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F I R S T A N A LY S I S O F M A C R O M O L E C U L A R C RY S T A L S 67

Cold gaseous

nitrogen stream

(a) (b)

(t = ⫺180˚C) Shock-cooled

crystal

(in propane) (e)

Goniometer head

assembled on X-ray

camera

(c) (d)

Procedure for transfer of shock-cooled macromolecular crystals to the X-ray camera. (a) The goniometer head is assembled on the

Figure 4.5

X-ray camera and the cold gaseous nitrogen stream is centred on the eucentric point of the camera. (b) For samples prepared with propane or

ethane, a cryovial containing a crystal shock-cooled in propane is selected and allowed to partially thaw (5–10 s). Once the external layer of

propane has thawed, a pair of forceps is used to lift the loop-mounted crystal, still encased in solid propane, out of the cryovial and placed on the

goniometer head. The solid propane is allowed to thaw completely to liquid, which falls away from the crystal. Excess liquid should be collected

and disposed of properly. (c) For samples prepared with liquid nitrogen, a cryovial is selected and placed in a bath of liquid nitrogen (held in a

shallow Dewar flask). The pin containing the loop-mounted crystal is removed from the cryovial and captured with the magnetic crystal wand as

shown above. The mounting pin (held by the magnetic crystal wand) is manoeuvred into the head of the cryotong. (d) With the cryotongs in its

locked position, the crystal is lifted out of the liquid nitrogen bath and the mounting pin positioned on the magnetic mount held on the goniometer

head. (e) The cryotongs are quickly unlocked to expose the crystal to the nitrogen stream.

A crystal lattice. Circles represent lattice points; heavy lines represent a unit cell.

Figure 4.6

68 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

Protocol 4.6 Recovery of shock-cooled crystals from the X-ray diffraction camera

(Protocols 4.4 and 4.5). Cool the cryotongs, cryovial, and

Materials vial clamp immersion in liquid nitrogen.

Cryotongs The cold cryotongs are used to rapidly capture the

2.

Shallow Dewar flask mounting pin/loop. This step should be carried out rapidly

Cane so that the crystal leaves the cold stream and enters the

Cryovial protected (cold) space within the cryotongs in a minimum

Vial clamp amount of time.

Storage Dewar The cryotongs (with enclosed crystal) is rapidly lifted off

3.

Liquid nitrogen the goniometer head and plunged into a liquid nitrogen

Gloves bath.

Face shield The magnetic crystal wand is used to capture the

4.

mounting pin.

While immersed in liquid nitrogen, the mounting pin

5.

(attached to crystal wand) is manoeuvred into a cryovial

Procedure (which is held with the cooled vial-clamp).

Recovery of crystals from the cold stream involves

1. The recovered crystal (in the cryovial) is secured to a

performing the procedure described for the mounting of 6.

cryocane, which is stored in a liquid nitrogen storage Dewar.

crystals to X-ray camera but with the steps reversed

Protocol 4.7 Storage and transport of macromolecular crystals

Dewar. Allow the ‘dry shipping’ Dewar to stand for several

Materials minutes upside down until it is completely empty of liquid

‘Dry Shipping’ Dewar (Taylor Wharton: Cryo Express and (commercial shipping companies will refuse to ship a

Cryo Flight) container that contains liquid).

Hard case Once dry, cryocanes containing cryocooled crystals are

4.

Insulated gloves transferred from the liquid nitrogen bath to the shipping

Face shield Dewar. Samples prepared with liquid propane or liquid

nitrogen are handled in the same manner. The temperature

inside the shipping Dewar should maintain the propane in

Procedure the cryovials in solid form.

A ‘dry shipping’ Dewar (at ambient temperature) is

1.

charged by filling it slowly with liquid nitrogen. Consult the Many commercial shipping companies will accept a properly

product literature for specific details. prepared shipping Dewar containers for transport around

Once filling is complete, the absorbent is thoroughly

2. the world. Each investigator should check with the shipping

cooled by the liquid nitrogen (approx 7–8 h). This will company of choice about requirements, paperwork,

require topping up of the Dewar several times. A properly and cost.

charged Dewar should be able to stably maintain liquid

nitrogen on standing.

In preparation for shipping, liquid nitrogen from the ‘dry

3.

shipping’ Dewar is decanted into an appropriate storage restrictions on its lattice constants, while the cubic

minimum symmetry, relevant to macromolecules, of system has four three-fold axes along the diagonal

the unit cell for each of the seven crystal systems and of the cube, three equal lattice lengths, and three lat-

the restrictions imposed on unit cell lattice constants. tice angles equal to 90 . The unit cell in each crystal

For example the triclinic system has no symmetry or ◦

F I R S T A N A LY S I S O F M A C R O M O L E C U L A R C RY S T A L S 69

system is chosen to contain at least one lattice point. (I; German for Innenzentrierte), where a unit cell

A unit cell that has only one lattice point at each of contains a second lattice point at the centre of the cell,

its corners is called primitive (P). Note that each cor- face-centred (F) having an additional lattice point

C-centred

ner of a unit cell in a crystal is shared by eight other at the centre of each of its six faces and

unit cells; therefore, a primitive cell contains only (C) having an additional lattice point at only one

ab

one lattice point. However, at times it is advanta- of the six faces (by definition the face). Centring

C-

geous to select a unit cell that contains more than expands the volume of the unit cell. Whereas

I-centring

one lattice point. These cells are called centred cells; and double the volume relative to that

P F-centring

there are three such cells. They are body-centred the cell, quadruples the volume. When

centring is included, the number of unique lattices

expands to 14 (Table 4.3 and Fig. 4.8). These 14 lat-

tices are known as the Bravais lattices; they were

first described by Frankheimer and Bravais in the

mid-nineteenth century.

A unique combination of crystal symmetry

c elements and centring is called a space group.

α β

γ There are only 230 possible space groups. However,

b this number is reduced to 65 for biological macro-

a molecules because the chirality of their biological

building blocks. The 65 biologically relevant space

A unit cell, showing the six parameters known as lattice

Figure 4.7

constants. groups are listed in Table 4.4.

Elements of crystal symmetry

Table 4.2

Symmetry element Description

Rotation axes Counterclockwise rotation of 360 about an axis, where is 1, 2, 3, 4 or 6

◦ /n n

2-fold axis is rotation by 180

3-fold axis is rotation by 120

4-fold axis is rotation by 90

6-fold axis is rotation by 60

Screw axes Same as rotation axis, but followed by a translation of along the rotation axis,

p/n

where is an integer <n

p

2 screw axis is rotation by 180 followed by translation of 1/2 of a unit cell

1

3 screw axis is rotation by 120 followed by translation of 1/3 of a unit cell

1

3 screw axis is rotation by 120 followed by translation of 2/3 of a unit cell

2

4 screw axis is rotation by 90 followed by translation of 1/4 of a unit cell

1

4 screw axis is rotation by 90 followed by translation of 1/2 of a unit cell

2

4 screw axis is rotation by 90 followed by translation of 3/4 of a unit cell

3

6 screw axis is rotation by 60 followed by translation of 1/6 of a unit cell

1

6 screw axis is rotation by 60 followed by translation of 1/3 of a unit cell

2

6 screw axis is rotation by 60 followed by translation of 1/2 of a unit cell

3

6 screw axis is rotation by 60 followed by translation of 2/3 of a unit cell

4

6 screw axis is rotation by 60 followed by translation of 5/6 of a unit cell

5

Inversion centre All points inverted through a centre of symmetry

Mirror plane Reflection through a plane

Glide plane Same as mirror plan, but followed by a translation of half the unit cell parallel to the plane; glide planes

are not relevant in macromolecular crystallography due to the chirality of the biological building blocks

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

70

Table 4.3 The seven crystal systems

System Bravais lattices Minimum symmetry of unit cell Restriction on lattice constants

Triclinic No symmetry ;

P a b c α β γ

= = = = ◦ ◦

Monoclinic , One 2-fold axis, parallel to ; 90 ; 90

P C b a b c α γ β

= = = = >

Orthorhombic Three mutually perpendicular 2-fold axes ; 90

P, C, I, F a b c α β γ

= = = = = ◦

Tetragonal , One 4-fold axis, parallel to ; 90

P I c a b c α β γ

= = = = =

◦ ◦

Trigonal/ One 3-fold axis, parallel to ; 90 ; 120

P c a b c α β γ

= = = = =

a ◦

rhombohedral ; 90

(or R) a b c α β γ

= = = = =

◦ ◦

Hexagonal One 6-fold axis, parallel to ; 90 ; 120

P c a b c α β γ

= = = = =

Cubic , Four 3-fold axes along the diagonal of ; 90

P I, F a b c α β γ

= = = = =

the cube

a Rhomobohedral is a subset of the trigonal system in which the unit cell can be chosen on either hexagonal or rhombohedral axes.

4.7 Lattice and space-group

determination from X-ray data

In the past, the traditional way of determining space-

Triclinic groups and cell dimensions was by analysing X-ray

precession photographs of the undistorted lattice for

P absences and measuring spot separations in order to

et al.,

determine lattice dimensions (Abdel-Meguid

1996). Nowadays, oscillation data collection is usu-

Monoclinic ally started on a cryocooled crystal mounted in a

loop at an undefined orientation and the space group

C

P determined ‘on the fly’ after 10–20 frames have been

collected.

Two processing packages are predominantly used

Orthorhombic by crystallographers in house and at synchrotrons

indexing of oscillation data. HKL2000 (and its pre-

decessor DENZO) written by Zbyszek Otwinowski

I

F

P C and Wladek Minor (Otwinowski and Minor, 1997

and 2001) and MOSFLM supported by CCP4 (Leslie,

Tetragonal 1993). Both have very powerful autoindexing rou-

tines. The code for that within HKL (Otwinowski

and Minor, 1997) is yet to be fully disclosed as it is a

I

P commercial package and MOSFLM has within it the

powerful algorithm DPS (open source) written by

Michael Rossman and Cees van Beek which uses a

similar method of Fourier indexing (Rossmann and

R

Trigonal P

Hexagonal van Beek, 1999; Powell, 1999; Rossmann, 2001) to

that of HKL. Both algorithms are extremely power-

Cubic ful but in the author’s experience they do not always

give the same result with difficult space-group deter-

I

F

P minations and it is often useful to run both when

initially indexing a crystal.

Figure 4.8 The 14 Bravais lattices. Black circles represent atoms or Other good processing packages are d*TREK

molecules. cells contain only one lattice point, while - and

P C incorporated in CrystalClear, written by Jim

cells contain two and -centred cells contain four.

I-centred F F I R S T A N A LY S I S O F M A C R O M O L E C U L A R C RY S T A L S 71

The 65 space groups that are possible for macromolecular crystals

Table 4.4 a b

Crystal system Diffraction symmetry Space groups

Triclinic 1̄ P1

Monoclinic 2/m P2, P2 , C2

1

Orthorhombic P222, P222 , P2 2 2, P2 2 2 , C222, C222 , F222, [I222,

mmm 1 1 1 1 1 1 1

I2 2 2 ]

1 1 1

Tetragonal 4/m P4, (P4 , P4 P4 , I4, I4

),

1 3 2 1

4/mmm P422, (P4 22, P4 22), P4 22, P42 2, (P4 2 2, P4 2 2),

1 3 2 1 1 1 3 1

P4 2 2, I422, I4 22

2 1 1

Trigonal 3̄ P3, (P3 , P3 R3

),

1 2

3̄m [P321, P312], [(P3 21, P3 21), (P3 12, P3 12)], R32

1 2 1 2

Hexagonal 6/m P6, (P6 , P6 (P6 , P6 P6

), ),

1 5 2 4 3

6/mmm P622, (P6 22, P6 22), (P6 22, P6 22), P6 22

1 5 2 4 3

Cubic P23, P2 3, F23, [I23, I2 3]

m3 1 1

P432, (P4 32, P4 32), P4 32, F432, F4 32, I432, I4 32

m3m 1 3 2 1 1

a The overbar indicates an inversion axis, while represents an mirror plane.

m

b Space groups in brackets and parentheses are indistinguishable from diffraction patterns. Those in parentheses are enantiomorphs.

which are hexagonal and tetragonal are easily recog-

Pflugrath (Pflugrath, 1997, 1999), which is marketed nizable from their external morphology and cubic

with MSC X-ray detectors (this program evolved crystals may be identified from their lack of polar-

from MADNESS) and XDS written by Wolfgang ization. Other rectangular habits turn out often to

Kabsch and incorporating the IDXREF autoindex- be either monoclinic or orthorhombic. Coming at

ing algorithm (Kabsch, 1988a, 1988b, 1993a, 1993b), an indexing problem armed with this morphological

which starts by calculating vectors between reflec- information is very helpful.

tions with low indices and building up to full

data indexing. Otwinowski and Minor have writ- 2. Collect a wedge of data (say 10 frames) and also

◦ ◦

ten the commercial, macromolecular autoindexing collect a frame at 45 and 90 away from the start-

routines within the PROTEUM which supports data ing oscillation position (for a crystal say whose habit

collection of Bruker detectors. appears to have faces at 90 to each other and whose

ELVES has been developed as an expert system, by space group could be monoclinic, orthorhombic, or

James Holton and Tom Alber, to go from data collec- cubic). Collecting frames away from the starting

tion frames to structure without human intervention oscillation position can save considerable time col-

and will obviate the need for intermediate space- lecting worthless data if these frames are found to

group determination described above. Very recently, be have pathologies such as very high mosaicity

12 different European sites have been collaborat- or splitting as they will be probably encountered

ing to develop a software package known as DNA later in a full data collection when the full oscillation

(automateD collectioN of datA) for the automatic range is swung through.

collection and indexing of macromolecular diffrac- 3. Make sure you have an accurate values for the

tion data. Further information is available at the web direct-beam position on the detector you are using

site www.dna.ac.uk. and the crystal-film distance. If these have been

recorded from a previous successful data collection

and processing, time can be saved by having them

4.7.1 Starting out – preliminary data as starting parameters for indexing. At many syn-

collection and indexing chrotrons, the prerecording of a wax ring will give

1. Look at the crystals carefully under a dissecting an accurate crystal to detector distance, sometimes

microscope equipped with polarizers. Often crystals the values displayed on the LED may be fallible.

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

72 Graphical display of the autoindexing of TRPV ankyrin diffraction data using MOSFLM.

Figure 4.9 Graphical display of the autoindexing of TRPV ankyrin diffraction data using MOSFLM with the calculated spot predictions

Figure 4.10

superimposed. F I R S T A N A LY S I S O F M A C R O M O L E C U L A R C RY S T A L S 73

Graphical display of the autoindexing of TRPV ankyrin diffraction data using DENZO.

Figure 4.11 Graphical display of the autoindexing of TRPV ankyrin diffraction data using DENZO with the calculated spot predictions

Figure 4.12

superimposed.


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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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