visualisation of a 2'-5' parallel stranded double helix at atomic resolution: crystal structure of cytidylyl-2',5'-adenosine. 
Nucleic Acids Research  1991;19(2):379-384.
X-ray crystallographic studies on 3'-5' oligomers have provided a great deal of information on the stereochemistry and conformational flexibility of nucleic acids and polynucleotides. In contrast, there is very little information available on 2'-5' polynucleotides. We have now obtained the crystal structure of Cytidylyl-2',5'-Adenosine (C2'p5'A) at atomic resolution to establish the conformational differences between these two classes of polymers. The dinucleoside phosphate crystallises in the monoclinic space group C2, with a = 33.912(4)A, b = 16.824(4)A, c = 12.898(2)A and beta = 112.35(1) with two molecules in the asymmetric unit. Spectacularly, the two independent C2'p5'A molecules in the asymmetric unit form right handed miniature parallel stranded double helices with their respective crystallographic two fold (b axis) symmetry mates. Remarkably, the two mini duplexes are almost indistinguishable. The cytosines and adenines form self-pairs with three and two hydrogen bonds respectively. The conformation of the C and A residues about the glycosyl bond is anti same as in the 3'-5' analog but contrasts the anti and syn geometry of C and A residues in A2'p5'C. The furanose ring conformation is C3' endo, C2' endo mixed puckering as in the C3'p5'A-proflavine complex. A comparison of the backbone torsion angles with other 2'-5' dinucleoside structures reveals that the major deviations occur in the torsion angles about the C3'-C2' and C4'-C3' bonds. A right-handed 2'-5' parallel stranded double helix having eight base pairs per turn and 45 degrees turn angle between them has been constructed using this dinucleoside phosphate as repeat unit. A discussion on 2'-5' parallel stranded double helix and its relevance to biological systems is presented.
Images
PMCID: PMC333605  PMID: 2014174
12.  A near-vision chart for children aged 3-5 years old: new designs and clinical applications 
AIM
To introduce a new near-vision chart for children aged 3-5 years old and its clinical applications.
METHODS
The new near-vision chart which combined the Bailey-Lovie layout with a newly devised set of symmetry symbols was designed based on Weber-Fechner law. It consists of 15 rows of symmetry symbols, corresponding to a visual acuity range from 1.3 to 0.1 logMAR. The optotypes were red against a white background and were specially shaped four basic geometric symbols: circle, square, triangle, and cross, which matched the preschool children's cognitive level. A regular geometric progression of the optotype sizes and distribution was employed to arrange in 15 lines. The progression rate of the optotype size between two lines was 1.2589 and two smaller groups of optotypes ranging from 0.7 to -0.1 logMAR were included for repetitive testing. A near visual acuity was recorded in logMAR or decimal, and the testing distance was 25 cm.
RESULTS
This new near-vision chart with pediatric acuity test optotypes which consists of 4 different symbols (triangle, square, cross, and circle) met the national and international eye chart design guidelines. When performing the near visual acuity assessment in preschoolers (3-5 years old). It overcame an inability to recognize the letters of the alphabet and difficulties in designating the direction of black abstract symbols such as the tumbling ‘E’ or Landolt ‘C’, which the subjects were prone to lose interest in. Near vision may be recorded in different notations: decimal acuity and logMAR. These two notations can be easily converted each other in the new near-vision chart. The measurements of this new chart not only showed a significant correlation and a good consistency with the Chinese national standard logarithmic near-vision chart (r=0.932, P<0.01), but also indicated good test-retest reliability (89% of retest scores were within 0.1 logMAR units of the initial test score) and a high response rate.
CONCLUSION
The results of this study support the validity and reliability of near visual acuity measurements using the new near-vision chart in children aged 3-5y over a wide range of visual acuities, and the new eye chart was especially suitable for the detection of amblyopia risk factors and low vision examination in children (3-5y of age). It can be applied in routine clinical practice.
doi:10.3980/j.issn.2222-3959.2014.03.13
PMCID: PMC4067659  PMID: 24967191
preschool children; Weber-Fechner law; Chinese national standard logarithmic near-vision chart
13.  Chemical conversion of cisplatin and carboplatin with histidine in a model protein crystallized under sodium iodide conditions 
Crystals of HEWL with cisplatin and HEWL with carboplatin grown in sodium iodide conditions both show a partial chemical transformation of cisplatin or carboplatin to a transiodoplatin (PtI2 X 2) form. The binding is only at the Nδ atom of His15. A further Pt species (PtI3 X) is also seen, in both cases bound in a crevice between symmetry-related protein molecules.
Cisplatin and carboplatin are platinum anticancer agents that are used to treat a variety of cancers. Previous X-ray crystallographic studies of carboplatin binding to histidine in hen egg-white lysozyme (HEWL) showed a partial chemical conversion of carboplatin to cisplatin owing to the high sodium chloride concentration used in the crystallization conditions. Also, the co-crystallization of HEWL with carboplatin in sodium bromide conditions resulted in the partial conversion of carboplatin to the transbromoplatin form, with a portion of the cyclobutanedicarboxylate (CBDC) moiety still present. The results of the co-crystallization of HEWL with cisplatin or carboplatin in sodium iodide conditions are now reported in order to determine whether the cisplatin and carboplatin converted to the iodo form, and whether this took place in a similar way to the partial conversion of carboplatin to cisplatin in NaCl conditions or to transbromoplatin in NaBr conditions as seen previously. It is reported here that a partial chemical transformation has taken place to a transplatin form for both ligands. The NaI-grown crystals belonged to the monoclinic space group P21 with two molecules in the asymmetric unit. The chemically transformed cisplatin and carboplatin bind to both His15 residues, i.e. in each asymmetric unit. The binding is only at the Nδ atom of His15. A third platinum species is also seen in both conditions bound in a crevice between symmetry-related molecules. Here, the platinum is bound to three I atoms identified based on their anomalous difference electron densities and their refined occupancies, with the fourth bound atom being a Cl atom (in the cisplatin case) or a portion of the CBDC moiety (in the carboplatin case).
doi:10.1107/S2053230X14013995
PMCID: PMC4157406  PMID: 25195879
cisplatin; carboplatin; sodium iodide crystallization; histidine; transiodoplatin (PtI2X2); PtI3X species
14.  Type II dehydroquinase: molecular replacement with many copies 
The type II dehydroquinase enzyme is a symmetrical dodecameric protein which crystallizes in either high-symmetry cubic space groups or low-symmetry crystal systems with multiple copies in the asymmetric unit. Both systems have provided challenging examples for molecular replacement; for example, a triclinic crystal form has 16 dodecamers (192 monomers) in the unit cell. Three difficult examples are discussed and two are used as test cases to compare the performance of four commonly used molecular-replacement packages.
Type II dehydroquinase is a small (150-amino-acid) protein which in solution packs together to form a dodecamer with 23 cubic symmetry. In crystals of this protein the symmetry of the biological unit can be coincident with the crystallographic symmetry, giving rise to cubic crystal forms with a single monomer in the asymmetric unit. In crystals where this is not the case, multiple copies of the monomer are present, giving rise to significant and often confusing noncrystallographic symmetry in low-symmetry crystal systems. These different crystal forms pose a variety of challenges for solution by molecular replacement. Three examples of structure solutions, including a highly unusual triclinic crystal form with 16 dodecamers (192 monomers) in the unit cell, are described. Four commonly used molecular-replacement packages are assessed against two of these examples, one of high symmetry and the other of low symmetry; this study highlights how program performance can vary significantly depending on the given problem. In addition, the final refined structure of the 16-dodecamer triclinic crystal form is analysed and shown not to be a superlattice structure, but rather an F-centred cubic crystal with frustrated crystallographic symmetry.
doi:10.1107/S0907444907054923
PMCID: PMC2394815  PMID: 18094474
multi-copy molecular replacement; superlattice structure; pseudo-cubic symmetry; type II dehydroquinases
15.  Crystallization and initial X-ray diffraction studies of the flavoenzyme NAD(P)H:(acceptor) oxidoreductase (FerB) from the soil bacterium Paracoccus denitrificans  
The flavin-dependent enzyme FerB from P. denitrificans has been purified and both native and SeMet-substituted FerB have been crystallized. The two variants crystallized in two different crystallographic forms belonging to the monoclinic space group P21 and the orthorhombic space group P21212, respectively. X-ray diffraction data were collected to 1.75 Å resolution for both forms.
The flavin-dependent enzyme FerB from Paracoccus denitrificans reduces a broad range of compounds, including ferric complexes, chromate and most notably quinones, at the expense of the reduced nicotinamide adenine dinucleotide cofactors NADH or NADPH. Recombinant unmodified and SeMet-substituted FerB were crystallized under similar conditions by the hanging-drop vapour-diffusion method with microseeding using PEG 4000 as the precipitant. FerB crystallized in several different crystal forms, some of which diffracted to approximately 1.8 Å resolution. The crystals of native FerB belonged to space group P21, with unit-cell parameters a = 61.6, b = 110.1, c = 65.2 Å, β = 118.2° and four protein molecules in the asymmetric unit, whilst the SeMet-substituted form crystallized in space group P21212, with unit-cell parameters a = 61.2, b = 89.2, c = 71.5 Å and two protein molecules in the asymmetric unit. Structure determination by the three-wavelength MAD/MRSAD method is now in progress.
doi:10.1107/S1744309110005099
PMCID: PMC2852337  PMID: 20383015
flavoenzymes; quinone reductases; Paracoccus denitrificans
16.  Cloning, expression, purification, crystallization and preliminary X-ray diffraction analysis of tetrahydrodipicolinate-N-succinyltransferase (Rv1201c) from Mycobacterium tuberculosis  
M. tuberculosis tetrahydrodipicolinate-N-succinyltransferase, the enzyme that catalyses the fifth reaction step of the lysine-biosynthesis pathway, has been cloned, expressed, purified and crystallized.
Tetrahydrodipicolinate-N-succinyltransferase from Mycobacterium tuberculosis (DapD, Rv1201c) has been cloned, heterologously expressed in Escherichia coli, purified using standard chromatographic techniques and crystallized in the cubic space group I23 or I213. Preliminary diffraction data analysis indicates the presence of five molecules per asymmetric unit. Furthermore, the data exhibit icosahedral point-group symmetry. One possible explanation for this is that the enzyme assembles into a 60-mer exhibiting 235 point-group symmetry and crystallizes as such in space group I23. In this case, the combination of crystallographic and noncrystallographic symmetry elements results in an arrangement of the icosahedrons in the cubic crystal with one pentamer in the asymmetric unit. Another explanation is that the packing of the molecules itself mimics icosahedral symmetry. In this case both space groups I23 and I213 would be possible.
doi:10.1107/S1744309108026559
PMCID: PMC2531272  PMID: 18765924
tetrahydrodipicolinate-N-succinyltransferase; Mycobacterium tuberculosis; DapD
17.  Crystallization and preliminary X-ray crystallographic analysis of dihydrouridine synthase from Thermus thermophilus and its complex with tRNA 
Crystals of dihydrouridine synthase from Thermus thermophilus and its complex with tRNA were obtained and X-ray diffraction data were collected to 1.70 and 3.51 Å resolution, respectively.
Dihydrouridine synthase (Dus) is responsible for catalyzing dihydrouridine formation in RNA by the reduction of uridine. To elucidate its RNA-recognition mechanism, Dus from Thermus thermophilus (TthDus) and its complex with tRNA were crystallized. Diffraction data sets were collected from crystals of native and selenomethionine-substituted TthDus to resolutions of 1.70 and 2.30 Å, respectively. These crystals belonged to space group P1. Preliminary X-­ray crystallographic analysis showed that two molecules of TthDus were contained in an asymmetric unit. In addition, diffraction data were collected to 3.51 Å resolution from a crystal of selenomethionine-substituted TthDus in complex with tRNA, which belonged to space group P41212. Preliminary structural analysis showed that the asymmetric unit contained two TthDus–tRNA complexes.
doi:10.1107/S1744309111012486
PMCID: PMC3107143  PMID: 21636912
dihydrouridine synthase; tRNA; flavin mononucleotide; Thermus thermophilus
18.  A C-terminal disulfide bond in the copper-containing amine oxidase from pea seedlings violates the twofold symmetry of the molecular dimer 
In a new crystal form of PSAO, the homodimeric molecules lack twofold symmetry and the C-termini of the two protomers are linked by a previously unrevealed disulfide bond.
The structure of a newly crystallized form of the copper-dependent amine oxidase from pea seedlings has been refined at a resolution of 2.2 Å to a final R factor of 0.181. The structure (form II) was originally discovered during a study of xenon binding to copper-dependent amine oxidases as a probe for dioxygen-binding sites [Duff et al. (2004 ▶), J. Mol. Biol. 344, 599–607]. The form II crystals belong to space group P21, with two dimers in the asymmetric unit. The overall structure is very similar to the crystals of form I in space group P212121 with a dimer in the asymmetric unit [Kumar et al. (1996 ▶), Structure, 4, 943–955]. In form I the last three residues (644–647) observable in the two subunits were apparently splayed apart. It was noted that the absence of a disulfide bond between the Cys647 residues of the two subunits was inconsistent with chemical evidence for the absence of free sulfhydryl groups. In both of the crystallographically independent dimers of form II the two subunits are clearly joined by a disulfide bridge between the C-terminal cysteine residues. This is only possible if the two polypeptide chains in the dimer adopt different conformations near the C-terminus so that the twofold symmetry is lost. A proline residue (645) two residues before the cysteine has a cis conformation in one chain and a trans conformation in the other. As a result, the disulfide bond lies more than 5 Å from the twofold axis. The loss of local twofold symmetry in form II can be explained by intermolecular contacts, which provide an asymmetric environment.
doi:10.1107/S1744309106043685
PMCID: PMC2225380  PMID: 17142890
copper amine oxidase; PSAO
19.  Mathematical aspects of molecular replacement. I. Algebraic properties of motion spaces 
Search spaces in the method of molecular replacement are shown to be coset spaces of the Lie group of rigid-body motions by the chiral space group of a crystal. The resulting ‘motion space’ can be endowed with a quasigroup operation that has interesting properties which are explored here.
Molecular replacement (MR) is a well established method for phasing of X-ray diffraction patterns for crystals composed of biological macromolecules of known chemical structure but unknown conformation. In MR, the starting point is known structural domains that are presumed to be similar in shape to those in the macromolecular structure which is to be determined. A search is then performed over positions and orientations of the known domains within a model of the crystallographic asymmetric unit so as to best match a computed diffraction pattern with experimental data. Unlike continuous rigid-body motions in Euclidean space and the discrete crystallographic space groups, the set of motions over which molecular replacement searches are performed does not form a group under the operation of composition, which is shown here to lack the associative property. However, the set of rigid-body motions in the asymmetric unit forms another mathematical structure called a quasigroup, which can be identified with right-coset spaces of the full group of rigid-body motions with respect to the chiral space group of the macromolecular crystal. The algebraic properties of this space of motions are articulated here.
doi:10.1107/S0108767311021003
PMCID: PMC3171898  PMID: 21844648
rigid-body motion; coset space; quasigroup; fundamental domain; molecular replacement
20.  Crystallization and X-ray data analysis of the 10 kDa C-terminal lid subdomain from Caenorhabditis elegans Hsp70 
Crystals of the C-terminal 10 kDa lid subdomain from the C. elegans chaperone Hsp70 have been obtained that diffract X-rays to ∼3.5 Å and belong to space group I212121. Analysis of X-ray data and initial heavy-atom phasing reveals 24 monomers in the asymmetric unit related by 432 non-crystallographic symmetry.
Hsp70 is an important molecular chaperone involved in the regulation of protein folding. Crystals of the C-terminal 10 kDa helical lid domain (residues 542–640) from a Caenorhabditis elegans Hsp70 homologue have been produced that diffract X-rays to ∼3.4 Å. Crystals belong to space group I212121, with unit-cell parameters a = b = 197, c = 200 Å. The Matthews coefficient, self-rotation function and Patterson map indicate 24 monomers in the asymmetric unit, showing non-crystallographic 432 symmetry. Molecular-replacement studies using the corresponding domain from rat, the only eukaryotic homologue with a known structure, failed and a mercury derivative was obtained. Preliminary MAD phasing using SHELXD and SHARP for location and refinement of the heavy-atom substructure and SOLOMON for density modification produced interpretable maps with a clear protein–solvent boundary. Further density-modification, model-building and refinement are currently under way.
doi:10.1107/S1744309106032064
PMCID: PMC2242859  PMID: 16946485
Hsp70; chaperone; C. elegans
21.  Crystallographic characterization of the radixin FERM domain bound to the cytoplasmic tails of adhesion molecules CD43 and PSGL-1 
The radixin FERM domain has been crystallized in complex with CD43 and PSGL-1 peptides. Diffraction data sets were collected from the complexes to 2.9 and 2.8 Å resolution, respectively.
Radixin is a member of the ERM proteins that cross-link plasma membranes and actin filaments. The FERM domains located in the N-terminal regions of ERM proteins are responsible for membrane association through direct interaction with the cytoplasmic tails of integral membrane proteins. Here, crystals of the radixin FERM domain bound to the cytoplasmic peptides of two adhesion molecules, CD43 and PSGL-1, have been obtained. Crystals of the radixin FERM domain bound to CD43 belong to space group P4322, with unit-cell parameters a = b = 68.72, c = 201.39 Å, and contain one complex in the crystallographic asymmetric unit. Crystals of the radixin FERM domain bound to PSGL-1 belong to space group P212121, with unit-cell parameters a = 80.74, b = 85.73, c = 117.75 Å, and contain two complexes in the crystallographic asymmetric unit. Intensity data sets were collected to a resolution of 2.9 Å for the FERM–CD43 complex and 2.8 Å for the FERM–PSGL-1 complex.
doi:10.1107/S1744309106054145
PMCID: PMC2330108  PMID: 17183174
ERM; CD43; PSGL-1
22.  Expression, purification, crystallization and preliminary X-ray diffraction analysis of nurse shark β2-microglobulin 
A crystal of nurse shark β2-microglobulin diffracted to 2.3 Å resolution and belonged to space group P3221, with unit-cell parameters a = b = 88.230, c = 67.146 Å and two molecules per asymmetric unit. The Matthews coefficient V M was calculated to be about 3.28 Å3 Da−1, corresponding to 62.5% solvent content.
β2-Microglobulin (β2m) is an essential subunit of the major histocompatibility complex (MHC) class I molecule that helps to stabilize the structure of peptide–MHC I (pMHC I). It is also one of the typical immunoglobulin superfamily (IgSF) molecules in the adaptive immune system (AIS). Sharks belong to the cartilaginous fish, which are the oldest jawed vertebrate ancestors with an AIS to exist in the world. Thus, the study of cartilaginous fish β2m would help in understanding the evolution of IgSF molecules. In order to demonstrate this, β2m from a cartilaginous fish, nurse shark (Ginglymostoma cirratum), was expressed, refolded, purified and crystallized. Diffraction data were collected to a resolution of 2.3 Å. The crystal belonged to space group P3221, with unit-cell parameters a = b = 88.230, c = 67.146 Å. The crystal structure contained two molecules in the asymmetric unit. The results will provide structural information for study of the evolution of β2m and IgSF in the AIS.
doi:10.1107/S1744309112006811
PMCID: PMC3325820  PMID: 22505420
nurse shark; β2-microglobulin
23.  The Crystallographic Structure of Panicum Mosaic Virus (PMV) 
Journal of structural biology  2012;181(1):37-52.
The structure of Panicum Mosaic Virus (PMV) was determined by X-ray diffraction analysis to 2.9 Å resolution. The crystals were of pseudo symmetry F23; the true crystallographic unit cell was of space group P21 with a=411.7 Å, b=403.9 Å and c=412.5 Å, with β=89.7°. The asymmetric unit was two entire T=3 virus particles, or 360 protein subunits. The structure was solved by conventional molecular replacement from two distant homologues, Cocksfoot Mottle Virus (CfMV) and Tobacco Necrosis Virus (TNV), of ~20% sequence identity followed by phase extension. The model was initially refined with exact icosahedral constraints and then with icosahedral restraints. The virus has Ca++ ions octahedrally coordinated by six aspartic acid residues on quasi threefold axes, which is completely different than for either CfMV or TNV. Amino terminal residues 1–53, 1–49 and 1-21 of the A, B and C subunits, respectively, and the four C-terminal residues (239-242) are not visible in electron density maps. The additional ordered residues of the C chain form a prominent “arm” that intertwines with symmetry equivalent “arms” at icosahedral threefold axes, as was seen in both CfMV and TNV. A 17 nucleotide hairpin segment of genomic RNA is icosahedrally ordered and bound at 60 equivalent sites at quasi twofold A–B subunit interfaces at the interior surface of the capsid. This segment of RNA may serve as a conformational switch for coat protein subunits, as has been proposed for similar RNA segments in other viruses.
doi:10.1016/j.jsb.2012.10.012
PMCID: PMC3525795  PMID: 23123270
Crystal structure; X-ray; icosahedral; NCS; tombusvirus; refinement; RNA; virus crystallography
24.  An ortho­rhom­bic polymorph of pyrazino­[2,3-f][1,10]phenanthroline-2,3-dicarbonitrile 
The title compound, C16H6N6, is a polymorph of the previously reported structure [Kozlov & Goldberg (2008 ▶). Acta Cryst. C64, o498–o501]. Unlike the previously reported monoclinic polymorph (space group P21/c, Z = 8), the title compound reveals ortho­rhom­bic symmetry (space group Pnma, Z = 4). The mol­ecule shows crystallographic mirror symmetry, while the previously reported structure exhibits two independent mol­ecules per asymmetric unit. In the title compound, adjacent mol­ecules are essentially parallel along the c axis and tend to be vertical along the b axis with dihedral angles of 72.02 (6)°. However, in the reported polymorph, the entire crystal structure shows an anti­parallel arrangement of adjacent columns related by inversion centers and the two independent mol­ecules are nearly parallel with a dihedral angle of 2.48 (6)°.
doi:10.1107/S1600536811047039
PMCID: PMC3238907  PMID: 22199760
25.  Preliminary X-ray crystallographic analysis of β-­carbonic anhydrase psCA3 from Pseudomonas aeruginosa  
Two crystal forms of β-carbonic anhydrase psCA3 from P. aeruginosa were grown. Crystal form A belonged to space group P212121, with unit-cell parameters a = 81.9, b = 84.9, c = 124.2 Å, and diffracted X-rays to 2.9 Å resolution; crystal form B belonged to space group P21212, with unit-cell parameters a = 69.9, b = 77.7, c = 88.5 Å, and diffracted X-rays to 3.0 Å resolution.
Pseudomonas aeruginosa is a Gram-negative bacterium that causes life-threatening infections in susceptible individuals and is resistant to most clinically available antimicrobials. Genomic and proteomic studies have identified three genes, pa0102, pa2053 and pa4676, in P. aeruginosa PAO1 encoding three functional β-carbonic anhydrases (β-CAs): psCA1, psCA2 and psCA3, respectively. These β-CAs could serve as novel antimicrobial drug targets for this pathogen. X-ray crystallographic structural studies have been initiated to characterize the structure and function of these proteins. This communication describes the production of two crystal forms (A and B) of β-CA psCA3. Form A diffracted to a resolution of 2.9 Å; it belonged to space group P212121, with unit-cell parameters a = 81.9, b = 84.9, c = 124.2 Å, and had a calculated Matthews coefficient of 2.23 Å3 Da−1 assuming four molecules in the crystallographic asymmetric unit. Form B diffracted to a resolution of 3.0 Å; it belonged to space group P21212, with unit-cell parameters a = 69.9, b = 77.7, c = 88.5 Å, and had a calculated Matthews coefficient of 2.48 Å3 Da−1 assuming two molecules in the crystallographic asymmetric unit. Preliminary molecular-replacement solutions have been determined with the PHENIX AutoMR wizard and refinement of both crystal forms is currently in progress.
doi:10.1107/S1744309113017594
PMCID: PMC3729167  PMID: 23908036
carbonic anhydrases; β-class carbonic anhydrases; Pseudomonas aeruginosa

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