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Space group validation using Zanuda

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Presentation on theme: "Space group validation using Zanuda"— Presentation transcript:

1 Space group validation using Zanuda
Andrey Lebedev, CCP4

2 non-crystallographic
Pseudosymmetry General non-crystallographic symmetry Crystallographic symmetry Pseudo-symmetry Global and exact Global and not exact Local and not exact Global <=> consistent with crystallographic translations <=> pseudosymmetry space/point groups can be defined 09/01/2015 CCP4 Study Weekend

3 Pseudosymmetry and twinning
twin axis Twinning + Generic NCS Twinning axis || NCS axis OR Single crystal 09/01/2015 CCP4 Study Weekend

4 Pointless: twinning and pseudo-symmetry
"The second best" point group may be correct Nelmt ... CC Rmeas ... operator ... 1 0.98 0.051 identity 2 0.071 *** 2-fold l ... 3 0.96 0.089 *** 2-fold h ... 4 0.97 *** 2-fold k ... Laue Group ... CC Rmeas = 1 P m m m *** 0.97 0.07 2 P 1 2/m 1 0.98 0.06 3 4 5 P -1 0.05 09/01/2015 CCP4 Study Weekend

5 Pseudotranslation Crystallographic translation: global and exact
global but not exact Pseudotranslation + crystallographic axes -> pseudosymmetry axes 09/01/2015 CCP4 Study Weekend

6 Pseudotranslation: what else can go wrong?
Cell and H-M symbol are the same P2 (a,b,c) P2 (a,b,c) Crystallographic and pseudosymmetry axes are confused True structure False structure Molecular Replacement: Two structures are globally very similar (e.g. rmsd = 0.5A) MR can in some cases pick up a wrong solution 09/01/2015 CCP4 Study Weekend

7 Example: anti-TRAP True structure False structure 09/01/2015
CCP4 Study Weekend

8 True and false structures: refinement and rebuilding
(P21 true and P21 false structures of anti-TRAP from B. licheniformis) True structure: R 26% R-free 32% complete model can be built False structure: R 38% R-free 44% partial model only can be built 09/01/2015 CCP4 Study Weekend

9 CCP4 online http://www.ccp4.ac.uk/ccp4online Zanuda 09/01/2015
CCP4 Study Weekend

10 Submitting Zanuda job mtz-file pdb-file 09/01/2015 CCP4 Study Weekend

11 Zanuda output Download of output pdb- and mtz-files Symmetry analysis
09/01/2015 CCP4 Study Weekend

12 A simple example (1yup) Space group and its relation
to the structure 1yup Crystallographic axes Pseudosymmetry axes Positions of molecules C2 Pseudo-symmetry space group False space group P2 P21 True space group 09/01/2015 CCP4 Study Weekend

13 Space group validation: step 1
Pseudosymmetry Space Group; here: a' = a/2 09/01/2015 CCP4 Study Weekend

14 Space group validation: step 2
True SG 2-axis axis C2 crystallographic crystallographic P2 crystallographic NCS P21 NCS crystallographic 09/01/2015 CCP4 Study Weekend

15 space group validation: step 3
Output (P21) 09/01/2015 CCP4 Study Weekend

16 Example from prof Daan Van Aalten, Dundee
Vicious cycle: good model is needed to clearly distinguish between correct and incorrect space groups knowledge of correct space group is needed (desirable) to improve model Availability of P1 MR solution can be beneficial data reduction in zanuda is not perfect reprocess data with aimless, if space group has to be changed (aimless job 88; zanuda jobs 17, 82; refmac jobs 74, 90) 09/01/2015 CCP4 Study Weekend

17 Example from Misha Isupov, Exeter
Four alternative origins (alternative P21) Even good programs can make mistakes, and even in simple cases: solved with Phaser corrected with Zanuda Simple expansion of an incorrect model into P1 and refinement may not work (China54: phaser job 1; refmac jobs 2; zanuda job 7; coot on any structure) 09/01/2015 CCP4 Study Weekend

18 CCP4I interface "REFINE" or "SAVE" only (quick) Do not "SYMMETRISE"
for MR solutions in P1! 09/01/2015 CCP4 Study Weekend

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22 Automatic space group assignment
ccp4 automatic space group assignment: - analysis of lattice symmetry - comparison of related reflections - analysis of systematic absences imosflm mosflm pointless aimless ctruncate structure solution model building and refinement PDB 09/01/2015 CCP4 Study Weekend

23 Manual space group assignment
ccp4 imosflm mosflm pointless pointless try second best space group aimless aimless ctruncate ctruncate twin Split into three slides? structure solution structure solution not solved model building and refinement model building and refinement PDB PDB 09/01/2015 CCP4 Study Weekend

24 Strong pseudo-symmetry
ccp4 Attempts at refinements in all relevant subgroups of pseudo-symmetry space group imosflm mosflm pointless aimless Zanuda ctruncate some model rebuilding R-free ≈ 40% density does not improve structure solution reiterate data reduction etc. model building and refinement PDB 09/01/2015 CCP4 Study Weekend

25 Pseudosymmetry: what can go wrong
Data reduction: Pseudosymmetry can be confused with the crystallographic symmetry Wrong space group assignment Problems with structure solution Most relevant cases Twin axis || pseudosymmetry axis Not unusual The first problem is quite usual and I will not show any example. 09/01/2015 CCP4 Study Weekend

26 An example of symmetry correction
PDB code: 1yup space group (PDB): P1 8 molecules per a.u. space group (true): P21 4 molecules per a.u. Pseudo-symmetry space group: C2 2 molecules per a.u. (because of pseudo-translation) 09/01/2015 CCP4 Study Weekend

27 Structure solution and symmetry validation
Data processing ( 2/m ) Molecular replacement ( P2 ) Refinement R-free ≈ 0.37 ( -1 ) ( P1 ) R / R-free = 0.24 / 0.31 PDB: 1yup ( P1 ) PDB: 1yup Zanuda ( P21 ) R-free = 0.33 09/01/2015 CCP4 Study Weekend

28 Zanuda protocol is not perfect
Assumptions: The pseudosymmetry is very strong (r.m.s.d. from exact symmetry ≈ 1A) The structure is almost correct (although it might have been refined / rebuilt in an incorrect space group) If it is not so, then it is unlikely to obtain the correct answer. Things went wrong way 09/01/2015 CCP4 Study Weekend

29 Summary Signs of incorrect interpretation of pseudo-symmetry:
Reasonable density for a part of the structure Large fragments of poor density with no useful features for further rebuilding High R-free However, model building and refinement in incorrect space group may improve model Then models with different symmetries can be generated, refined and compared Split into three slides? 09/01/2015 CCP4 Study Weekend

30 Zanuda: space group validation
Algorithm: From input model: determine pseudosymmetry space group (PSSG) From PSSG: select subgroups with observed unit cell For each such subgroup: Convert model and data into the subgroup Restrained refinement Repeat refinements of the best (R-free) model Starting from P1 Adding the best (r.m.s.d.) symmetry element at each refinement Terminate if there is no symmetry elements to be added Terminate and cancel the last symmetry element if R-free jumps 09/01/2015 CCP4 Study Weekend

31 Zanuda: limitations Assumptions:
The pseudosymmetry is very strong (r.m.s.d. from exact symmetry ≈ 1A) The structures of individual molecules are almost correct although they might have been refined / rebuilt in an incorrect space group If assumptions are not satisfied, the results will likely to be wrong. 09/01/2015 CCP4 Study Weekend

32 Common supergroup: P4222 with twice smaller c
Algorithm Model preparation; determination of pseudosymmetry space group including pseudotranslation Subgroup/supergroup graph Test refinements in subgroups with observed unit cell (blue) Best structure expanded into P1 Refinements followed by adding symmetry elements Returns model and data in the "best" SG Common supergroup: P4222 with twice smaller c - ... - Chains aligned and truncated to have identical content in the AU of the PSSG - Internal representation of the sub/super group graph - Procedure is repeated in another 09/01/2015 CCP4 Study Weekend

33 Possible development  Options:
- currently available: refinement in all possibilities SGs (auto) - to be added: refinements in subgroups selected by a user - to be added: transformations only, no refinements - Appropriate interface - Will be OK for distribution  Using unmerged data as an option - this is the right way - using Pointless for data conversion  Starting from incomplete model (completion by e.g. Molecular Replacement) 09/01/2015 CCP4 Study Weekend

34 Four alternative solutions in two space groups
GAF (N-terminal) domain of CodY protein from Bacillus subtilis Levdikov, V. M. et al. (2006). J Biol Chem 281, MR solution 09/01/2015 CCP4 Study Weekend

35 Comparison of possible structures
09/01/2015 CCP4 Study Weekend

36 Twinned crystal with pseudo-symmetric substructure
Human macrophage receptor CLEC5A for dengue virus Watson, A. A. et al. (2011). J Biol Chem 286, pseudo-P3121 (a' b' c) P31 (a b c) a' b' a b a b 3-fold axes with respect to the true structure: crystallographic pseudosymmetry for (A) + (A) (B) Substructure (A) is common for twin individuals Substructure (B) is not even approximately symmetric relative to and The choice of correct origin was essential for structure completion 09/01/2015 CCP4 Study Weekend

37 Attempt at structure solution
Conventional MR (4 monomers) Phased MR (2 monomers) Refinement R = 0.45 R-free = 0.48 Something is wrong False origin? a b 09/01/2015 CCP4 Study Weekend

38 Pseudo-symmetry space group
Hermann Mauguin symbol P3121 translation base vectors b' = (a - b)/ 3 a' = (a + 2b)/ 3 c' = c in the true large cell three alternative origins associated with the three types of 3-fold axes shown in the figure a b a' b' 3-fold axes with respect to MR solution: crystallographic pseudosymmetry 09/01/2015 CCP4 Study Weekend

39 Transformation of MR solution
structure reference (unit cell) This picture fixed moves Refinement program (A)  (C) a b We need to move structure to tell the program that we want to select different set of axes Select the AU, in which 3 pairs of molecules are related by pseudotranslation save required symmetry equivalents coot merge them into a single PDB-file text editor Shift the whole AU by (a + b)/ 3 lsqkab 09/01/2015 CCP4 Study Weekend

40 Using Zanuda Table form Zanuda: comparison of refinements
R-factors are still too high Incomplete structure? (C) a b P1 0.430 0.466 P31 0.460 0.498 0.459 0.495 C2 0.441 0.481 P3112 0.455 0.480 09/01/2015 CCP4 Study Weekend

41 size of ordered domains
Crystal disorder Twinning, partial disorder: Missing global periodicity size of ordered domains Single crystal (Single ordered domain) Twinned crystal (Two or more ordered domains) Partially disordered crystal (Many ordered domains) Coherence length of X-rays 09/01/2015 CCP4 Study Weekend

42 09/01/2015 CCP4 Study Weekend

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45 Guidelines In most cases high R-free means wrong solution or insufficiently good model Zanuda can also make mistakes especially with poor starting model Hence: Firstly, improve model Secondly, improve model If nothing works, then perhaps space group or origin assignment is a problem Model from MR: R-free 50-60%: first of all, this structure needs to be solved (or model rebuilt) R-free around 40% after model rebuilding and no obvious ways for further improvement of the model: try Zanuda In words: difference in R-free may be 3% for refined models in different space groups, which are not subgroups of each other. 09/01/2015 CCP4 Study Weekend

46 Zanuda CCP4I default & YSBL server
- Automatic detection of pseudosymmetry space group - Refinement in subgroups of PSSG - Selection and validation of the best model CCP4I option - no refinement, save all transformed models and data When can be used: (1) Space group validation for twinning || pseudosymmetry (2) False origin correction (as in the CodY example) (3) Structure solver in P1 09/01/2015 CCP4 Study Weekend

47 Pseudotranslation Pseudotranslation C/2
Crystallographic translation Pseudo-translation Pseudotranslation C/2 Planes 2L+1 contain weak reflections Limiting case, C' = C/2 • Weak reflections vanish Two times larger reciprocal lattice spacing Crystallographic translation 09/01/2015 CCP4 Study Weekend

48 Statistics of one intensity are strongly affected by pseudotranslation
1jjk: Pseudotranslation results in alteration of strong and weak reflections > Acentric moments of E for k=1,3,4 > 4th moments of E ... 09/01/2015 CCP4 Study Weekend

49 Monoclinic groups related to 1yup
Crystallographic axes Pseudosymmetry axes Pseudo-symmetry space group False structure True structure Remove drawings with molecules? C2 P2 P21 09/01/2015 CCP4 Study Weekend

50 CCP4I interface CCP4I > Validation & Deposition > Validate space group Modes REFINE all transformed models and save the best model SAVE all transformed models and data without refinement Option SYMMETRYSE input model before further refinements 09/01/2015 CCP4 Study Weekend

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