Resolution and data formats. Andrea Thorn

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1 Resolution and data formats Andrea Thorn

2 RESOLUTION

3 Motivation Courtesy of M. Sawaya

4 Map resolution

5 Data quality indicators Resolution accounts for the amount of data available in data/parameter ratio. How to decide at which resolution to cut the data? What is the actual resolution of a data set? Are these two the same?

6 QUALITY INDICATORS

7 <I/σ(I)> <I/σ(I)> <I/σ(I)>* in the highest resolution shell signal to noise level at cut off most popular cut off values: 2 or 3 much lower nowadays * Scalepack may give <I>/<σ(I)> instead.

8 R merge R merge (a.k.a. R sym, R linear ) widely used the higher the multiplicity, the higher R merge If I hkl and I h k l regarded independent, unique reflections: R merge lower.

9 Redundancy independent merging R factor R rim = R meas gives precision of intensity measurements independent of multiplicity (how broad distribution?) low resolution data qualityboth R meas and R merge measure the uncertainty before data are merged.

10 Precision indicating merging R factor R pim precision of the averaged intensity measurements = standard error of the mean (How precisely the center of the distribution is known). Like R meas needs to be calculated before symmetry equivalents are merged.

11 CC 1/2 CC 1/2 data set is randomly split in half Pearson s correlation coefficient is determined: Results from Student's t test can indicate where statistical significance ends (as a rule of thumb data are usually insignificant if CC 1/2 < 0.15 or lower).

12 Data quality indicators Importance of data cut off is overstated. Employed data cut off does not necessarily indicate the map quality. It is also bad practice to cut resolution after refinement to improve refinement R values. A general shortcoming of R values is that they are not statistically established as, for example, CC.

14 Literature Einspahr, H. M. & Weiss, M. S. (2012). International Tables for Crystallography, Vol. F, Crystallography of Biological Macromolecules, 2nd ed., ch. 2.2, pp Heidelberg: Springer. Weiss, M. S. & Hilgenfeld, R. (1997). J. Appl. Cryst. 30, Diederichs, K. & Karplus, P. A. (1997). Nat. Struct. Biol. 4, (& Diederichs, K. & Karplus, P. A. (1997b). Erratum. Nat. Struct. Biol. 4, 592.) Karplus, P. A. & Diederichs, K. (2012). Science, 336,

15 ANISOTROPY

16 Motivation Courtesy of M. Sawaya

17 Motivation Crystal structures are inherently anisotropic. X ray diffraction reflects this fact. Account for it by: Anisotropic scaling Anisotropic atomic displacement parameters (ADPs) TLS (+ isotropic B factor) refinement In cases of severe anisotropy, an ellipsoidal rather than spherical boundary for the reflections can be used. Sheriff & Hendrickson (1987). Acta Cryst. A43, Strong et al. (2010). Comp. Cryst. Newsletter 1,

18 DATA FORMATS

19 SHELX HKL ASCII text format Columns: h, k, l, I, σ(i)

20 Scalepack SCA There are two scalepack formats, both called.sca, and both ASCII text: The unmerged one looks like this: p

21 CCP4 MTZ Binary format Needs to be read/edited with dedicated programs, such as MTZDMP. Typically contains: Unit cell, space group Crystal & Project name Columns: h,k,l, I, σ(i) or F, σ(f) or I+, σ(i+), I-, σ(i-) Column types

22 How to manage? When processing: Output data in various formats. Do not cut or merge data unless necessary. Use a lab journal and a good naming scheme. Do not confuse refinement output with input. Note which data statistics belong to which data set. Keep data statistics in lab journal.

23 Protein Data Bank format.pdb ASCII text format: The old PDB format. HEADER OXIDOREDUCTASE 18-JUL-07 2V6G TITLE STRUCTURE OF PROGESTERONE 5BETA-REDUCTASE FROM DIGITALIS ( ) REMARK 900 STRUCTURE OF PROGESTERONE 5BETA-REDUCTASE FROM REMARK 900 DIGITALIS LANATA CRYST P ATOM 1 N SER A N ANISOU 1 N SER A N ATOM 2 CA SER A C ANISOU 2 CA SER A C ATOM 3 C SER A C ANISOU 3 C SER A C ATOM 4 O SER A O

24 mmcif mmcif became the standard PDB archive format in 2014 loop atom_site.group_pdb _atom_site.id _atom_site.auth_atom_id ( ) _atom_site.pdbx_auth_alt_id _atom_site.b_iso_or_equiv ATOM 1 N N GLN A ATOM 2 CA C GLN A ATOM 3 C C GLN A ATOM 4 O O GLN A ATOM 5 CB C GLN A ATOM 6 N N VAL A ATOM 7 CA C VAL A

25 CCP4 map format Binary format. Contains a grid With grid spacing And one or multiple values for each grid point Same as MRC format (3D for movies). Used in Coot, Pymol, cryo EM and others.

26 Summary Resolution is not one thing and overrated as a concept. CC(1/2) is currently the best criterion for the cut off. Anisotropy needs to be accounted for. X ray crystallographers use a plethora of data formats: For example for data.hkl,.mtz,.sca,.mmcif For example for coordinates:.psb,.mmcif For example for maps:.map,.ccp4,.mrc (all the same)

27 Next lecture (Twinning, Thursday, 13th of October, 11:30, Diamond) Low resolution refinement and model building Tuesday, 25th of October, 16:00, Strubi

28 PHS format Weird order map format in ASCII

Anisotropy in macromolecular crystal structures. Andrea Thorn July 19 th, 2012

Anisotropy in macromolecular crystal structures Andrea Thorn July 19 th, 2012 Motivation Courtesy of M. Sawaya Motivation Crystal structures are inherently anisotropic. X-ray diffraction reflects this