Cryo-EM

Question 1

Cryo EM characteristics

Answer 1

Vitrified material in vitreous ice by EM

Atomic resolution 1,2A
Better preservation (native state)
Less damage
Scarce and heterogenous samples
Resolution limitations
Diff sample preparation
Polymers and viruses diff
90% particles discarded
Limit above 60kDA

Question 2

Process (6)

Answer 2

1- overexpression and purify protein/complex (affinity or size exclusion)
2- grid: hydrophilic to hydrophobic
3- plunge freeze: so fast there is no time to rearrange - <2pm pressure, >10^5°C/sec
4- preservation liquid N2
5- cryo-EM: 1000s micrographs mvies - more inf less noise, less dose imaging (less damage)
6- GPU cluster (process data) ot grid optimisation/sample

Question 3

Types of Cryo

Answer 3

Single particle (78,6%)
Subtomogram averaging (7,9%)
Tomography (6,8%)
Helical reconstruction (5,4%)
Electron (1,2%)

Question 4

Cryo-EM tomography

Answer 4

Structures in situ (not purified protein) - native environment
pleomorphic samples (enveloped viruses and cells)

sub tomogram averaging: tilt samples to get different orientations (+/- 60 degrees) recorded regular intervals - 3D tomogram

Question 5

detectors for the TEM

Answer 5

Film: larger area, good DQE but only 50 exposures, needs devoloping-scanning and adds moisture to microscope

CCD: easy to use, instant but low DQE (0,1)

direct electron: high DQE, fast frame rates movies but expensive

Question 6

DQE (formula)

Answer 6

Detective quantum efficiency
signal to noise ratio

(S/N)^2 out / (S/N)^2 in
output signal to noise ratio / input signal to noise ratio
ideal - DQE=1

• DQE - + noise- - resolution

Question 7

Radiation damage

Answer 7

low dose of electrons to prevent structure deformation
20-50electrons/A2 per exposure/micrograph

Question 8

Single Particle Analysis

Answer 8

motion correction: 1-60secs movies
align movies - 3D (removes junk)
refinement - Gold Standard FSC: slipt in 2, refine individually and compare maps
we know the protein sequence and structures of aa - build amino acid chain into 3D map one residue at a time

Resolution
15A - overall shape
<8A - a helices and b sheets
<4A- b begin separate
<3,5A - aa side chains visible

maps- anisotropic (different resolutions, more rigid better)