Reciprocal-Space Refinement Panel

Perform reciprocal space refinements. These can include minimization of R-factors for the coordinates, B-factors and grouped occupancies; adjustment of parts or all of the protein as rigid bodies, and simulated annealing.

To open this panel, click the button for the task under Reciprocal-Space Refinement in the PrimeX Panel.

  • Using
  • Features
  • Additional Resources

Using the Reciprocal-Space Refinement Panel

Noncrystallographic symmetry restraints

You can define restraints on the optimization to satisfy noncrystallographic symmetry. The restraints are applied to specified groups of chains. Noncrystallographic symmetry restraints are generally used only in the early stages of refinement.

Noncrystallographic symmetry (NCS) is imposed at the chain level. To impose this symmetry, you create groups of chains or chain segments that are related by NCS. The parts of the chains that are considered to be related must have identical atom composition, so you must omit residues that differ between the chains, including differences in protonation or in residue name. The terminal residues of each chain are unique in PrimeX, and must be excluded from the restraint system. Restraints can be applied optionally to just the backbone as well as to whole residues. You can use the sequence viewer in the Workspace to compare the chains, and can click on residues to select them in the Workspace. You should check that the residues have the same atoms: if they do not, they must be omitted from the group.

Rigid Body Refinement

Rigid-body refinement is usually performed with only the low-resolution data. Each rigid body is defined by a group of atoms, which you can select at will. You can also decide what to do with atoms that are not part of any explicitly chosen rigid body. The overall positioning of the protein by rigid-body refinement is often the first step in a refinement. Before you submit the job, you might therefore want to increase the high-resolution cutoff for the reflection data in the Calculation Settings Dialog Box.

Simulated Annealing

Simulated annealing is generally used after the larger changes in positions have been completed with rigid-body refinement. Usually the initial temperature is decreased with successive runs. This method is useful for moving across low energy barriers.

When a simulated annealing job fails with the error message “Too many unreasonably large velocities detected”, the cause of the failure can be that the X-ray weight is too large. This problem is most likely to be seen with the approximate energy model. The error message suggests other possible solutions: minimizing the starting structure or reducing the MD time step. Early in the refinement of a structure, either reducing the X-ray weight or performing a minimization is probably the most useful solution. Later in the refinement, reducing the X-ray weight or decreasing the time step is probably the most useful solution. Reducing the time step to 0.006 usually fixes the problem.

Minimization

Minimization is usually done in the last stages of the refinement. This method is often the best for placement of water molecules.

You can minimize the R factors for the coordinates, the B-factors, grouped B-factors, and grouped occupancies, and you can perform the minimizations on selected atoms.

There are also some settings that you should make in the General tab: Use current values should be selected under Isotropic B-factor values, and the X-ray term weight setting should be set to Constant weight, with a value of 1.0. With this weight for the X-ray term and a B-factor restraint weight of 1.0, the restraints on the B-factors will be very strong. This setting is appropriate for the beginning of individual isotropic B-factor refinement of a moderate-resolution structure. Approaching the end of the refinement, you might reduce this restraint weight by factors of 2 or more, until R-free no longer decreases.

After each refinement job is done, maps are automatically generated. You can select the type of map and set up the parameters of the map in the Maps tab.

 

To write out the input file and a script for running the job from the command line, click the arrow next to the Settings button and choose Write. For information on command usage and options, see primex Command Help and Running PrimeX Tasks from the Command Line.

Reciprocal-Space Refinement Panel Features

Refinement method option menu

There are three refinement methods available:

  • Minimization—Minimize the R factors for the coordinates, B-factors, and grouped occupancies. Useful for water placement and small changes to coordinates.

  • Rigid Bodies—Adjust the overall positioning of the protein by moving it or user-defined pieces as rigid bodies. Usually used at the beginning of a refinement with only the low-resolution data.

  • Simulated Annealing—Perform a simulated annealing calculation. Useful for moving across low energy barriers.

Tabs

There are four tabs, which contain controls for the refinement. Three of these are common to all refinement methods. The fourth tab changes according to the method chosen.

  • General
  • NCS
  • Maps
  • Minimization
  • Rigid Bodies
  • Simulated Annealing

General Tab Features

In this tab, you specify the target for refinement, the weighting of X-ray and energy terms in the target function, and how to set the isotropic B factors.

Target options

There are two choices of refinement target: Maximum likelihood and Least squares. If you choose Least squares, the Unweighted option in the Weighting options group of the Maps tab is selected and the controls are then disabled so that you cannot change the option. This is done because you cannot create a weighted map with the least squares target. These options are enabled again if you choose Maximum likelihood.

X-ray term weight options

The two options provide a way of controlling the weighting of the gradient of the electron density fit relative to the energy gradient in the refinement.

  • Calculated weight multiplied by—The optimal weight calculated by PrimeX can be multiplied by a factor that weights the X-ray terms (density fit). A value of 1.0 selects the calculated weight.
  • Constant weight—Specify the weight of the X-ray terms. The default is 0.5.

If the energy term is too large, R and R(free) might not change very much, or might increase. If the energy term is too small, R is likely to decrease but R(free) will remain the same.

Weighting of the X-ray terms against geometric restraints is a complicated balancing act. Choices must be made based on the stage of the refinement process, the deviation of molecular geometry from expected values, and the current values for R and R-free. The complexity of these choices in PrimeX can be greatly reduced with the application of the following guidelines.

The Constant weight option with a value of 2.0 is usually an appropriate starting point for reciprocal-space minimization and simulated annealing refinement for most molecular replacement cases. If the divergence between R and R-free is becoming unacceptably high, or if the molecular geometry deviates too far from expected values, then this value should be decreased to 1 or lower. Values less than 0.1 are rarely productive. Insufficient progress in refinement can sometimes be improved with a larger constant weight. Also, if the values for R and R-free both increase, a higher weight is indicated. The weight will not need to be adjusted very often once an appropriate value is found.

Although PrimeX estimates an appropriate value for weighting the X-ray term upon selection of the Calculated weight option, this value tends to be conservatively low, especially for early stages of refinement. The same considerations described above apply to adjusting the multiplier with this option.

Isotropic B-factor values options

There are two options for setting the isotropic B-factors: Use current values and Set to constant value of N Å2. The current maximum, minimum, and mean values are listed below the option.

NCS Tab Features

The features in this tab are a Groups table that lists the parts of the structure that are to be restrained, and Add and Delete buttons, to add groups to the table or delete groups from the table.

To add a group to the table, click Add. A new row is added, and the Chains column is populated with the chains that have not been used previously. The B-factor weight and coordinate weight are set to the default values. If all chains have been used in previous groups, the group is initialized with all chains, so that you can make other connections between chains than have already been defined.

To delete a group, select it in the table and click Delete.

To edit a table cell, click in the cell, make changes, then press ENTER.

# Group number
Chains Chains that are included in the group. The chains need not be identical, but if they are not, you must omit the residues that differ. The chains must have the same numbering for corresponding residues.
Residues to omit Comma-separated list of residue numbers to be omitted from the specified chains. The list is a comma-separated list of residue numbers (including insertion codes) or ranges, such as 223-226a. If the chains are not identical, you must omit residues that differ between chains or are missing from some chains in the group.
Backbone only If the box is checked, apply restraints to the backbone only, not to the side chains.
B-factor weight Weight of the restraint on the B-factors
Coord weight Weight of the restraint on the coordinates

Maps Tab Features

The Maps tab provides options for defining the parameters and extent of the maps that will be created with a refinement job.

Coefficients options

Four options are provided for the coefficients to be used in the map: Fo, Fo-Fc (difference map), 2Fo-Fc, and 3Fo-2Fc. You can select any number of these options. For fitting rotamers with the Rotamers Dialog Box, you must generate either a 2Fo-Fc or a 3Fo-2Fc map.

Weighting options

The two options are SigmaA and Unweighted. If you select the Least squares target in the General tab, you can only create an unweighted map. In this case, the Unweighted option is selected and the options are disabled, so that you cannot change the selection. To reenable the controls, select Maximum likelihood in the General tab.

Grid size options

Two preset options for the grid spacing are provided: 0.33 Å, 0.25 Å. If you want to specify your own grid spacing, select Other and enter a value in the text box.

Scale map to sigma units option

This option allows you to scale the map to sigma units, instead of the default units. The units of the default map depends on the units of Fobs.

Extent options

The Extent options allow you to specify the extent of the map. There are four options:

  • Molecule, plus N Å — Limit the map to the region within the specified distance of the selected molecule.

  • Unit cell — Calculate the map for the entire unit cell.

  • Asymmetric unit — Calculate the map for the asymmetric unit.

  • Box — Calculate the map for the box defined in the text boxes. There are two options for the box:

    Fractional space
    Define the map as a fraction of the unit cell parameters. The fractional values can be entered in the min and max text boxes for each crystallographic direction.
    Orthogonal space
    Define the extent of the map in Cartesian (x, y, z) coordinates. The coordinate origin is that of the structure in the Workspace.

Minimization Tab Features

Minimizer options

Select the method for minimization, from Truncated Newton, Conjugate gradient, Quasi-Newton (LBFGS), or Optimal. For the Optimal method choice, the conjugate gradient method is selected for the calculation if the initial gradients are large, and the quasi-Newton method is selected otherwise. The default is Optimal.

Number of cycles text box

Specify the maximum number of cycles to use. Each cycle consists of a number of minimizer steps. At the beginning of each cycle, the second derivatives and scaling factors are recalculated. This reduces the buildup of rounding errors that can affect the course of the minimization.

Maximum steps per cycle text box

Specify the maximum number of steps to take per cycle.

Select atoms to minimize section

This section provides the standard picking controls to select the atoms to minimize. The default is to minimize all atoms.

If you have calculated a density fit (with the Density Fit Panel), you can select residues whose density fit exceeds a certain threshold, by clicking the +button and using the Atom tab of the Atom Selection Dialog Box.

Minimize options

These options specify the quantities whose error (represented by the target specified in the General tab) is to be minimized. You can choose only one of these options for a given minimization job. The options are:

  • Coordinates—Minimize the error in the coordinates.

  • B-factors—Minimize the error in the isotropic B-factors for each atom. B-factor minimization is usually performed with anisotropic overall B-factor scaling.

  • Grouped B-factors—Minimize the error in the isotropic B-factors for groups of atoms. Each residue consists of two groups, the side chain atoms and the backbone atoms (with the exception of glycine, which has only one group). A common B factor is used for the atoms within a group. This approach allows you to refine the B-factors at low resolution where otherwise the number of variables being optimized might exceed the number of data points.

  • Grouped occupancies—Minimize the error by optimizing the fractional occupancies of groups of atoms that have occupancies less than 1. You must select the atoms for this task, in a single occupancy group, in the Select atoms to minimize section. The atoms with partial occupancy can be in alternate conformations or a ligand or other molecule that is not present in saturating amounts. In the first case, the partial occupancies must sum to one; in the second the partial occupancy must be less than one.

    Occupancy refinement should be applied only after the atomic positions and B-values have been thoroughly refined, near the end of the refinement process. Reciprocal-space refinement refines the coordinates of both of a pair of alternate conformations. Note also that only at high resolution is enough information available for refinement to converge at independent values for both B-factors and occupancy.

Isotropic B-factor refinement section

In this section you can set parameters that control the refinement of isotropic B-factors. This section is only available when you choose one of the B-factor options from the Minimize options.

  • B-factor limits — Specify the smallest and largest permissible B-factor values in the Low and High text boxes.

  • B-factor restraint weighting factor — Specify the weight of the restraint of B-factor values to the target sigma values. Not used for grouped B-factor refinement.

  • Target sigma values for B-factor restraints — Specify the target standard deviation of the B-factors for geometric parameters in each residue. The restraints are applied to each residue separately. The four geometric parameter types are Backbone bonds, Backbone angles, Side-chain bonds, and Side-chain angles.

Rigid Bodies Tab Features

Minimizer options

Select the method for minimization, from Truncated Newton, Conjugate gradient, Quasi-Newton (LBFGS), or Optimal. For the Optimal method choice, the conjugate gradient method is selected for the calculation if the initial gradients are large, and the truncated Newton method is selected otherwise. The default is Optimal.

Number of cycles text box

Specify the maximum number of cycles to use. Second derivatives and scaling factors are calculated at the beginning of each cycle. Cycling reduces the chance of accumulated error building up to the point at which it affects the results.

Maximum steps per cycle text box

Specify the maximum number of steps to take per cycle.

Define rigid body group section

In this section you can define the content of a rigid body group, using the standard picking controls. You can pick objects in the Workspace, or use the Atom Selection Dialog Box (opened by the + button) to define the atoms in the rigid body group. When you have made your selection, click Add to create a rigid body group with the selected atoms. The new group is then listed in the Rigid body groups table.

Rigid body groups table

This table lists the rigid body groups that have been defined. The Group column lists the index of the group, and the Residues column lists the residues that belong to the group.

To delete groups, select the groups and click Delete. To delete all groups, click Delete All.

Atoms not explicitly included in a group options

These options allow you to decide what to do with atoms that are not in a group. There are two choices:

  • Fix coordinates for these atoms—Keep the coordinates of the atoms that are not in a group fixed at their initial values.
  • Form an implicit group with these atoms—Include all the atoms that are not assigned to a group in a default group, which is treated as another rigid body to be moved during the optimization. The coordinates of this group can change.

Simulated Annealing Tab Features

Select atoms to minimize section

This section provides the standard picking controls to select the atoms to minimize. The default is to minimize all atoms.

If you have calculated a density fit (with the Density Fit Panel), you can select residues whose density fit exceeds a certain threshold, by clicking the +button and using the Atom tab of the Atom Selection Dialog Box.

Energy model options

Select the model used to compute molecular mechanics energies:

  • Approximate—Neglect solvation and electrostatic terms. Runs faster, and useful for early stages of refinement.

  • Complete—Use all molecular mechanics terms. Slower, but safer for later stages of refinement, and more appropriate if you wish to use simulated annealing to help improve hydrogen positions.

Initial minimization steps text box

Specify the number of steps to take in the initial minimization (at the initial temperature).

MD energy scale estimation steps text box

Specify the number of steps to use in estimating the MD energy scale.

Temperatures text boxes

Specify the initial temperature, the high temperature for the start of the annealing process, and the final temperature. The high temperature should be at least 500 K for sampling of conformations outside the local minimum.

Heating steps text box

Specify the number of steps used to heat the system to the high temperature.

Cooling steps text box

Specify the number of steps used to cool the system from the high temperature to the final temperature.

Final minimization steps text box

Specify the number of steps used for the final minimization.

Molecular dynamics section

This section provides text boxes to set the MD time step in ps and the cutoff for nonbonded interactions in angstroms.

Calculation settings button

This button opens the Calculation Settings Dialog Box, in which you can set the resolution limit, overall B-factor scaling, choose a bulk solvent correction, set limits for rejecting reflections, and choose whether to include hydrogen atoms in the output.

Job toolbar

Manage job submission and settings. See Job Toolbar for a description of this toolbar.

The Job Settings button opens the Reciprocal-Space Refinement - Job Settings Dialog Box, where you can make settings for running the job.

Status bar

The status bar displays information about the current job settings and status for the panel. The settings includes the job name, task name and task settings (if any), number of subjobs (if any) and the host name and job incorporation setting. The job status can include messages about job start, job completion and incorporation.

Use the Reset button to reset the panel to its default settings and clear any data from the panel. You can also reset the panel from the Job toolbar.

The status bar also contains the Help button , which opens the help topic for the panel in your browser. If the panel is used by one or more tutorials, hovering over the Help button displays a button, which you can click to display a list of tutorials (or you can right-click the Help button instead). Choosing a tutorial opens the tutorial topic.

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