Labeling particles with heavy atom clusters Brian Gibbons Kornberg - - PowerPoint PPT Presentation

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Labeling particles with heavy atom clusters Brian Gibbons Kornberg Lab Stanford Localization / identification Tissue / cells Tomograms Cryo-EM Negative stain Alignment - HEAVEM Proposal Proc. Natl. Acad. Sci. USA Vol. 95,

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Labeling particles with heavy atom clusters

Brian Gibbons Kornberg Lab Stanford

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SLIDE 2

Localization / identification

 Tissue / cells  Tomograms  Cryo-EM  Negative stain

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SLIDE 3
  • Proc. Natl. Acad. Sci. USA Vol. 95, pp. 9262–9267, August

1998 Biophysics

Single-particle selection and alignment with heavy atom cluster-antibody conjugates

GRANT J. JENSEN AND ROGER D. KORNBERG*

Alignment - HEAVEM Proposal

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 (i) Synthesis of appropriate organothiolate

monolayer-protected gold clusters (MPCs)

 (ii) selection, generation and labeling of adaptor

molecules

 linkers between MPCs and targeted particles  (iii) labeling of particles, removal of excess label  (iv) data collection and processing.

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Monolayer Protected Cluster (MPC) synthesis

Parameters: Ligand type Ligand to gold ratio Amount of BH4

  • Au(III) + 3RSH (-Au(I)/SR-)n + RS-SR

(-Au(I)/SR-)n + BH4

  • Aux(SR)y

Au SR SR SR RS

Maia Azubel

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Organothiolate monolayer-protected gold culsters (MPCs)

  • Stable
  • Water soluble
  • Reactive (on - off)

3MBA/Au 3

Maia Azubel

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Uniformity of MPCs as synthesized

Chris Ackerson

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SLIDE 8

Au102(p-MBA)44 at 1.1 Å

P .D. Jadzinski, G. Calero, C.J. Ackerson, D.A. Bushnell and R.D. Kornberg, Science 318, 430 (2007).

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SLIDE 9

Adaptor molecule

scFv

Cys

Cys

Au SR SR SR

Cys

Au SR SR SR RS

+

SR’

1) Labeling 2) Passivation Cys

Au SR’ SR’ SR’

Fv Fab Fc Antigen-binding site Antigen-binding site

Murray place exchange

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SLIDE 10

Adaptor molecule

Maia Azubel

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Complex formation and purification

Pol II Cys

Au SR’ SR’ SR’

Cys

Au SR’ SR’ SR’

Cys

Au SR’ SR’ SR’

Gel filtration

Pol II Cys

Au SR’ SR’ SR’

Cys

Au SR’ SR’ SR’

Maia Azubel

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SLIDE 12

Cryo EM

Pol II + Au_dsDNA Pol II +Au_scFv aRpb3 & aRpb4

Maia Azubel

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SLIDE 13

Fv Fab Fc Antigen-binding site Antigen-binding site

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SLIDE 14

Fc

trypsin

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Fc

TCEP

HS-

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SLIDE 16

Fc

Fc-Au

Au SR SR SR RS

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CBP affinity purification

Prot

  • tein

n A Prot

  • tein

n A

CBP y.f. Protein calmodulin + Calcium sepharose

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SLIDE 18

CBP affinity purification

Prot

  • tein

n A Prot

  • tein

n A

CBP y.f. Protein + EDTA

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TAP AP t tag - Fc in c interact ctio ion

Prot

  • tein

n A Prot

  • tein

n A Fc Fc

Au SR SR SR RS Au SR SR SR RS

CBP y.f. Protein

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Cryo-EM of TAP- RNA polymerase II - Fc - gold

Chris Ackerson

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SLIDE 21

Prot

  • tein

n A Prot

  • tein

n A

CBP y.f. Protein

TEV EV

sepharose IgG IgG IgG IgG IgG IgG IgG IgG

IgG affinity purification

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CBP

y.f. Protein

calmodulin

Au SR SR SR RS

calmodulin

Au SR SR SR RS

calmodulin

Au SR SR SR RS

calmodulin

Au SR SR SR RS

calmodulin

Au SR SR SR RS

Label with calmodulin-Au

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CBP

y.f. Protein

calmodulin

Au SR SR SR RS

SEC or density gradient

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TFIIH is essential for transcription and DNA repair

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RNA Polymerase II pre-initiation complexes

Bushnell et al. 2004

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TFIIH holoenzyme – 10 subunits

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core core + Ssl2 holo TFIIH

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Localization of Tfb3 with calm-Au

  • C-CBP

Tfb3

calmodulin

Au SR SR SR RS

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Acknowledgments

Roger Kornberg Maia Azubel Phil Robinson Kenji Murakami Xin Liu Dave Bushnell Barbara Davis Ralph Davis Craig Kaplan Yael Kalisman Francisco Asturias Ed Brignole Neil Voss John McCaferty Consuelo Garcia Grant Jensen Chris Ackerson Pablo Jadzinski

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SLIDE 30

Chris Ackerson

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SLIDE 31

Adaptor molecule

scFv

Cys

Cys

Au SR SR SR

Cys

Au SR SR SR RS

+

SR’

1) Labeling 2) Passivation Cys

Au SR’ SR’ SR’

Fv Fab Fc Antigen-binding site Antigen-binding site

Murray place exchange

Maia Azubel

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SLIDE 32
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SLIDE 33

HEAVEM Proposal

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 Relies on derivatization of biological

sample with heavy atom clusters

scatter electron strongly

 Requires discrete particles

specific attachment rigid attachment

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  • Imaging under conditions required for high resolution
  • low dose
  • close to focus
  • Overcome technical limitations
  • detection of drift, astigmatism & magnification

variation (microscope aberrations)

  • CTF determination
  • Resolve particle heterogeneity
  • Allows faster & automated data collection and particle

picking

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SLIDE 36
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scFv-gold for heavy atom electron microscopy

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3-D crystals of 2 nm 4-mercaptobenzoic acid MPCs

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Sample preparation

H.R. Saibil 2000

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Cryo-EM of neuraminadase-scFv-gold

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Cryo-EM of neuraminadase-scFv-gold

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Summary

 Synthesis of defined Gold MPC  Selection and generation of adaptor molecule-

cojugates that provide Specificity & Rigidity

 Feasibility, Stability and visualization of

complexes

Stage is set for empirical test of the HEAVEM Proposal.

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Acknowledgments

Roger Kornberg Kornberg Lab Phil Robinson Xin Liu Barbara Davis Ralph Davis Andy Erhensberger Craig Kaplan John McCaferty Consuelo Garcia Grant Jensen Chris Ackerson Pablo Jadzinski