Introduction to phasing.
When collecting X-ray diffraction data from a crystal, we measure the intensities of the diffracted waves scattered from a series of planes that we can imagine slicing through the crystal in all directions. From these intensities we derive the amplitudes of the scattered waves, but in the experiment we lose the phase information; that is, how we offset these waves when we add them together to reconstruct an image of our molecule. This is generally known as the 'phase problem'. We can only derive the phases from some knowledge of the molecular structure. In small-molecule crystallography, some basic assumptions about atomicity give rise to relationships between the amplitudes from which phase information can be extracted. In protein crystallography, these ab initio methods can only be used in the rare cases in which there are data to at least 1.2 A resolution. For the majority of cases in protein crystallography phases are derived either by using the atomic coordinates of a structurally similar protein (molecular replacement) or by finding the positions of heavy atoms that are intrinsic to the protein or that have been added (methods such as MIR, MIRAS, SIR, SIRAS, MAD, SAD or combinations of these). The pioneering work of Perutz, Kendrew, Blow, Crick and others developed the methods of isomorphous replacement: adding electron-dense atoms to the protein without disturbing the protein structure. Nowadays, methods from small-molecule crystallography can be used to find the heavy-atom substructure and the phases for the whole protein can be bootstrapped from this prior knowledge. More recently, improved X-ray sources, detectors and software have led to the routine use of anomalous scattering to obtain phase information from either incorporated selenium or intrinsic sulfurs. In the best cases, only a single set of X-ray data (SAD) is required to provide the positions of the anomalous scatters, which together with density-modification procedures can reveal the structure of the complete protein.
Taylor
Garry L
GL
Centre for Biomolecular Sciences, University of St Andrews, St Andrews, Fife KY16 9ST, Scotland. glt2@st-andrews.ac.uk
D003198:Computer Simulation; D018360:Crystallography, X-Ray / Q000379:methods*; D008958:Models, Molecular; D011336:Probability; D017434:Protein Structure, Tertiary; D011506:Proteins / Q000032:analysis* / Q000737:chemistry
D011506:Proteins
Acta Crystallogr D Biol Crystallogr. 2003 Mar;59(Pt 3):615-7
12595742
Acta Crystallogr D Biol Crystallogr. 2007 Oct;63(Pt 10):1069-74
17881824
Acta Crystallogr D Biol Crystallogr. 2005 Jul;61(Pt 7):960-6
15983419
Nat Protoc. 2008;3(7):1171-9
18600222
Acta Crystallogr D Biol Crystallogr. 2000 Sep;56(Pt 9):1137-47
10957632
Acta Crystallogr D Biol Crystallogr. 2003 Oct;59(Pt 10):1801-8
14501121
Acta Crystallogr D Biol Crystallogr. 1996 Jan 1;52(Pt 1):30-42
15299723
Nature. 1958 Mar 8;181(4610):662-6
13517261
Acta Crystallogr D Biol Crystallogr. 2003 Nov;59(Pt 11):1966-73
14573951
Nat Struct Biol. 2000 Nov;7(11):1068-74
11062565
Nature. 1981 Mar 12;290(5802):107-113
28769131
Acta Crystallogr D Biol Crystallogr. 2003 Nov;59(Pt 11):1943-57
14573949
J Mol Biol. 2009 Feb 27;386(3):828-40
19166860
J Mol Biol. 1999 May 28;289(1):83-92
10339407
Acta Crystallogr D Biol Crystallogr. 2005 Nov;61(Pt 11):1533-40
16239732
Acta Crystallogr D Biol Crystallogr. 1998 Sep 1;54(Pt 5):905-21
9757107
Acta Crystallogr D Biol Crystallogr. 2007 Mar;63(Pt 3):366-80
17327674
Methods Enzymol. 1997;276:307-26
27754618
Acta Crystallogr D Biol Crystallogr. 2002 Mar;58(Pt 3):494-506
11856836
Acta Crystallogr D Biol Crystallogr. 2004 Jan;60(Pt 1):22-7
14684888
Proc Natl Acad Sci U S A. 1993 Nov 1;90(21):9852-6
8234325
Acta Crystallogr D Biol Crystallogr. 2002 Nov;58(Pt 11):1937-40
12393925
Acta Crystallogr D Biol Crystallogr. 2008 Jan;64(Pt 1):49-60
18094467
Methods Enzymol. 1997;276:472-494
27799110
Acta Crystallogr D Biol Crystallogr. 2002 Nov;58(Pt 11):1948-54
12393927
Acta Crystallogr D Biol Crystallogr. 2003 Nov;59(Pt 11):1958-65
14573950
J Appl Crystallogr. 2007 Aug 1;40(Pt 4):658-674
19461840
Methods Enzymol. 1985;115:90-112
4079800
Prog Biophys Mol Biol. 1999;72(3):245-70
10581970
Acta Crystallogr A. 2008 Jan;64(Pt 1):112-22
18156677