Calculates the neutron and x-ray scattering length density (SLD), contrast and I(0) values for a single molecule or complex consisting of proteins and/or nucleic acids, as well as other compounds such as polymers, lipids and carbohydrates, based only on sequence or chemical formula information.
Link to PDF file of a step-by-step tutorial using SASSIE-GUI (stand-alone)
The Contrast Calculator module is accessible from the Tools section of the main menu.
Basic Usage
The purpose of the module is to calculate neutron and x-ray SLD, contrast and I(0) values from biomolecular complexes to assist in experiment planning. The module handles complexes consising not only of proteins and nucleic acids but also polymers, lipids, surfactants, carbohydrates, etc. Single species not in complexes can also be handled.
Notes
Typical usage us to input protein and/or nucleic acid in formation in either FASTA or PDB format to calculate neutron and x-ray SLD, contrast and I(0) values for protein/nucleic acid complexes or protein/deuterated protein complexes. Note that using PDB files may not give the same answer as when using FASTA-formatted sequences if the PDB files have missing residues.
Polymers, lipids, surfactants, carbohydrates, etc. can also be part of the complex. Checmical formulas and corrresponding mass densities are entered manually.
The neutron SLD, contrast and I(0) values are calculated as a function of the %D2O in the solvent. The fraction of deuteration for the proteins and/or nucleic acids is considered in the calculation. D atoms are inserted into chemical formulas manually.
Non-water solvent components, such as salts, Tris, glycerol, etc. can also be considered if desired.
There is no limit to the number of proteins, nucleic acids or other components that comprise the complex. However, plots of the neutron SLD, contrast and I(0) as a function of %D2O are only provided if the total number of components is less than 3.
The SLD, contrast and I(0) information are output in 3 separate files. The output files contain the input information as well as the output parameters for both x-rays and neutrons.
Case 1: Protein/DNA Complex in water with 0.15 M NaCl
This is an example of a protein-DNA complex consisting of two synapsis-deficient Cre(A36V) recombinase molecules bound to a single loxP site. Two FASTA files are available; one contains the sequence of a single Cre(A36V) protein and the other contains the sequence of the loxP DNA.
Screen Shots and Description of Input Fields
The top of the Contrast Calculator screen contains the main user input.
run name: user defined name of folder that will contain the results.
output file name prefix: The main part of the name of three files that will contain the SLD, contrast and I(0) calculations.
number of protein, DNA or RNA input files: The number of FASTA and/or PDB files that will be used in the complex. In this case, there is one protein and one DNA file, so the number of protein, DNA or RNA input files is 2. Additional information regarding the input files then must be provided as follows.
FASTA? [1]: Select ON since the Cre(A36V) protein sequence is in FASTA format.
input file name [1]: Input FASTA file for the Cre(A36V) protein.
number of units [1]: The number of Cre(A36V) proteins in the complex, which is 2 in this case since the FASTA file contains the sequence of a single Cre(A36V) protein.
fraction deuterated [1]: The fraction of the Cre(A36V) non-exchangeable hydrogen atoms, i.e., those bound to carbon atoms, that have been replaced with deuterium, which is 0 in this case.
molecular type [1]: Choose protein from the pull-down menu.
FASTA? [2]: Select ON since the loxP DNA sequence is in FASTA format.
input file name [2]: Input FASTA file for the loxP DNA.
number of units [2]: The number of loxP DNAs in the complex, which is 1 in this case.
fraction deuterated [2]: The fraction of the DNA non-exchangeable hydrogen atoms, i.e., those bound to carbon atoms, that have been replaced with deuterium, which is 0 in this case.
molecular type [2]: Choose DNA from the pull-down menu.
number of additional components: The number of additional components in the complex that are not protein, DNA or RNA, such as polymers, lipids, carbohydrates, etc. If this number is not zero, then additional input fields will be provided to enter the desired chemical formula, mass density and other pertinent information. (If the number of protein, DNA or RNA input files is zero, then this number cannot be zero.)
The Optional Input is located below the main user input
total solute concentration (mg/ml): The concentration of the complex (both protein and DNA in this case). This information is needed to calculate the x-ray and neutron I(0) values. The default value of 1 mg/ml is convenient since the calculated I(0) values can easily be multiplied by a different concentration value to easily obtain the I(0) values for the new concentration.
solvent % D2O step: The step increment in the % D2O in the solvent for which the neutron SLD, contrast and I(0) values will be calculated.
fraction exchangeable H (protein): The fraction of exchangeable hydrogen atoms, i.e., those bound to nitrogen and oxygen, in the protein components in the complex that actually do exchange in solvents containing D2O. This value applies to all protein molecules in the complex and is automatically adjusted according to the % D2O in the solvent.
fraction exchangeable H (nucleic acid): The fraction of exchangeable hydrogen atoms, i.e., those bound to nitrogen and oxygen, in the DNA and/or RNA components in the complex that actually do exchange in solvents containing D2O. This value applies to all DNA and RNA molecules in the complex and is automatically adjusted according to the % D2O in the solvent.
number of non-water solvent components: The number of components in the solvent that are not water, i.e., sugars, salts, glycerol, components of Tris, HEPES, phosphate buffers, etc. In this case, since the water contains 0.15 M NaCl, the number of components is 1 and additional information must be provided as follows.
component atomic formula [1]: The atomic formula for the component in the solvent.
concentration (M) [1]: The molar concentration of the component.
Example Output
The output screen lists the match points for the protein and DNA components in the complex as well as for the entire complex. The output also shows plots of the calculated neutron SLDs, contrasts, I(0) and sqrt[I(0)] as a function of % D2O in the solvent since the complex doesn't contain more than 2 components. Note that roll-over help will indicate options to resize, zoom and reset the view of the plots. Resultant files containing the SLD, contrast and I(0) values are written to a new directory, "contrastcalculator", within the run0 directory.
From the SLD and contrast plots as well as the match point values, it is clear that the protein match point is very close to that of the entire complex. Therefore, it will not be possible to make a measurement at the protein match point in order to observe the scattering from the DNA component, as this will be too close to the match point for the entire complex and there will be no measurable signal. A measurement can be made at the DNA match point of 62% D2O in order to observe the scattering from the protein component. However, the I(0) value at 62% D2O is very low at 1 mg/ml and a higher concentration would be needed to obtain a reasonable signal.
Case 2: Protein/Deuterated Protein Complex in water with no additional components
This is an example of a protein-deuterated protein complex consisting of a skp chaperone trimer and a 50% deuterated unfolded outer membrane protein, ompA. Two PDB files are available; one contains the structure of the skp trimer and the other contains the structure of the ompA membrane protein.
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Screen Shots and Description of Input Fields
The top of the Contrast Calculator screen contains the main user input.
run name: user defined name of folder that will contain the results.
output file name prefix: The main part of the name of three files that will contain the SLD, contrast and I(0) calculations.
number of protein, DNA or RNA input files: The number of FASTA and/or PDB files that will be used in the complex. In this case, there is one protein and one deuterated protein file, so the number of protein, DNA or RNA input files is 2. Additional information regarding the input files then must be provided as follows.
FASTA? [1]: Select OFF since the skp trimer protein structure is in PDB format.
input file name [1]: Input PDB file for the skp protein.
number of units [1]: The number of skp proteins in the complex, which is 1 in this case since the PDB file contains the structure of the entire skp trimer.
fraction deuterated [1]: The fraction of the skp non-exchangeable hydrogen atoms, i.e., those bound to carbon atoms, that have been replaced with deuterium, which is 0 in this case.
molecular type [1]: Choose protein from the pull-down menu.
FASTA? [2]: Select OFF since the ompA protein structure is in PDB format.
input file name [2]: Input PDB file for the ompA protein.
number of units [2]: The number of ompA proteins in the complex, which is 1 in this case.
fraction deuterated [2]: The fraction of the protein non-exchangeable hydrogen atoms, i.e., those bound to carbon atoms, that have been replaced with deuterium, which is 0.5 in this case since the protein is 50% deuterated.
molecular type [2]: Choose protein from the pull-down menu.
number of additional components: The number of additional components in the complex that are not protein, DNA or RNA, such as polymers, lipids, carbohydrates, etc. If this number is not zero, then additional input fields will be provided to enter the desired chemical formula, mass density and other pertinent information. (If the number of protein, DNA or RNA input files is zero, then this number cannot be zero.)
The Optional Input is located below the main user input
total solute concentration (mg/ml): The concentration of the complex (both proteins in this case). This information is needed to calculate the x-ray and neutron I(0) values. The default value of 1 mg/ml is convenient since the calculated I(0) values can easily be multiplied by a different concentration value to easily obtain the I(0) values for the new concentration.
solvent % D2O step: The step increment in the % D2O in the solvent for which the neutron SLD, contrast and I(0) values will be calculated.
fraction exchangeable H (protein): The fraction of exchangeable hydrogen atoms, i.e., those bound to nitrogen and oxygen, in the protein components in the complex that actually do exchange in solvents containing D2O. This value applies to all protein molecules in the complex and is automatically adjusted according to the % D2O in the solvent.
fraction exchangeable H (nucleic acid): The fraction of exchangeable hydrogen atoms, i.e., those bound to nitrogen and oxygen, in the DNA and/or RNA components in the complex that actually do exchange in solvents containing D2O. This value applies to all DNA and RNA molecules in the complex and is automatically adjusted according to the % D2O in the solvent. Since there are no DNA or RNA molecules in this complex, this number will be ignored.
number of non-water solvent components: The number of components in the solvent that are not water, i.e., sugars, salts, glycerol, components of Tris, HEPES, phosphate buffers, etc. In this case, the number of components is 0 and no additional information is required.
Example Output
The output screen lists the match points for the protein and 50% deuterated protein components in the complex as well as for the entire complex. The output also shows plots of the calculated neutron SLDs, contrasts, I(0) and sqrt[I(0)] as a function of % D2O in the solvent since the complex doesn't contain more than 2 components. Note that roll-over help will indicate options to resize, zoom and reset the view of the plots. Resultant files containing the SLD, contrast and I(0) values are written to a new directory, "contrastcalculator", within the run1 directory.
From the SLD and contrast plots as well as the match point values, it is clear that both the protein and deuterated protein match point are well separated from that of the entire complex. Therefore, it is possible to make measurements at both contrast points, provided that the I(0) values are sufficient to obtain a measurable signal at those match points. In this case, concentrations higher than 1 mg/ml would be needed to obtain a reasonable signal at these match points.
Case 3: Protein/lipid Complex (Nanodisc) in water with no additional components
This is an example of a protein/lipid nanodisc consisting of 2 MSP1D1 proteins and 130 POPC lipid molcules. A FASTA file containing the sequence of MSP1D1 is available. The chemical formula for POPC, C42H82NO8P, will be used for the lipid component of the complex.
Screen Shots and Description of Input Fields
The top of the Contrast Calculator screen contains the main user input.
run name: user defined name of folder that will contain the results.
output file name prefix: The main part of the name of three files that will contain the SLD, contrast and I(0) calculations.
number of protein, DNA or RNA input files: The number of FASTA and/or PDB files that will be used in the complex. In this case, there is one protein, so the number of protein, DNA or RNA input files is 1. Additional information regarding the input files then must be provided as follows.
FASTA? [1]: Select ON since the MSP1D1 protein sequence is in FASTA format.
input file name [1]: Input FASTA file for the MSP1D1 protein.
number of units [1]: The number of MSP1D1 proteins in the complex, which is 2 in this case since the FASTA file contains the sequence of a single MSP1D1 protein.
fraction deuterated [1]: The fraction of the MSP1D1 non-exchangeable hydrogen atoms, i.e., those bound to carbon atoms, that have been replaced with deuterium, which is 0 in this case.
molecular type [1]: Choose protein from the pull-down menu.
number of additional components: The number of additional components in the complex that are not protein, DNA or RNA, such as polymers, lipids, carbohydrates, etc. Since POPC lipid is in this complex, the number of additional components is 1. Additional information regarding the component must be provided as follows.
chemical formula [1]: The chemical formula for POPC is entered as (C42H82NO8P)130 since there are 130 POPC molecules in the complex.
number of exchangeable H [1]: The number of exchangeable hydrogen atoms, i.e., those bound to nitrogen and oxygen, per POPC molecule. Since POPC has no exchangeable hydrogen atoms, 0 is entered in this case.
fraction exchangeable H [1]: The fraction of exchangeable hydrogen atoms in POPC that actually do exchange in solvents containing D2O. This applies to all POPC molecules in the complex and is automatically adjusted according to the % D2O in the solvent. Since POPC has no exchangeable hydrogen atoms, 0 is entered here as well.
mass density [1]: The mass density of POPC (g/cm3)
The Optional Input is located below the main user input
total solute concentration (mg/ml): The concentration of the complex (both protein and lipid in this case). This information is needed to calculate the x-ray and neutron I(0) values. The default value of 1 mg/ml is convenient since the calculated I(0) values can easily be multiplied by a different concentration value to easily obtain the I(0) values for the new concentration.
solvent % D2O step: The step increment in the % D2O in the solvent for which the neutron SLD, contrast and I(0) values will be calculated.
fraction exchangeable H (protein): The fraction of exchangeable hydrogen atoms, i.e., those bound to nitrogen and oxygen, in the protein components in the complex that actually do exchange in solvents containing D2O. This value applies to all protein molecules in the complex and is automatically adjusted according to the % D2O in the solvent.
fraction exchangeable H (nucleic acid): The fraction of exchangeable hydrogen atoms, i.e., those bound to nitrogen and oxygen, in the DNA and/or RNA components in the complex that actually do exchange in solvents containing D2O. This value applies to all DNA and RNA molecules in the complex and is automatically adjusted according to the % D2O in the solvent. Since there are no DNA or RNA molecules in this complex, this number will be ignored.
number of non-water solvent components: The number of components in the solvent that are not water, i.e., sugars, salts, glycerol, components of Tris, HEPES, phosphate buffers, etc. In this case, the number of components is 0 and no additional information is required.
Example Output
The output screen lists the match points for POPC (Molecule 1) and protein components in the complex as well as for the entire complex. The output also shows plots of the calculated neutron SLDs, contrasts, I(0) and sqrt[I(0)] as a function of % D2O in the solvent since the complex doesn't contain more than 2 components. Note that roll-over help will indicate options to resize, zoom and reset the view of the plots. Resultant files containing the SLD, contrast and I(0) values are written to a new directory, "contrastcalculator", within the run2 directory.
