Specimen preparation for high-resolution cryo-EM · continuous film grid bar Amorphous carbon Gold Foil materials-"!-"/ '"-"-">? 1 1 Lacey carbon Holey carbon Types of specimen supports

Specimen preparationfor high-resolution cryo-EM

Christopher J Russo

Copyright C. J. Russo, MRC Laboratory of Molecular Biology, 2016

All rights reserved.

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The specimen is the limiting factor

Passmore & Russo MiE 2016

Protein Preparation

Negative stain

Diagnostic cryo-EM

Initial cryo-EM data collection

•Composition•Purity, homogeneity•Stability (buffer composition)

•Biochemical activity

•Discrete particles•Stability•Particle size and shape

•Stability•Particle size and shape•Particle distribution vs. concentration

•High resolution 2D classes•Initial 3D model•Orientation distribution•Particle yield

High res cryo-EM data collection

180°

90°

0°

-180° 0 180°


Example data Evaluation criteria3

2

1

0

Abs.

(AU)

30 Volume (mL)

66–

31–

6.5–

45–

14–

M.W

. (kD

a)

Step

•Tilt-pairs / validation•Motion statistics•Angular accuracy•Local / overall resolution•Conformational states

Entry levelEntry levelM

id-rangeM

id-rangeHigh End

High End

TEM1

2

3

4

5

metal grid bar

amorphous carbon membrane

ice embedded protein particles

electron microscope grid80 μm

1 μm

Standard cryo-EM specimen


Protein Preparation

Negative stain

Diagnostic cryo-EM








180°

90°

0°

-180° 0 180°



2

1

0

Abs.

(AU)

30 Volume (mL)

66–

31–

6.5–

45–

14–

M.W

. (kD

a)

Step



id-rangeM

id-rangeHigh End

High End

TEM1

2

3

4

5

Protein Preparation

Negative stain

Diagnostic cryo-EM








180°

90°

0°

-180° 0 180°


2

1

0Ab

s. (A

U)30 Volume (mL)

66–

31–

6.5–

45–

14–

M.W

. (kD

a)Step



id-rangeM

id-rangeHigh End

High End

TEM1

2

3

4

5


Homogeneity• Looks homogeneous on SDS-PAGE and native PAGE• Biochemically active

A. Easter

SEC-MALS Superose 6

SEC MALS(Size exclusion chromatography coupled to multi-angle light scattering)

DLS(Dynamic light scattering)

Size exclusion chromatography

Homogeneity

*Optimise buffer conditions (salt, pH,

detergent, etc)

Quantity2 – 4 µl / grid50 nM – 5 µM concentration– Make sure your complex is stable at these

concentrations (e.g. consecutive SEC runs)

Vinothkumar & Henderson 2016

Protein Preparation

Negative stain

Diagnostic cryo-EM








180°

90°

0°

-180° 0 180°


2

1

0

Abs.

(AU)

30 Volume (mL)

66–

31–

6.5–

45–

14–

M.W

. (kD

a)Step



id-rangeM

id-rangeHigh End

High End

TEM1

2

3

4

5


Negative stain• Sample is embedded in a layer of heavy metal salts (e.g.

uranium, molybdenum, tungsten) which surrounds the protein like a shell

• Reveals the solvent-excluded surface and shape of the molecule

Carbon support

Electron beam

Negative Stain

Protein

Crowther et al. (1970) Nature 226, 421

Tomato bushy stunt virus

Schreiber et al (2011) NatureDa Fonseca et al (2011) Nature

Negative stain• Advantages– Quick to screen lots of conditions (10-20 in a day)• Evaluate homogeneity and size, presence of binding partners

– Very high contrast – small molecules– Mildly radiation sensitive– Relatively straightforward

• Disadvantages– Limited resolution (~20 Å)– Protein damage or distortion (high salt, low pH, dehydrated)

– often proteins are flattened– Uneven staining leads to difficulties with processing/

classification

Protein Preparation

Negative stain

Diagnostic cryo-EM








180°

90°

0°

-180° 0 180°


2

1

0

Abs.

(AU)

30 Volume (mL)

66–

31–

6.5–

45–

14–

M.W

. (kD

a)

Step



id-rangeM

id-rangeHigh End

High End

TEM1

2

3

4

5


Jacques Dubochet

Vitreous = glass, amorphousNOT crystalline

1 μm

200 Å

80 μm

grid bar

grid bar

3 mm

Copper!"#$%&

'"()*"+,

-"&"#.*

Gold/+01

2.&345%*+,

6&+,"*+,

'+*78(%*

Grid materials Film materials

Amorphous carbon9:);1%*%

9:);1%*%<.="5%

'"-"

grid

support/substrate

foil foilice embedded protein particles

continuous film

grid bar

Amorphous carbonGold

Foil materials

-"!

-"/

'"-"

-">?

1

1

Types of specimen supports

Russo & Passmore Current Op. 2016

1 μm

200 Å

80 μm

grid bar

grid bar

3 mm

Copper!"#$%&

'"()*"+,

-"&"#.*

Gold/+01

2.&345%*+,

6&+,"*+,

'+*78(%*



9:);1%*%<.="5%

'"-"

grid

support/substrate


continuous film

grid bar


Foil materials

-"!

-"/

'"-"

-">?

1

1

Lacey carbon

Holeycarbon



1 μm

200 Å

80 μm

grid bar

grid bar

3 mm

Copper!"#$%&

'"()*"+,

-"&"#.*

Gold/+01

2.&345%*+,

6&+,"*+,

'+*78(%*



9:);1%*%<.="5%

'"-"

grid

support/substrate


continuous film

grid bar


Foil materials

-"!

-"/

'"-"

-">?

1

1



gold grid bar

gold membrane



1 μm

250

200

150

100

50

0Verti

cal d

isplac

emen

t (Å)

6050403020100Fluence e/Å2

Grid vertical movement

Quantifoil

16 e/Å2/s30° tilt real time Russo & Passmore Science 2014

250

200

150

100

50

0Verti

cal d

isplac

emen

t (Å)

6050403020100Fluence e/Å2

Gold



Quantifoil250

200

150

100

50

0Verti

cal d

isplac

emen

t (Å)

6050403020100Fluence e/Å2

250

200

150

100

50

0Verti

cal d

isplac

emen

t (Å)

6050403020100Fluence e/Å2

Gold



Without ice

Carbon

Gold

No ice

228 Å vs. 3.8 Å

With ice

With ice

76 Å vs. 1.9 Å

Russo & Passmore Science 2014

4k particles on new gold grids 8.0 Å gold-std FSC

4k particles on carbon grids ~25 Å gold-std FSC

Apo ferritin Russo & Passmore Science 2014

Russo & Passmore Science 2014

Grid handling

100 µm scale bar1 mm scale bar

A B

C D

Handling of supports• Always check in

optical microscope before use– Discard any grids with

defects• Clean tweezers,

glass slides and storage dishes

1 mm 100 µm


Storing grids

BA

glass dishplastic dish

glovedhand

no glove

wrist grounding strap


Store in glass dish in desiccator cabinet ideally in dry nitrogen

Plasma treatment

• Plasma created by ionisation of a low pressure gas– E.g. in air (glow discharge), oxygen, argon, hydrogen

• Ions interact with surface to remove organic contamination and render it hydrophillic

• Other molecules can be introduced to alter the surface, e.g. Amylamine

Ted Pella easyGlow (c. 2015) Edwards S150B (c. 1995) Edwards 12E6 (c. 1962)



As made after 20 second 9:1 Ar:O2 plasma

Cryo-EM grid preparationPlunge freezing1. Apply sample to grid2. Blot away excess buffer (control humidity/temperature)3. Plunge grid into liquid ethane

For water to vitrify, the temperature has to drop faster than 105-106 K/s (Dubochet et al 1988) - Liquid nitrogen boils on contact - poor cooling

capacity- Water is a poor thermal conductor so sample

must be thin < 3 µm- Plunge at > 1 m/s

Cryo-EM grid preparation

Evaluate:1. Ice thickness and uniformity2. Phase of the ice3. Protein concentration and stability

1. Ice pathology

• Vitreous• Free of

contamination

Devitrification occurs in 5 min at -120 °C

Dubochet et al (1988)

1. Ice thickness and uniformity

Ribosomes, Chris Russo Vinothkumar

Optimise plasma exposure time and blot force.

1. Ice thickness and uniformity

Dobro et al (2010)

Incomplete wetting

Vinothkumar & Henderson 2016

Expected particle distribution on grids Given the concentration of the molecule of interest in mg/ml and the molecular weight (MW), how many particles should you see in the image if the frozen specimen has the

same concentration of molecules that you expect in free solution?

What if I don’t see the expected number of particles?

If it is stable in solution, the following parameters can be optimised:• Buffer optimisation (pH, salt), crosslink– change charge on protein

• Addition of small amounts of detergents, lipids– Protect protein from air-water interface

• Blotting and plasma conditions – Is the ice too thick? Too thin?

• Use of a thin film of carbon or graphene – Provides another surface for proteins to adsorb to

1 μm

200 Å

80 μm

grid bar

grid bar

3 mm

Copper!"#$%&

'"()*"+,

-"&"#.*

Gold/+01

2.&345%*+,

6&+,"*+,

'+*78(%*



9:);1%*%<.="5%

'"-"

grid

support/substrate


continuous film

grid bar


Foil materials

-"!

-"/

'"-"

-">?

1

1


Continuous carbon films

metal grid bar




1 μm

continuous am. carbon film

Depositing thin carbon onto grids

siphon

thin amorphous carbon

stainless steel ring

glass crystallization

dish

supports stainless steel mesh

filter paper

flow

A B


10 Å

Graphene to control protein adsorption

gold grid bar




1 μm

graphene

Pantelic, Stahlberg, Plitzko, BaumeisterRosenthal

Partial Hydrogenation: Russo & Passmore 2014 Nat MethodsPassmore & Russo 2016 Methods in Enzymology

Graphene oxide: Pantelic et al 2010https://figshare.com/articles/Graphene_Oxide_Grid_Preparation/3178669

no graphene graphene +20 s hydrogen

graphene +40 s hydrogen

Controlled adsorption of proteins to graphene

Human 20S proteasome

on graphene no graphene


Protein Preparation

Negative stain

Diagnostic cryo-EM








180°

90°

0°

-180° 0 180°



2

1

0

Abs.

(AU)

30 Volume (mL)

66–

31–

6.5–

45–

14–

M.W

. (kD

a)

Step



id-rangeM

id-rangeHigh End

High End

TEM1

2

3

4

5

Reference free class averages

no imposed symmetryRusso & Passmore Science 2014

30°

60°

90°

120°

150°

0°-90°-180° 90° 180°

30°

60°

90°

120°

150°70S amC70S graphene

80S amC80S graphene80S unsuppored ice

Russo & Passmore Nature methods 2014


Protein Preparation

Negative stain

Diagnostic cryo-EM








180°

90°

0°

-180° 0 180°



2

1

0

Abs.

(AU)

30 Volume (mL)

66–

31–

6.5–

45–

14–

M.W

. (kD

a)

Step



id-rangeM

id-rangeHigh End

High End

TEM1

2

3

4

5

Preparing for a trip to Diamond• Prepare a large batch of grids (same conditions) • Screen some from each batch in house

• Grid storage

Doryen A Bubeck

Shipping samples

• Dry cryo shipper• Shipping label

NOT RESTRICTED AS PER IATA SPECIAL PROVISION A152

Protein samples for researchnon-toxic, non-hazardous

Plan a data collection strategyTable 4 Currently Recommended Data Collection Settings at the MRC LMB

Mode SourceMicroscopetype Energy (keV) Detector

Pixel size(Å/px)

Flux(e2/Å2/s)

Exp.Time (s)

Fluence(e2/Å2)

Negative stain W-thermal Entry level 80–120 a CCD 3.3 9 2 18

Diagnostic cryo W-thermal Entry level 80–120 a CCD 3.3 9 2 18

Diagnostic cryo FEG Mid-range 200 Falcon 2 2.1 10 2 20

Medium-resolution cryo (!3.5 A) FEG Mid-range/high-end

300 Falcon 2 1.7 17 3 51

High-resolution ("3.5 A) !400 kDa FEG High-end 300 Falcon 2 1.3 28 2 56

High-resolution ("3.5 A) <400 kDa FEG High-end 300 (#5 eV) K2 1.8 1.5 40 60

Very high-resolution (<2.8 A) FEG High-end 300 (#5 eV) K2 0.90 6.2 10 62

Cryo-tomography cellular (>30 A) FEG High-end 300 (#5 eV) K2 3.5 b 0.65 1.25 pertilt angle

100

Cryo-tomography high-resolutionsubtomogram avg. (<15 A)

FEG High-end 300 (#5 eV) K2 2.2 b 1.5 1 per tiltangle

60

aDQE is maximum for a phosphor coupled CCD at approximately 80 keV but the effects of specimen charging and mean free path are lower at 120 keV.bPixel size is limited by flux instead of spatial resolution.

ARTICLE

INPRESS


McMullan et al. 2013

Back-thinned direct electron detectors

The quality of your data is at least directly proportional

to the quality of your specimen.

-D.B. Williams

Israel Fernandez Venki Ramakrishnan

Tanmay BharatGreg McMullan Shaoxia Chen Christos SavvaTony Crowther

LMB workshops Steve Scotcher

LMB computationToby Darling

Jake Grimmett

Thanks!Lori Passmore

Richard Henderson

Documents

Specimen preparation for high-resolution cryo-EM · continuous film grid bar Amorphous carbon Gold Foil materials-"!-"/ '"-"-">? 1 1 Lacey carbon Holey carbon Types of specimen supports