DALI and the importance of DALI searches

The DALI server is an advanced tool for protein structure alignment that is widely used in
computational biology and structural genomics. It allows users to compare protein structures
based on their 3D coordinates, providing insights into structural similarities and evolutionary
relationships. Here is a detailed explanation of how the DALI server works:

1. Overview of DALI Server

The DALI (Distance Matrix Alignment) server is designed to perform pairwise structural
alignments of protein structures. Unlike sequence-based comparison methods, DALI compares
the 3D spatial arrangement of atoms in the protein structures, making it effective even for
proteins with low sequence identity. The server uses a distance matrix approach to compare
protein structures by focusing on the geometric similarity between their atomic coordinates,
rather than the sequence.

2. Input Structure

• Protein Structure File: The primary input to the DALI server is the 3D structure of a
protein, usually in the form of a PDB (Protein Data Bank) file. This file contains the atomic
coordinates of the protein, typically including all atoms, but DALI focuses mainly on the Cα
atoms (backbone atoms) for comparison.

• Multiple Models: The server can also accept structures with multiple models (e.g., NMR
ensembles), and it will select the most representative model for comparison.

3. Preprocessing the Structure

• Cα Atom Selection: In the first preprocessing step, DALI focuses only on the Cα atoms,
which represent the backbone of the protein. This reduction in complexity is done because the
Cα atoms provide sufficient information to compare the overall 3D shape of proteins, and this
simplification helps to reduce computational complexity.

• Removing Water and Ligands: Any bound water molecules, ligands, or heteroatoms are
typically removed during preprocessing, as they are not crucial for the overall protein fold
comparison.

4. Distance Matrix Calculation

• Distance Matrix Construction: Once the Cα atoms are selected, the program calculates a
distance matrix for the protein. This matrix contains the pairwise distances between each pair of
Cα atoms. The distance between two Cα atoms iii and jjj is computed as:
D(i,j)=(xi−xj)2+(yi−yj)2+(zi−zj)2D(i,j) = \sqrt{(x_i - x_j)^2 + (y_i - y_j)^2 + (z_i -
z_j)^2}D(i,j)=(xi−xj)2+(yi−yj)2+(zi−zj)2 where x,y,zx, y, zx,y,z are the 3D Cartesian
coordinates of atoms iii and jjj.
• Structural Information Capture: This matrix captures the spatial relationships between
atoms, reflecting the protein’s three-dimensional conformation.

5. Alignment Procedure

• Rigid Body Transformation: DALI employs a rigid-body transformation method to align

the structures. This involves rotating and translating the protein structures so that the best
possible alignment is achieved while minimizing the structural deviation (root-mean-square
deviation, or RMSD) between the Cα atoms of the query and the reference protein.

o Rotation: The program uses rotational matrices to adjust the orientation of the two
structures in 3D space.

o Translation: The translation ensures that the structures are moved so that their centers of
mass align.

• Optimal Alignment Search: The algorithm searches for the optimal transformation that
minimizes the RMSD between the aligned structures. This is done by systematically trying
different possible alignments and selecting the one that results in the lowest RMSD.

6. Scoring the Alignment

• Z-score: Once the alignment is completed, DALI assigns a Z-score to the structural
alignment. The Z-score is a statistical measure that compares the alignment score to a
distribution of random alignments. A high Z-score indicates that the observed alignment is
unlikely to occur by chance, suggesting significant structural similarity between the proteins.
Typically, a Z-score greater than 2.0 indicates a meaningful structural match.

• RMSD: The Root Mean Square Deviation (RMSD) is calculated to quantify the overall
structural deviation between the aligned structures. A lower RMSD indicates that the structures
are more similar in terms of their 3D geometry.

7. Database Search and Comparison

• Reference Database: DALI performs comparisons not only with the input structure but
also with a database of known protein structures. This database contains thousands of protein
structures, including many solved by X-ray crystallography, NMR spectroscopy, and cryo-EM.
DALI compares the query protein with all proteins in the database and identifies those that show
the greatest structural similarity.

• Multiple Sequence and Structure Search: DALI can generate alignments with multiple
proteins from the database, helping users identify structural homologs even when sequence
identity is low. This feature is especially useful for discovering evolutionary relationships and
potential functional similarities between proteins.

8. Output Results
The output from the DALI server consists of several key components:

• Z-scores and RMSD: For each alignment, the server provides the Z-score and RMSD
values. These scores help evaluate the significance and quality of the alignment.

• Aligned Structures: The program provides a superposition of the query and the aligned
structure. This allows users to visually inspect the regions of the proteins that align well.

• List of Structural Homologs: DALI outputs a list of proteins from the database that show
significant structural similarity to the query protein. This list includes the Z-scores, RMSD, and
other relevant alignment metrics.

• Graphical Representations: Users can view graphical depictions of the structural

superpositions, often as 3D models or distance matrices, helping them understand the degree of
alignment.

9. Advanced Features

• Iterative Search: DALI can perform iterative searches, where it refines the alignment by
considering the top matches from initial rounds of comparison.

• Superimposition: The aligned structures can be overlaid in 3D to show the conserved and
variable regions. This visualization helps researchers understand the evolutionary conservation
of protein folds and identify functional domains.

• Multiple Alignment: DALI can also be used for multiple structure alignments to compare
several proteins at once. This helps in understanding the structural variability within a protein
family.

10. Applications of DALI

DALI is widely used for various purposes in structural biology and bioinformatics:

• Protein Fold Recognition: DALI can be used to predict the fold of a protein by comparing
it with known folds in the database.

• Structural Homology Search: The server helps identify proteins with similar 3D
structures, aiding in the discovery of new functional relationships between proteins.

• Functional Annotation: By comparing a query protein with a set of proteins whose

functions are already known, DALI can suggest potential functions for the query protein based
on structural similarity.

• Molecular Evolution: DALI is useful in evolutionary studies, helping researchers trace

the structural evolution of protein families and domains.
11. Advantages of DALI

• Effective at Low Sequence Identity: DALI is particularly powerful when comparing

proteins with low sequence similarity but similar 3D structures. This makes it ideal for structural
genomics projects.

• Robust and Reliable: DALI has been extensively used in the scientific community for
more than two decades and is considered a standard tool for protein structure comparison.

• Comprehensive Database: The DALI database contains a vast collection of protein

structures, making it a valuable resource for identifying structural homologs.

12. Limitations

• Focus on Cα Atoms: DALI primarily uses the Cα atoms for comparison, which means it
may not capture finer details related to side-chain interactions or other structural features.

• Rigid-Body Alignment: The rigid-body alignment approach may not work as well for
proteins that undergo significant conformational changes or have flexible regions.

13. Usage and Access

• Web Interface: DALI is accessible via a web interface, where users can upload their
protein structures and perform the alignment. The results are typically available in a few minutes,
depending on the complexity of the comparison.

• Automated Tools: The DALI server also provides automated tools for integrating the
alignment results into other workflows, such as structural analysis and functional annotation.

In summary, the DALI server is a powerful tool for comparing protein structures, providing
insights into structural similarities and evolutionary relationships that may not be apparent from
sequence alone. Its focus on 3D spatial alignment makes it an indispensable tool in the study of
protein structure-function relationships.

DALI Lite

The DALI Lite program is a lightweight version of the DALI (Distance Matrix Alignment)
server, designed to perform protein structure alignment using a fast and efficient method. DALI
Lite works by comparing the 3D structures of proteins and identifying similar structural features,
even when the sequence identity between them is low. Here’s a detailed explanation of how it
works:

1. Input Structure
• Protein Structure: Users input a protein structure in the Protein Data Bank (PDB) format
or its equivalent. The structure can be in the form of a single model or a set of models (e.g., from
a crystallographic or NMR structure).

• Alignment Type: DALI Lite can perform pairwise structure comparisons, typically
between a query structure and a database of known protein structures.

2. Preprocessing the Structure

• Reduction to Cα Atoms: The program first reduces the complexity of the protein structure
by focusing only on the backbone Cα atoms (the carbon atoms of the amino acid backbone). This
simplifies the representation of the protein, reducing computational time.

• Spatial Coordinates: The spatial coordinates of the Cα atoms are extracted for further
calculations. This step ensures that the program compares only the spatial arrangement of atoms,
rather than their chemical or biological properties.

3. Pairwise Structural Alignment

• Distance Matrix Calculation: For each input protein, DALI Lite computes a distance
matrix based on the positions of the Cα atoms. The matrix contains the pairwise distances
between the Cα atoms in the structure. This matrix serves as the foundation for comparing
different protein structures.

• Rigid Body Transformation: To align the two structures, the program employs a rigid-
body transformation that adjusts for rotations and translations of the protein structures. The
transformation allows for a best-fit alignment of the structures in 3D space, minimizing the root-
mean-square deviation (RMSD) between corresponding atoms.

• Pairwise Comparison of Structures: The program compares the structure of the input
protein with the structures in a reference database using the distance matrix. The comparison is
based on the spatial arrangement of atoms rather than sequence similarity, making it effective
even when sequence homology is low.

4. Scoring the Alignment

• Z-score: The program assigns a Z-score to each alignment. The Z-score quantifies how
well the two structures align compared to a random alignment. A high Z-score indicates a good
structural match, while a low Z-score suggests a poor alignment. Typically, a Z-score greater
than 2.0 suggests a significant structural similarity.

• RMSD: The RMSD between the aligned structures is also calculated. A lower RMSD
value indicates a closer match between the structures.

5. Result Interpretation
• Structural Superposition: The results include a visual representation of the aligned
structures. The user can examine the degree of structural similarity and the regions of the protein
that align well.

• Homology and Functional Insights: By comparing the structural alignment results with
those of known proteins, DALI Lite can provide insights into potential functional similarities
between the query protein and proteins with known functions.

• Database Search: If the user has selected a database of proteins, the program outputs a list
of structures from the database that show the highest structural similarity to the input protein,
with detailed Z-scores and RMSD values.

6. Optimized for Speed

• Fast Execution: DALI Lite is optimized for speed and efficiency. By focusing on
backbone atoms and using fast matrix calculations, it provides quick results compared to the
original, more computationally intensive DALI method.

• Limited Database: The database used for comparisons in DALI Lite may be more limited
compared to the full DALI server, which contains a larger set of protein structures. This trade-off
ensures that results are generated quickly without the need for extensive computational
resources.

7. Output Results

• Alignment Summary: The final output includes a detailed summary of the structural
alignment, including the alignment score, Z-score, RMSD, and a list of aligned proteins.

• Graphical Output: Users can also access a graphical representation of the aligned
structures to visually inspect the structural similarities and differences.

• Sequence Annotations: While DALI Lite focuses on structure rather than sequence, it
may also include sequence information to help users correlate structural features with known
protein domains or functional regions.

Use Cases of DALI Lite:

• Structural Homology Searching: It is used for identifying proteins with similar 3D

structures, even when sequence identity is low, which can provide insights into evolutionary
relationships and functional similarities.

• Protein Structure Validation: It helps in validating the predicted structures by comparing

them against known protein structures in the database.
• Comparative Structural Genomics: Researchers can use DALI Lite to explore the
structural diversity of protein families, especially when sequence-based methods do not provide
conclusive results.

Overall, DALI Lite is a powerful tool for quick and efficient structural comparison, particularly
in large-scale studies where speed is essential, yet the need for accurate 3D structural alignment
is still crucial.

Importance of performing DALI searches for annotating protein domains

Performing a DALI search plays a crucial role in annotating protein domains, particularly in the
context of structural genomics and functional annotation. Here’s why DALI is valuable for domain
annotation:

1. Identification of Structural Homologs

Structural Comparison Beyond Sequence: Traditional sequence-based methods (e.g., BLAST) can
identify homologous proteins that share sequence similarity. However, many proteins that share
similar 3D structures may have little sequence identity. DALI is particularly effective at identifying
these structural homologs by comparing the 3D spatial arrangement of atoms. This ability is crucial
for domain annotation when sequences are too divergent to provide reliable results via sequence
homology alone.

Discovering Remote Homologs: DALI can reveal remote homologs (proteins that share a similar fold
but have diverged significantly in sequence), which can be crucial for identifying conserved
functional domains that might not be obvious through sequence alignment methods.

2. Accurate Domain Mapping

Structural Alignment for Domain Identification: DALI aligns protein structures based on their 3D
coordinates, and this alignment can highlight structurally conserved domains across different
proteins. These domains are often functionally significant, and their identification is key to
understanding protein function.

Revealing Novel Domains: By comparing an uncharacterized protein with known structures in the
DALI database, researchers can identify novel domains or domain architectures that may not have
been previously annotated. DALI can link these novel domains to known structural families,
suggesting their potential function and evolutionary history.

3. Functional Annotation Based on Structure

Function Inference from Structure: The 3D structure of a protein often carries significant functional
information. Proteins that share similar structural domains tend to have related functions, even if
their sequences differ. By identifying structural homologs using DALI, one can infer potential
functions for uncharacterized proteins or regions of proteins. This is especially useful when
sequence-based functional annotation is inconclusive or unavailable.

Mapping of Active Sites and Functional Regions: In addition to domain identification, DALI's
alignment provides a way to identify active sites and functional regions conserved across related
proteins. This can assist in understanding the mechanisms of enzymatic activities, binding sites, or
other functional roles that are encoded in the protein structure.

4. Evolutionary Insights into Domain Evolution

Tracing Domain Evolution: DALI can be used to study the evolution of protein domains across
different species or protein families. By aligning structures from different organisms, DALI can help
identify how specific domains evolved and adapted to different functional needs. This evolutionary
insight is crucial for annotating the functional diversification of domains and understanding how
certain domains may have acquired new functions through evolution.

Domain Duplication and Divergence: DALI’s ability to detect structural similarity can help track
domain duplication events, where a domain has been copied within a protein or across different
proteins, leading to functional specialization in different contexts.

5. Improving Domain Databases

Enhancing Domain Classification: DALI's ability to identify structural homologs also contributes to
the improvement of domain classification databases, such as Pfam, SCOP, or CATH. DALI helps in
refining domain boundaries, detecting new domain families, and updating existing domain
classifications. This aids in curating domain annotations that can be used for functional
predictions.

Support for Domain Architecture Predictions: By aligning structures with known domain databases,
DALI can assist in predicting domain architectures in newly sequenced proteins. For example, if a
domain is structurally similar to a known catalytic domain or a binding domain, it can be annotated
accordingly, helping researchers make accurate predictions about protein function.

6. Integration with Other Structural and Functional Resources

Integration with Structural Databases: DALI can be integrated with other databases like PDB,
UniProt, or InterPro to cross-reference structural alignments with available sequence and
functional data. This integration enhances the reliability of domain annotations and ensures that
structural annotations are consistent with known functional information.

Comparing Multiple Proteins: DALI can also perform multiple structure alignments, allowing the
comparison of several proteins simultaneously. This feature is useful for understanding the
diversity of domain architectures within a family and can reveal conserved functional regions that
are shared by multiple members of a protein family.

7. Resolving Ambiguities in Domain Boundaries

Refining Domain Boundaries: DALI’s structural alignment approach helps resolve ambiguities in
defining the boundaries of protein domains, especially in cases where sequence-based methods
might not be able to detect subtle structural differences. By comparing protein structures, DALI can
highlight boundaries of domains and their linkers, refining domain annotation.

8. Handling Structural Variability

Capturing Conformational Changes: Some domains may exhibit conformational flexibility or
induced fit, where their structures change depending on the environment or interaction partners.
DALI can compare different conformations of the same domain to detect such variability and help
annotate domains in a way that accounts for these structural changes.

9. Enhancing Accuracy in Structural Prediction

Validation of Predicted Structures: DALI can be used to validate predicted protein structures,
including models generated by homology modeling or computational predictions. By comparing the
predicted structure to a known structural template, DALI can help confirm or refine the predicted
domain boundaries and functions.