Cryo-EM is revolutionizing structure determination

Cryo-electron microscopy (aka electron cryo-microscopy)

Don’t have to

Protein needs to be pure, but

you don’t need quite as much
compared to X-ray and NMR.

Recent advances in
technology enable

Limitations: So far, only works well for big proteins and protein complexes
(Ribosomes work really well!)

Equipment is very expensive right now, not yet widely available.

Cryo-EM data from your TA, Ryan Feathers

500 nm
 CHESS = Cornell’s High Energy Synchrotron Source

THE DIFFRACTION EXPERIMENT

Collect dozens (hundreds) of images like this, use computers to crunch the data

This is real data collected

by my lab at CHESS
electron density from a protein

The Protein DataBank: A public depository of all protein structures ever determined!
(In academia)
X-ray: ~132,000 deposited structures
NMR: ~12,500 deposited structures
EM/cryo-EM: ~2,800 deposited structures (growing fast)
Check it out: www.rcsb.org
 Announcements / review
Lecture 4

Remember:

Keep current with the textbook reading (see syllabus online).

Reading for today: 115-124
Reading for tomorrow: 125-133, 137-142

Yesterday:
Experimental methods for studying proteins

Today:
Protein secondary structure

Bonds and interactions in solution

For extra reading on this topic, see textbook pages 43-50, 114-115 (5th) or 47-54,116-117 (6th)

In general:
Types of chemical bonds:
Covalent bond – Typical “bond” between atoms (CO2)
Covalent bonds are

Ionic bond – Bonds dissociated by

Hydrogen-bond or “H-bond” (10-40kJ/mol)

Other important interactions:

van der Waals / London dispersion forces -
“Atoms like to touch” or “Nature abhors a vacuum”
This is why

Hydrophobic interaction (~5kJ/mol)

Critical for
 Protein structure is dictated by weak forces

In proteins:
All of these bonds and forces are important in determining protein structure.

WHAT LEVEL(S) OF STRUCTURE ARE COVALENT BONDS IMPORTANT FOR?

Instead of ionic bonds, we consider:

Electrostatic interactions - (10-40kJ/mol) called:

Most important “weak” force for determining protein structure:

The hydrophobic effect causes peptides to fold into proteins

Simplified schematic:

Polypeptides – low solubility in water

Driving force: Black atoms:
See pages ENTROPY of
52-53 hydrophobic
water
of textbook molecules Red/blue atoms:
hydrophilic

Folded proteins – high solubility in water

 Hydrogen bonds (H-bonds)

The folded protein must allow virtually

H-bonds have an optimal

o
2.8 - 3.2 A
Straight line through
electronegative atoms and
hydrogen

About

1 angstrom = 0.1 nm

Hydrogen bonds

The peptide bond provides both

Donate: Ser, Thr, Cys, Tyr, Trp, Asn, Gln, His, Lys*, Arg*
Sidechains: *electrostatic interaction is more
Accept: Ser, Thr, Cys, Tyr, Asn, Gln, His, Glu*, Asp* likely

Don’t memorize this list – instead, go look at the sidechain structures, and the
types of groups that donate or accept to understand why!

Be able to identify potential hydrogen bonds if given chemical structures.

The polypeptide backbone is limited to certain conformations
Two main constraints: Torsion angles:
F = PHI Y = PSI

Peptide bond:

Y = 1800

For fully
extended
polypeptide:

Carbon names: mainchain: a-carbon, carbonyl-carbon

sidechain: b-carbon, ….
Peptide bonds =
Single bonds allowing free rotation:

6 atoms form a plane that rotates: ca(i) - ca(i+1)

Steric hindrance can be plotted on a phi / psi graph

STIPPLED REGIONS SHOW THE “PROHIBITED” STERIC CONTACTS
CALCULATED FOR POLYGLYCINE CALCULATED FOR POLYALANINE

Steric hindrance:

FOR A TYPICAL PROTEIN, ONLY

THIS SIMPLE STERIC HINDRANCE FACTOR CONTROLS WHICH PROTEIN STRUCTURES

ARE POSSIBLE
 The Ramachandran plot
Showing allowed phi, psi combinations.
Calculated, for all non-glycine residues in proteins
REALITY: COMPOSITE F, Y FOR
A FEW HUNDRED PROTEINS
+180o

Y
0o

-180o
-180o 0o +180o
f
(includes glycines)
Allowed regions shown in blue.
Darkest blue regions =
Lighter blue =

Defining secondary structure

The sequence of phi, psi, and omega values defines the path of the mainchain, and is referred to
as the secondary structure. Can describe either the entire polypeptide or shorter segments.

Secondary structures allow proteins to

The “regular” secondary structural elements consist of repeating phi/psi values:

“poly-pro” or “left-handed” helices (textbook => collagen)

Another major secondary structure element: turns (common, but not

What else can the polypeptide chain look like? (~50% of protein residues)
coil: structured, but
loops: “unstructured” because of
Recently appreciated – functional role of some “unstructured” parts of proteins

All of these elements also make all possible H-bonds!

 The a-helix (“alpha-helix”)

REPEATING : F ~ -57o, Y ~ -47o

ALL R GROUPS POINT

H-BONDS PARALLEL TO
(rotated 1800 from
the figure in your
text) EVERY PEPTIDE CARBONYL IS ACCEPTOR FOR
PEPTIDE N-H DONOR 4 AA AWAY; but…
TERMINAL AA NEED
HELIX ALWAYS
HELIX ROTATES 360o EVERY 3.6 AA

ANY AA CAN BE IN AN a-HELIX, HOWEVER:

• RUNS OF POSITIVE OR NEGATIVE CHARGE
UNFAVORABLE;
• AA VARY IN TENDENCY TO BE IN HELIX AND
End-on view

Many a-helices are “amphipathic”

COLOR SCHEME:
BLUE - POLAR/CHARGED
YELLOW - NONPOLAR

HELIX TURNS 360o

EVERY 3.6 AA;
IF POLAR RESIDUES (Myoglobin)
ARE SPACED ~ EVERY 3
- 4 AA, THEY ARE ALL
ON ONE SIDE OF THE
HELIX!
AA 125 - 146
Ala-Asp-Ala-Gln-Gly-Ala-Met-Asn-
Lys-Ala-Leu-Glu-Leu-Phe-Arg-Lys-
Asp-Ile-Ala-Ala-Lys-Tyr-

THESE AMPHIPATHIC HELICES ARE AT THE

THEY FOLLOW THE “RULE OF THE

HYDROPHOBIC INTERACTION”:
AA 1 - 17
Val-Leu-Ser-Glu-Gly-Glu-Trp-Gln-
Leu-Val-Leu-His-Val-Trp-Ala-Lys-Val-
The “helical wheel” allows prediction of a-helix structure

Is this helix amphipathic?

b-Sheets (“beta-sheets”) and b-strands

REPEATING:
F: -60o to -150o;
Y: +90o to +180o

C N C C
ALL POSSIBLE H-BONDS
ARE
N C N N

STRANDS ARE SO-CALLED

“

R GROUPS alternate
ANTIPARALLEL PARALLEL

RUNS OF b-SHEET ARE

SIDE-BY-SIDE STRANDS,
NOT