Neutron structure determination

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Ambrose Price
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1 Neutron structure determination An X-ray structure is always available and is used as a starting model for neutron structure determination, therefore once the neutron data is available, the main steps to get final refined model are: - model preparation (add H or D, or H/D; library files for ligands), - model refinement and completion (adding DOD, OD or O), - model validation

available, the main steps to get final refined model are: - model preparation (add H or D, or

2 Model preparation Model preparation (add H or D, or H/D; library files for ligands), - add H or D, or H/D, - library files for ligands (CIF file) Solution in PHENIX: - add H to everything and create ligand CIF file (if any) phenix.ready_set model.pdb - add D to everything and create ligand CIF file (if any) phenix.ready_set model.pdb perdeuterate=true - add H and exchangeable H/D: phenix.ready_set model.pdb neutron_exchange_hydrogens=true - To see all options of phenix.ready_set: phenix.ready_set --show-defaults - To remove all H and D atoms: phenix.reduce -trim model_h.pdb > model_noh.pdb

pdb - add D to everything and create ligand CIF file (if any) phenix.ready_set model.pdb perdeuterate=true - add H and exchangeable H/D: phenix.

3 Model preparation phenix.pdbtools set of tools for PDB file manipulations For any selected model part: - shake coordinates, ADP, occupancies - rotation-translation shift of coordinates - shift, scale, set ADP (add, multiply, assign a constant) - converting to isotropic / anisotropic - removing selected part of a model Easy to run: % phenix.pdbtools model.pdb set_b_iso=30 selection="chain A This will sell all B-factors to 30 for all atoms in chain A. Also: - complete model statistics (geometry, B-factors) - geometry regularization - output MTZ with complete calculated structure factors (that may include all scales and bulk solvent)

- shift, scale, set ADP (add, multiply, assign a constant) - converting to isotropic / anisotropic - removing selected part of a model Easy to run: % phenix.

4 Model refinement Designed to be very easy to use phenix.refine model.pdb data.hkl [parameters]

5 Some basic examples of running phenix.refine from the command line Refinement of individual coordinates, B-factors, and occupancies for some atoms: phenix.refine model.pdb data.hkl Add water picking and Simulated Annealing to default run above: phenix.refine model.pdb data.hkl simulated_annealing=true ordered_solvent=true Refinement of individual coordinates and B-factors using neutron data: phenix.refine model.pdb data.hkl main.scattering_dictionary=neutron To see all parameters (more than 300): phenix.refine --show_defaults=all

6 Running phenix.refine % phenix.refine model.pdb data.hkl parameter_file where parameter_file contains following lines: refinement.main { high_resolution = 2.0 low_resolution = 15.0 simulated_annealing = True ordered_solvent = True number_of_macro_cycles = 5 } refinement.refine.adp { tls = chain A tls = chain B } For typing enthusiasts, the equivalent command line run would be: % phenix.refine model.pdb data.hkl xray_data.high_resolution=2 xray_data.low_resolution=15 simmulated_annealing=true ordered_solvent=true adp.tls= chain A adp.tls= chain B main.number_of_macro_cycles=5

refine.adp { tls = chain A tls = chain B } For typing enthusiasts, the equivalent command line run would be: % phenix.refine model.pdb data.

7 Typical way of phenix.refine run from the command line 1. Get the file with all parameters: % phenix.refine --show-defaults=all > parameter_file 2. Edit the file parameter_file: - Remove all parameters that you are not planning to change (make sure to have all { } matched) - Change the rest of parameters 3. Run phenix.refine as following: % phenix.refine model.pdb data.hkl parameter_file or (If model.pdb and data.hkl are included into parameter_file file) % phenix.refine parameter_file Useful tip: to compare the set of parameters in your parameter_file file against the set of all default parameters, type: % phenix.refine --diff-params parameter_file

Run phenix.refine as following: % phenix.refine model.pdb data.hkl parameter_file or (If model.pdb and data.hkl are included into parameter_file file) % phenix.

8 Refinement runs required two steps: hydrogens and ligands When running: % phenix.refine model.pdb data.hkl each item in model.pdb is matched against the CCP4 Monomer Library to extract the topology and parameters and to automatically build corresponding restraints. If model.pdb contains an item not available in CCP4 Monomer Library, e.g. a novel ligand, use ReadySet! program to generate topology and parameter definitions for refinement: % phenix.ready_set model.pdb This will produce the file LIG.cif and updated PDB file model.updated.pdb with all H atoms added which can be used for refinement: % phenix.refine model.pdb data.hkl LIG.cif

pdb contains an item not available in CCP4 Monomer Library, e.g. a novel ligand, use ReadySet!

9 % phenix.reel LIG.cif Restraints and novel ligands: REEL

10 Refinement runs required two steps: twinning Two steps to perform twin refinement: - run phenix.xtriage to get twin operator (twin law): % phenix.xtriage data.mtz - run phenix.refine: % phenix.refine model.pdb data.mtz twin_law="-h-k,k,-l"

11 Running joint X/N refinement: Individual neutron and joint X+N refinement % phenix.refine model.pdb parameter_file Where the parameter_file defines where the X-ray and neutron data are: refinement.input.neutron_data { file_name = data.mtz labels = "F-obs-neutron,SIGF-obs-neutron" r_free_flags { file_name = data.mtz label = "R-free-flags-neutron" } } refinement.input.xray_data { file_name = data.mtz labels = "F-obs,SIGF-obs" r_free_flags { file_name = "data.mtz" label = "R-free-flags" } }

$neutron_data { file_name = data.mtz labels = "F-obs-neutron,SIGF-obs-neutron" r_free_flags { file_name = data.$

12 Important remark about free-r flags in joint X+N refinement Free-R flags in both datasets (X-ray and neutron) must match, that is: what is test reflection in X-ray dataset, must be a test reflection in neutron dataset. Same for work reflections. phenix.refine automatically checks for this, and if there is a mismatch, phenix.refine stops and offers two options: - keep going with existing flags (but keep in mind that R free will be unreliable); - create new free-r flags that will be consistent for both datasets.

refine automatically checks for this, and if there is a mismatch, phenix.

13 Model refinement - output Input command Output files phenix.refine model.pdb data.mtz [parameters] model_refine_001.eff model_refine_001.geo model_refine_001.log model_refine_001.pdb summary of all input parameters summary of all restraints used complete information about refinement refined structure model_refine_001_map_coeffs.mtz Fourier map coefficients model_refine_002.def parameters for the next run If data file is not in MTZ format, or there are multiple data files at input (example: one with Fobs and the other one with free-r flags), then phenix.refine will combine them into one MTZ data file called: model_data.mtz and this file should be used in all subsequent runs.

pdb summary of all input parameters summary of all restraints used complete information about refinement refined structure model_refine_001_map_coeffs.

14 Model validation Inspect.log file created by phenix.refine. From PHENIX GUI run Comprehensive validation From the command line type: phenix.model_vs_data model.pdb data.mtz If data.mtz contains several data columns, then phenix.model_vs_data model.pdb data.mtz --f-obs-label=fo In case of neutron data: phenix.model_vs_data model.pdb data.mtz --scattering-table=neutron Simply type phenix.model_vs_data to get list of options.

pdb data.mtz If data.mtz contains several data columns, then phenix.model_vs_data model.pdb data.mtz --f-obs-label=fo In case of neutron data: phenix.

15 Reporting bugs, problems, asking questions Something didn t work as expected?... program crashed?... missing feature?... Bad: silently give up and run away looking for alternative software. Good: report us a problem, ask a question, request a feature (explain why it s good to have), ask for help (send data). Reporting a bug / problem: Bad: Hi! phenix.refine crashed and I don t know why and what to do. Goodbye. Good: Hi! phenix.refine crashed. Here are: 1) PHENIX version; 2) The exact command I used; 3) Input and output files (at least logs). PHENIX:

Good: report us a problem, ask a question, request a feature (explain why it s good to have), ask for help (send data).

Automated TLS group determination in Phenix

Computational Crystallography Initiative Automated TLS group determination in Phenix Pavel Afonine Computation Crystallography Initiative Physical Biosciences Division Lawrence Berkeley National Laboratory,