UCSF-Chimera

Short Playlist:

So you need to visualize protein structures, analyze binding sites, or understand why a mutation causes disease? Welcome to Chimera — your molecular visualization workhorse.

What is Chimera?

UCSF Chimera — a free molecular visualization program from UC San Francisco. It lets you:

• Visualize 3D protein/DNA/RNA structures
• Analyze protein-ligand interactions
• Measure distances and angles
• Compare structures (superposition)
• Color by various properties (charge, hydrophobicity, conservation, flexibility)
• Generate publication-quality images

Getting Started

Opening a Structure

From PDB (online):

File → Fetch by ID → Enter PDB code (e.g., 1a6m) → Fetch

From file:

File → Open → Select your .pdb file

Representation Styles

The Main Styles

How to Change Representation

Actions → Atoms/Bonds → [stick/ball & stick/sphere/wire]
Actions → Ribbon → [show/hide]

Selection: The Most Important Skill

Everything in Chimera starts with selection. Select what you want, then do something to it.

Selection Methods

Useful Selection Menu Options

Select → Chain → [A, B, C...]           # Select by chain
Select → Residue → [ALA, HIS, IHP...]   # Select by residue type
Select → Structure → Protein            # All protein
Select → Structure → Ligand             # All ligands
Select → Chemistry → Side chain         # Just sidechains
Select → Clear Selection                # Deselect everything
Select → Invert (all models)            # Select everything NOT selected

Zone Selection (Within Distance)

Select everything within X Å of current selection:

Select → Zone...
  → Set distance (e.g., 6 Å)
  → OK

This is super useful for finding binding site residues!

Coloring

Color by Element (Default)

Actions → Color → by element

Color by Hydrophobicity

Tools → Depiction → Render by Attribute
  → Attribute: kdHydrophobicity
  → OK

Why use this? To see the hydrophobic core of proteins — nonpolar residues hide inside, polar residues face the water.

Color by Electrostatic Potential (Coulombic)

This is the red-white-blue coloring from your exercise!

Step 1: Generate surface first

Actions → Surface → Show

Step 2: Color by charge

Tools → Surface/Binding Analysis → Coulombic Surface Coloring → OK

What to look for:

• Binding pockets often have complementary charge to ligand
• DNA-binding proteins have positive (blue) surfaces to attract negative DNA
• Negatively charged ligands (like phosphates) bind in positive (blue) pockets

Color by B-factor (Flexibility)

B-factor = temperature factor = how much an atom "wiggles" in the crystal.

Tools → Depiction → Render by Attribute
  → Attribute: bfactor
  → OK

What to expect:

• Protein core: Blue (rigid)
• Loops and termini: Red (floppy)
• Active sites: Often intermediate

Color by Conservation

When you have multiple aligned structures:

Tools → Sequence → Multialign Viewer
  → (structures get aligned)
Structure → Render by Conservation

Conserved residues = functionally important (active sites, structural core)

Molecular Surfaces

Show/Hide Surface

Actions → Surface → Show
Actions → Surface → Hide

Transparency

Actions → Surface → Transparency → [0-100%]

Use ~50-70% transparency to see ligands through the surface.

Cross-Section (Clipping)

To see inside the protein:

Tools → Depiction → Per-Model Clipping
  → Enable clipping
  → Adjust plane position

Or use the Side View panel:

Tools → Viewing Controls → Side View

Measuring Distances

Method 1: Distance Tool

Tools → Structure Analysis → Distances

Then Ctrl+Shift+Click on first atom, Ctrl+Shift+Click on second atom.

Distance appears as a yellow dashed line with measurement.

Method 2: Command Line

distance :169@OG :301@O34

What Distances Mean

Hydrogen Bonds

What is a Hydrogen Bond?

Donor—H · · · · Acceptor
         ↑
    H-bond (~2.5-3.5 Å)

• Donor: Has hydrogen to give (—OH, —NH)
• Acceptor: Has lone pair to receive (O=, N)

Find H-Bonds Automatically

Tools → Structure Analysis → FindHBond

Options:

• ✓ Include intra-molecule (within protein)
• ✓ Include inter-molecule (protein-ligand)

H-bonds appear as blue/green lines.

Common H-Bond Donors in Proteins

Common H-Bond Acceptors

Salt Bridges (Ionic Interactions)

A salt bridge = electrostatic attraction between opposite charges.

Typical distance: ~2.8–4.0 Å between charged atoms

Coordination Bonds (Metals)

Metals like Fe, Zn, Mg are coordinated by specific atoms:

Example: In myoglobin (1a6m), the proximal histidine coordinates Fe at ~2.1 Å.

Ramachandran Plot

Shows allowed backbone angles (φ/ψ) for amino acids.

Tools → Structure Analysis → Ramachandran Plot

Regions of the Plot

Why Glycine is Special

Glycine has no sidechain → no steric clashes → can be in "forbidden" regions (positive φ).

Select → Residue → GLY

Glycines often appear in the right half of the Ramachandran plot where other residues can't go.

Structural Superposition

Compare two similar structures by overlaying them.

Method 1: MatchMaker (Sequence-based)

Tools → Structure Comparison → MatchMaker
  → Reference: structure 1
  → Match: structure 2
  → OK

Output tells you:

• RMSD (Root Mean Square Deviation): How well they align
  • < 1 Å = very similar
  • 1–2 Å = similar fold

  • 3 Å = significant differences

• Sequence identity %: How similar the sequences are

Method 2: Match (Command)

match #1 #0

Restricting Alignment to a Region

To align just the active site (e.g., within 4 Å of ligand):

sel #1:hem #0:hem zr < 4
match sel

Working with Chains

Delete Unwanted Chains

Select → Chain → B
Actions → Atoms/Bonds → Delete

Select Specific Chain

Select → Chain → A

Or command:

select #0:.A

AlphaFold Structures and pLDDT

What is pLDDT?

AlphaFold stores its confidence score (pLDDT) in the B-factor column.

Color by pLDDT

Since pLDDT is in B-factor column, use:

Tools → Depiction → Render by Attribute → bfactor

Or select low-confidence regions:

select @@bfactor<70

The Hydrophobic Core

Soluble proteins organize with:

• Hydrophobic residues (Leu, Ile, Val, Phe, Met) → inside (core)
• Polar/charged residues (Lys, Glu, Ser, Asp) → outside (surface)

Visualizing the Core

1. Color by hydrophobicity
2. Use cross-section/clipping to see inside
3. Orange/tan inside, blue/cyan outside = correct fold

Protein-Ligand Interaction Analysis

General Workflow

1. Isolate the binding site:

   Select → Residue → [ligand name]
   Select → Zone → 5-6 Å
   

2. Delete or hide everything else:

   Select → Invert
   Actions → Atoms/Bonds → Delete (or Hide)
   

3. Show interactions:

   Tools → Structure Analysis → FindHBond
   

4. Measure specific distances:

   Tools → Structure Analysis → Distances
   

5. Look at electrostatics:

   Actions → Surface → Show
   Tools → Surface/Binding Analysis → Coulombic Surface Coloring
   

What to Report

For protein-ligand interactions, describe:

Example: Analyzing a Binding Site (3eeb)

This is the exercise you did!

The Setup

1. Fetch 3eeb
2. Delete chain B (Select → Chain → B, then Delete)
3. Show surface, color by electrostatics

Result: Blue (positive) binding pocket for the negative IHP (6 phosphates).

The Details

1. Hide surface
2. Select IHP, then Zone 6 Å
3. Invert selection, Delete
4. Show sidechains, keep ribbon
5. Measure distances

Result:

• Ser 169 OG ↔ IHP O34: ~2.8 Å = hydrogen bond (Ser donates H)
• His 55 NE2 ↔ IHP O22: ~2.9 Å = hydrogen bond (His donates H)

The Interpretation

"IHP binding is driven by electrostatic attraction (positive pocket, negative ligand) and stabilized by specific hydrogen bonds from Ser 169 and His 55 to phosphate oxygens."

Cancer Mutations in p53 (1tup)

Example from your lectures showing how to analyze mutation hotspots:

The Hotspot Residues

Analysis Approach

1. Open 1tup, keep chain B
2. Show R175, R248, R273, H179 in spacefill
3. Color surface by electrostatics

Result:

• R248 and R273 are right at the DNA interface (positive surface touching negative DNA)
• R175 and H179 are buried, maintaining the fold
• Mutations here → lose DNA binding → lose tumor suppression → cancer

Common Chimera Workflows

Quick Look at a Structure

1. File → Fetch by ID
2. Actions → Ribbon → Show
3. Presets → Interactive 1 (ribbons)
4. Rotate, zoom, explore

Analyze Active Site

1. Select ligand
2. Select → Zone → 5 Å
3. Actions → Atoms/Bonds → Show (for selection)
4. Tools → Structure Analysis → FindHBond

Compare Two Structures

1. Open both structures
2. Tools → Structure Comparison → MatchMaker
3. Check RMSD and sequence identity

Make a Figure

1. Set up your view
2. Presets → Publication 1
3. File → Save Image

Command Line Quick Reference

The command line is at the bottom of the Chimera window. Faster than menus once you know commands.

Keyboard Shortcuts

Troubleshooting Common Issues

"Nothing selected"

You tried to do something but nothing happened:

• Check: Is anything actually selected? (Green highlighting)
• Fix: Select → [what you want] first

Surface coloring disabled

• Check: Does a surface exist?
• Fix: Actions → Surface → Show first

Can't see ligand

• Check: Is it hidden?
• Fix: Select → Residue → [ligand], then Actions → Atoms/Bonds → Show

Structure looks weird after operations

• Fix: Presets → Interactive 1 to reset to default view

Atoms showing when you want ribbon only

Actions → Atoms/Bonds → Hide
Actions → Ribbon → Show

External Resources for Structure Analysis

TL;DR

Key distances:

• ~2.0 Å = coordination bond
• ~2.5–3.5 Å = hydrogen bond
• ~2.8–4.0 Å = salt bridge

Electrostatic colors:

• Blue = positive
• Red = negative
• White = neutral

Now go visualize some proteins! 🧬