Space group validation using Zanuda Andrey Lebedev, CCP4.

Space group validation using Zanuda Andrey Lebedev, CCP4

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

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

Pointless: twinning and pseudo-symmetry 09/01/2015CCP4 Study Weekend4 Nelmt...CC...Rmeas... operator... 1...0.98...0.051identity 2...0.98...0.071*** 2-fold l... 3...0.96...0.089*** 2-fold h... 4...0.97...0.089*** 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 P 1 2/m 1...0.97...0.07... 4 P 1 2/m 1...0.97...0.07... 5 P -1...0.98...0.05... "The second best" point group may be correct

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

Pseudotranslation: what else can go wrong? 09/01/2015CCP4 Study Weekend6 Molecular Replacement: Two structures are globally very similar (e.g. rmsd = 0.5A) MR can in some cases pick up a wrong solution Crystallographic and pseudosymmetry axes are confused P2 (a,b,c) Cell and H-M symbol are the same True structureFalse structure

True structureFalse structure Example: anti-TRAP 09/01/2015CCP4 Study Weekend7

True structure: R26% R-free32% complete model can be built False structure: R38% R-free44% partial model only can be built True and false structures: refinement and rebuilding (P21 true and P21 false structures of anti-TRAP from B. licheniformis) 09/01/2015CCP4 Study Weekend8

http://www.ccp4.ac.uk/ccp4online Zanuda CCP4 online 09/01/2015CCP4 Study Weekend9

mtz-file pdb-file Submitting Zanuda job 09/01/201510CCP4 Study Weekend

Download of output pdb- and mtz-files Symmetry analysis Zanuda output 09/01/201511CCP4 Study Weekend

False space groupP2 P2 1 True space group C2Pseudo-symmetry space group Space group and its relation to the structure 1yup Crystallographic axes Pseudosymmetry axes Positions of molecules A simple example (1yup) 09/01/201512CCP4 Study Weekend

Space group validation: step 1 09/01/2015CCP4 Study Weekend13 Pseudosymmetry Space Group; here: a' = a/2

Space group validation: step 2 09/01/2015CCP4 Study Weekend14 2-axis2 1 -axis C2crystallographic crystallographic P2crystallographic NCS P2 1 NCS crystallographic True SG

space group validation: step 3 09/01/2015CCP4 Study Weekend15 Output (P2 1 )

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) 09/01/2015CCP4 Study Weekend16

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) 09/01/2015CCP4 Study Weekend17

CCP4I interface 09/01/2015CCP4 Study Weekend18 "REFINE" or "SAVE" only (quick) Do not "SYMMETRISE" for MR solutions in P1!

09/01/2015CCP4 Study Weekend19

mosflm pointless aimless ctruncate structure solution model building and refinement PDB imosflm ccp4 automatic space group assignment: - analysis of lattice symmetry - comparison of related reflections - analysis of systematic absences Automatic space group assignment 09/01/2015CCP4 Study Weekend22

mosflm pointless aimless ctruncate structure solution PDB imosflm ccp4 pointless aimless ctruncate structure solution PDB twin not solved try second best space group Manual space group assignment 09/01/2015CCP4 Study Weekend23 model building and refinement model building and refinement

mosflm pointless aimless ctruncate structure solution PDB imosflm ccp4 R-free 40% density does not improve Zanuda Attempts at refinements in all relevant subgroups of pseudo-symmetry space group Strong pseudo-symmetry 09/01/2015CCP4 Study Weekend24 model building and refinement some model rebuilding reiterate data reduction etc.

Pseudosymmetry: what can go wrong 09/01/2015CCP4 Study Weekend25 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

PDB code:1yup space group (PDB):P18 molecules per a.u. space group (true):P2 1 4 molecules per a.u. Pseudo-symmetry space group:C22 molecules per a.u. (because of pseudo-translation) An example of symmetry correction 09/01/201526CCP4 Study Weekend

Structure solution and symmetry validation 09/01/2015CCP4 Study Weekend27 Data processing ( 2/m ) Molecular replacement ( P2 ) Refinement ( P2 ) R-free 0.37 Data processing ( -1 ) Molecular replacement ( P1 ) Refinement ( P1 ) R / R-free = 0.24 / 0.31 PDB: 1yup ( P1 ) PDB: 1yup Zanuda ( P2 1 ) R-free = 0.33

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. 09/01/2015CCP4 Study Weekend28 Things went wrong way

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 09/01/2015CCP4 Study Weekend29

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 09/01/2015CCP4 Study Weekend30

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. 09/01/2015CCP4 Study Weekend31

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 Algorithm Common supergroup: P4 2 22 with twice smaller c 09/01/201532CCP4 Study Weekend

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) 09/01/201533CCP4 Study Weekend Possible development

09/01/2015CCP4 Study Weekend Four alternative solutions in two space groups 34 GAF (N-terminal) domain of CodY protein from Bacillus subtilis Levdikov, V. M. et al. (2006). J Biol Chem 281, 11366-73. MR solution

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

Twinned crystal with pseudo-symmetric substructure 09/01/2015CCP4 Study Weekend36 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 (A)(B) + 3-fold axes with respect to the true structure: crystallographic pseudosymmetry for (A) pseudo-P3 1 21 (a' b' c)P3 1 (a b c) Human macrophage receptor CLEC5A for dengue virus Watson, A. A. et al. (2011). J Biol Chem 286, 24208-18.

Attempt at structure solution 09/01/2015CCP4 Study Weekend37 Conventional MR (4 monomers) Phased MR (2 monomers) Refinement R = 0.45 R-free = 0.48 Something is wrong False origin?

Pseudo-symmetry space group 09/01/2015CCP4 Study Weekend38 Hermann Mauguin symbol P3 1 21 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 3-fold axes with respect to MR solution: crystallographic pseudosymmetry

(A) (C) Transformation of MR solution 09/01/2015CCP4 Study Weekend39 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 structurereference (unit cell) This picturefixedmoves Refinement program movesfixed

Using Zanuda 09/01/2015CCP4 Study Weekend40 Table form Zanuda: comparison of refinements R-factors are still too high Incomplete structure? (C) P10.4300.466 P3 1 0.4600.498 P3 1 0.4590.495 P3 1 0.4300.466 C20.4410.481 P3 1 120.4550.480

Crystal disorder Twinning, partial disorder:Missing global periodicity 09/01/2015CCP4 Study Weekend41 Single crystal(Single ordered domain) Twinned crystal(Two or more ordered domains) Partially disordered crystal(Many ordered domains) size of ordered domains Coherence length of X-rays

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 09/01/2015CCP4 Study Weekend45

Zanuda 09/01/2015CCP4 Study Weekend46 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

Pseudotranslation 09/01/2015CCP4 Study Weekend47 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

> Acentric moments of E for k=1,3,4 > 4th moments of E... 1jjk: Pseudotranslation results in alteration of strong and weak reflections Statistics of one intensity are strongly affected by pseudotranslation 09/01/201548CCP4 Study Weekend

False structure P2P2 1 True structure C2 Pseudo-symmetry space group Crystallographic axes Pseudosymmetry axes Monoclinic groups related to 1yup 09/01/2015CCP4 Study Weekend49

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 09/01/2015CCP4 Study Weekend50

Space group validation using Zanuda Andrey Lebedev, CCP4.

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