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M A C R O M O L E C U L A R C RY S T A L L O G R A P H Y

88 IA VIIA VIIIA

1 1 2

H IIA IIIA IVA VA VIA H He

3 4 5 6 7 8 9 10

Li Be B C N 0 F Ne

11 12 VIII 13 14 15 16 17 18

Na Mg IIIB IVB VB VIB VIIB IB IIB Al Si P S Cl Ar

19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36

K Ca Sc Ti V Cr Mn Fe Ni Cu Zn Ga Ge As Br

Co Se Kr

37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54

Rb Y Zr Nb Tc In Sb Te

Rh Pd Ag Cd Xe

Sr Mo Ru Sn I

55 56 57 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86

Cs Hf Bi Po At Rn

#La Ta W Re Os Ir Pt Au Hg TI Pb

Ba

87 88 89 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118

Fr Ra *Ac Rf Db Sg Bh Hs Mt Ds Rg Uub Uut Uuq Uup Uuh Uus Uuc

58 59 60 61 62 63 64 65 66 67 68 69 70 71

#Lanthanides Pm Lu

Ce Pr Nd Eu Gd Tb Dy Ho Er Tm Yb

Sm

90 91 92 93 94 95 96 97 98 99 100 101 102 103

*Actinides Pa Np Pu Am Cm Bk Cf Es Fm Md No Lr

U

Th

The Periodic table showing the elements used successfully as heavy-atom derivatives in bold and underlined. The rest of the

Figure 6.1

elements are shown only for completeness. Thus, each measured intensity

the soaking process causes the crystal to become can be reduced

(I )

hkl

non-isomorphous. to structure factor amplitude with unknown

(F )

hkl

phases, where is proportional to the square root

F

hkl

of . Each structure factor amplitude and its

I (F )

hkl hkl

6.2 Theoretical basis associate phase can be described in terms of a

(α )

hkl

vector quantity, the structure factor For every

(F ).

hkl

Originally, isomorphous replacement phasing of hkl, native and derivative structure factors (Fig. 6.2)

biological macromolecules requires the measure- are related as shown in Eq. 1:

ment of at least three X-ray diffraction data sets,

a native and two or more derivatives. Therefore, (1)

F F F

= +

PH P H

the method was more commonly referred to as where , , and are the structure factors of the

F F F

the method of multiple isomorphous replacement PH P H

derivative, the native protein, and the heavy atom,

(MIR). However, the introduction of area detectors respectively.

and synchrotron radiation allowed for significant Once the heavy-atom position has been deter-

improvements in data quality and the ability to use mined, its structure factor amplitude and phase

only a single isomorphous derivative if its heavy F

H

can be calculated. Since the structure factor

atom is an anomalous scatterer. The latter is referred α H

amplitudes for the native and derivative

to as single isomorphous replacement with anoma- (F ) (F )

P PH

are experimentally measured quantities, it is thus

lous scattering (SIRAS), where the anomalous data possible to calculate the protein phase angle from

is used to break the phase ambiguity. α

P

the following equations:

The theoretical basis of isomorphous replacement

can be found in Blundell and Johnson (1976), Drenth 2

2

2 2F cos(α (2)

F

F

F

F )

+ −

+

= α

P H P H

H

P

PH

(1999), and was recently summarized by Taylor

(2003). Here, I will only give a brief overview. As or

indicated above, an X-ray diffraction experiment

only gives us intensities of waves scattered from 2 2 2

−1

cos F F F

)/2F )]

= + [(F − −

α α

P H PH P H P H

planes (hkl) in the crystal, but the phase shift associ- ′ (3)

= ±

α α

H

ated with each hkl is lost during data measurement. ISOMORPHOUS REPLACEMENT 89

(a)

Imaginary Imaginary

F F

PH H F

PH

F F

P P

α Pa

α P F Real

α

H Pb

F

Real P

F PH

(b) Imaginary

Vector (Argand) diagram showing the relationships

Figure 6.2

between heavy-atom derivative native protein and heavy

(F ), (F )

PH P

atom is the phase angle for the native protein. The vectors

(F ); α

H P

are plotted in the complex plane. F

P

α

P

From Eq. 3 and Fig. 6.3a it is clear that with F Real

H1 F

only one heavy-atom derivative (single isomor- H2

phous replacement; SIR) the resultant phase will

have two values (α and ); one of these phases

α

Pa Pb

will represent that of one structure and the other of

its mirror image. But, since proteins contain only

acids, this phase ambiguity must be elim-

l-amino

inated using a second derivative, the anomalous

component of the heavy atom or by solvent level- Isomorphous replacement phase determination (Harker

Figure 6.3

ling (Wang, 1985), as shown diagrammatically in construction). (a) Single isomorphous replacement. The circle with

Fig. 6.3b. radius represents the heavy-atom derivative, while that with

F

PH

Once the phase angle has been determined for radius represents the native protein. Note that the circles intersect

F

α P P

at two points causing an ambiguity in the phase angle; and

every hkl, a Fourier synthesis is used to compute the α α

Pa Pb

represent the two possible values. (b) Double isomorphous

electron density at each position (xyz) in the unit

(ρ) replacement. The same construction as that in single isomorphous

cell (the repeating unit forming the crystal lattice) (vector

replacement except that an additional circle with radius F

PH2

using Eq. 4: not shown for simplicity) has been added to represent a second

heavy-atom derivative. Note that all three circles (in the absence of

errors) intersect at one point thus eliminating the ambiguity in the

−2πi(hx+ky+lz)

1/V (4)

F

(hkl)e

=

ρ(xyz) P

h k l protein phase angle . and represent the heavy-atom

F F

α

P H1 H2

vectors for their respective derivatives.

i

where V is the volume of the unit cell, is the

imaginary component -1, and The electron density map can then be

(ρ(xyz))

P

F F cos

F (hkl) (hkl)e (hkl) (hkl)

= = α

P P P P interpreted in terms of a three-dimensional atomic

iF sin (5)

(hkl) (hkl)

+ α

P P model.

90 M A C R O M O L E C U L A R C RY S T A L L O G R A P H Y

6.3 Selection of heavy-atom reagents with proteins in a similar fashion, except for those

2−

containing (Pt(CN) which bind to positively

)

2

Both the size and chemical composition of the charged residues. Mercurial compounds are the sec-

molecule under investigation are important criteria ond most successful group of reagents in derivative

to consider when selecting heavy-atom reagents for preparation; most mercurials either bind to cysteine

derivatization. Ones choice must insure that the sulphurs or histidine nitrogens. In addition to plat-

differences in diffraction amplitudes due to heavy- inum and mercury, Fig. 6.1 show the many elements

atom contributions are larger than the errors in successfully used in isomorphous replacement

data measurement. The size of the heavy atom phasing.

(atomic number) and the number of sites required

for successful phasing are proportional to the size

(molecular weight) of the macromolecule. Larger 6.4 Heavy atoms and their ligands

macromolecules may require not only atoms of high

atomic number, but also more than one heavy atom The preparation of heavy-atom derivatives and

per molecule. For example the structure determina- selection of reagents have been extensively reviewed

tion of the ribosome required heavy-atom clusters (Abdel-Meguid, 1996; Blundell and Johnson, 1976;

+2 et al.,

such as Ta Br (Ban 2000). Therefore, when

6 12 et al.,

Petsko, 1985; Kim 1985; Holbrook and Kim,

studying larger macromolecules it may be useful 1985; Garman and Murray, 2003). Historically, heavy

to calculate the magnitude of the change in the atoms have been grouped into class A and class

diffraction signal before deciding which heavy atom B elements based on their ligand preference. Class

to try. Crick and Magdoff (1956) showed that the A elements prefer ‘hard’ ligands such as carboxy-

average fractional intensity change for acen-

(∆I/I) lates and other oxygen containing groups. These

tric reflections can be estimated from the following ligands are electronegative and form electrostatic

equation: interactions with the derivatives; they include the

carboxylates of aspartic and glutamic acids and the

1/2 (6)

∆I/I (2N /N ) (Z /Z )

= H P H P hydroxyl of serine and threonine. On the other hand,

N N class B elements prefer ‘soft’ ligands such are those

where and are the number of heavy atoms

H P Z containing sulphur, nitrogen, and halides. These

and non-hydrogen protein atoms, respectively; H include methionine, cysteine, and histidine. The lat-

is the atomic number of the heavy atom and

Z ter two amino acids are the most reactive amongst

is 6.7 (the average atomic number of protein

P all 20 naturally occurring amino acid residues. The

atoms). cysteine’s sulphur is an excellent nucleophile; it will

In the case of small proteins, inspection of the react irreversibly with mercuric ions and organomer-

amino acid composition can give valuable insights curials at wide range of pHs. The thiolate anion

into which reagents should be tried first. For exam- also forms stable complexes with class B metals,

ple if the protein contains no free cysteines or but since cysteines are almost totally protonated at

histidines it may be best to start soaking with com- pH 6 or below, this reaction is more sensitive to

pounds other than mercurials, or to genetically pH than that with mercurials. The imidizole side

engineer heavy-atom binding sites as was done chain of histidine is also highly reactive, particu-

with the catalytic domain of resolvase (Abdel-

γδ

et al., et al., larly above pH 6 where it is unprotonated. It reacts

Meguid 1984; Hatfull 1989). However, well with reagents containing platinum, mercury,

assuming a normal distribution of amino acids, one and gold.

should begin with platinum compounds such as In addition to the amino acids described above,

K PtC1 (the most widely successful heavy-atom

2 4 several other amino acid residues are also reac-

reagent), which binds mainly to methionine, his-

et al. tive toward compounds containing heavy atoms.

tidine, and cysteine residues. Petsko (1978) These are the side chains of arginine, asparagine,

have described the chemistry of this reagent in a glutamine, lysine, tryptophan, and tyrosine. Those

variety of crystal mother liquors. They also con- that are not reactive are alanine, glycine, isoleucine,

cluded that most other platinum compounds react 91

ISOMORPHOUS REPLACEMENT

Cationic: This group includes heavy-atom

leucine, phenylalanine, proline, and valine. Argi- reagents that predominantly bind to acidic regions

nine forms electrostatic interactions with anionic lig- on the protein through their overall positive

ands, while asparagine and glutamine may weakly 2+ 3+

charge, such as , , and

coordinate to simple metal complexes through their (Pt(NH ) ) (Ir(NH ) )

3 4 3 6

2+ .

side chain’s amide nitrogen. Like arginine, lysine (Hg(NH ) )

3 2

Hydrophobic: This group includes the nobel gases

can form electrostatic interactions with anionic lig-

ands at pHs below its pKa. Near or above its pKa krypton and xenon, which bind to hydrophobic

pockets in the protein. The main impediment to

of about 9, it can also react with platinum and

gold complexes. The indole ring nitrogen of trypto- the use of these gases has been the technical chal-

phan can be mercurated, but tryptophan is usually lenge in derivatization under pressure, particu-

buried in the protein and rarely accessible to sol- larly since pressurized capillaries of glass or quartz

vent. While the phenol hydroxyl oxygen of tyrosine are explosion hazards. A special device to make

is expected to be a good nucleophile, its pKa is nobel gas derivatives has been described by Schiltz

et al.

greater than 10. Thus, the tyrosine utility as a lig- (1994), and a commercial one is now being

sold by Molecular Structure Corporation for use in

and for heavy atoms has been in the substitution of cryocrystallography.

the phenolate hydroxyl with iodine (Sigler, 1970). Others:

In addition to the side chains of amino acids, the This includes iodine that can be used to

N-terminal amine and the C-terminal carboxylate mono- or di-iodinate tyrosine residues.

of a protein are potentially ligands for heavy-atom

derivatives. 6.5 Preparation of heavy-atom

Dr Bart Hazes (University of Alberta) has fur- derivatives

ther grouped heavy-atom derivatives in six different

categories as follow: Macromolecular crystals grow in an equilibrium

state with their mother liquor. Disrupting this equi-

Class A: This class consists of the alkaline earth librium can often destroy the crystals or their ability

2+ 2+ 3+

metals (Sr and Ba ), the lanthanides (La , to diffract X-rays. This situation can be exacerbated

3+ 3+ 3+

3+ 3+ 3+ 3+ 3+

Ce , Pr , Nd , Sm , Eu , Gd , Tb , Dy , by the transfer of the crystal to a solution contain-

3+

3+ 3+ 3+

Ho , Er , Tm , and Yb ) and the actinide ing a heavy atom. Therefore, it is important, once

2+ . As indicated above, these elements prefer

(UO )

2 crystals are removed from their sealed environment,

carboxylates and other oxygen containing ligands. to first transfer them to a stabilizing solution and

They withstand low pH and ammonium sulphate, let them re-equilibrate before further transfer to the

but they have lower solubility at higher pH and in heavy atom solution. Usually, a stabilizing solution

the presence of phosphates. is identical to the mother liquor in which the crys-

Class B: As described above, this group con- tal was grown, but with a higher concentration of

tains many of the most popular heavy-atom deriva- precipitant.

tives containing mercury and platinum, such as The mechanics of derivative preparation is

p-chloromercuribenzoate, HgC1 , mercuric acetate, simple; it involves the transfer of one or more

2

ethylmercury chloride, K PtC1 , K Pt(NO , and

)

2 4 2 2 4 native crystals from the stabilizing solution to a

K PtC1 . In general, these reagents prefer ligands

2 6 solution differing only in the presence of a com-

containing sulphur and nitrogen such as cysteine pound containing the desired heavy atom. However,

and histidine. This group also consists of many before attempting to prepare derivatives, it is impor-

silver, gold, palladium, iridium, osmium, and cad- tant to recognize that heavy-atom reagents are very

mium containing reagents. toxic and must be handled with utmost care. These

Anionic: This group includes heavy-atom reagents reagents are selected for their strong affinity for

that predominantly binds to basic regions on the biological molecules. Thus, they present real and

protein through their overall negative charge, such serious danger to their users. Once crystals have

2− 3−

as iodide, (HgI , , , and

) (Pt(CN) ) (IrC1 )

4 6 been transferred to the heavy-atom solution, they

3

− .

(Au(CN) )

2 can be soaked in that solution for a period of time.


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DETTAGLI
Corso di laurea: Corso di laurea in scienze geologiche
SSD:
Università: Pisa - Unipi
A.A.: 2011-2012

I contenuti di questa pagina costituiscono rielaborazioni personali del Publisher Atreyu di informazioni apprese con la frequenza delle lezioni di Cristallografia e studio autonomo di eventuali libri di riferimento in preparazione dell'esame finale o della tesi. Non devono intendersi come materiale ufficiale dell'università Pisa - Unipi o del prof Bonaccorsi Elena.

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