Cryo-electron tomography (CET) produces three-dimensional images of cells in a near-native state at macromolecular resolution, but identifying structures of interest can be challenging. Here we describe a correlated cryo-PALM (photoactivated localization microscopy)-CET method for localizing objects within cryo-tomograms to beyond the diffraction limit of the light microscope. Using cryo-PALM-CET, we identified multiple and new conformations of the dynamic type VI secretion system in the crowded interior of Myxococcus xanthus

Chang, Yi-Wei

Chen, Songye

Jensen, Grant J.

Löbach, Stephanie

Søgaard-Andersen, Lotte

Tocheva, Elitza I.

Treuner-Lange, Anke

PubMed

Chang, Y.

Chen, S.

Tocheva, E.

Treuner-Lange, A.

Loebach, S.

Sogaard-Andersen, L.

Jensen, G.

MPG.PuRe

Correlated cryogenic photoactivated localization microscopy and cryo-electron tomography

Caltech Authors - Main

Supplementary	  Information	  
	  
Correlated	  cryogenic	  photoactivated	  localization	  microscopy	  
and	  cryo-­‐electron	  tomography	  	  
	  
Yi-­‐Wei	  Chang1,2,	  Songye	  Chen1,2,	  Elitza	  I.	  Tocheva1,	  Anke	  Treuner-­‐Lange3,	  	  
Stephanie	  Löbach3,	  Lotte	  Søgaard-­‐Andersen3	  &	  Grant	  J.	  Jensen1,2,4	  	  
	  
1Division	  of	  Biology	  and	  Biological	  Engineering,	  California	  Institute	  of	  Technology,	  Pasadena,	  USA.	  
2Howard	  Hughes	  Medical	  Institute,	  Pasadena,	  USA.	  
3Max	  Planck	  Institute	  for	  Terrestrial	  Microbiology,	  Marburg,	  Germany.	  
4Correspondence	  should	  be	  addressed	  to	  G.J.J.	  (jensen@caltech.edu).	   	  
Nature Methods: doi:10.1038/nmeth.2961
	   2	  
Supplementary	  Figure	  1	  |	  Photoactivation/photoconversion	  of	  different	  PA-­‐FPs	  at	  80	  K.	  
	   	   	  	  	  	  	  	   	  
	  
The	  two	  columns	  in	  the	  left	  panel	  show	  the	  photoactivation/photoconversion	  of	  PA-­‐FPs	  in	  
E.	  coli	  at	  room	  temperature.	  The	  two	  columns	  in	  the	  right	  panel	  show	  the	  results	  of	  
photoactivation/photoconversion	  tests	  using	  the	  same	  samples	  at	  80	  K.	  The	  image	  
brightness	  of	  different	  PA-­‐FPs	  was	  optimized	  for	  signal	  visibility	  for	  each	  fluorophore,	  but	  
is	  the	  same	  within	  each	  before/after	  pair.	  Note	  only	  PA-­‐GFP	  maintained	  substantial	  
photoactivatable	  properties	  at	  80	  K.	  Scale	  bar	  1	  μm.	  Representative	  images	  of	  five	  trials	  on	  
each	  sample.	  	   	  
Nature Methods: doi:10.1038/nmeth.2961
	   3	  
Supplementary	  Figure	  2	  |	  Histograms	  of	  total	  numbers	  of	  photons	  detected	  from	  
activated	  single	  PA-­‐GFP	  molecules	  in	  the	  cryo-­‐PALM	  images.	  
	  
	  
	  
(a)	  Figure	  1a,	  (b)	  Figure	  1d,	  (c)	  Figure	  2a,	  Supplementary	  Movie	  2,	  (d)	  Figure	  2e	  and	  (e)	  
Supplementary	  Movie	  1.	  The	  average	  number	  of	  photons	  collected	  per	  blinking	  PA-­‐GFP	  
molecule	  is	  206.	  
	   	  
Nature Methods: doi:10.1038/nmeth.2961
	   4	  
Supplementary	  Figure	  3	  |	  Preventing	  laser-­‐induced	  ice	  crystallization.	  	  
	  
	  
	  
Cryo-­‐EM	  images	  of	  M.	  xanthus	  cells	  (a)	  frozen	  on	  an	  EM	  grid	  in	  CTT	  culture	  medium	  and	  
exposed	  to	  the	  cryo-­‐PALM	  laser	  for	  150	  seconds;	  (b)	  frozen	  on	  an	  EM	  grid	  in	  CTT	  medium	  
containing	  5%	  Ficoll	  PM	  70	  and	  exposed	  to	  the	  laser	  for	  300	  seconds;	  (c)	  frozen	  on	  an	  EM	  
grid	  in	  CTT	  medium	  containing	  10%	  Ficoll	  PM	  70	  and	  10%	  ethylene	  glycol	  and	  exposed	  to	  
the	  laser	  for	  300	  seconds	  total,	  with	  60	  seconds	  rest	  after	  every	  60	  seconds	  exposure.	  In	  all	  
cases,	  the	  laser	  was	  adjusted	  to	  an	  intensity	  of	  300	  W/cm2	  on	  the	  sample	  and	  spread	  over	  
an	  area	  of	  ~100	  μm2.	  Scale	  bar	  1	  μm.	   	  
Nature Methods: doi:10.1038/nmeth.2961
	   5	  
Supplementary	  Figure	  4	  |	  Immunoblot	  analysis	  of	  Hcp	  and	  fluorescently-­‐labeled	  VipA	  
accumulation	  in	  total	  cell	  extracts	  and	  supernatants	  of	  the	  indicated	  genotypes.	  
	  
	  
	  
Total	  cell	  extract	  (T)	  from	  2.5x107	  cells	  and	  cell-­‐free	  supernatant	  (S)	  from	  1.5x1010	  cells	  
were	  loaded.	  The	  blots	  were	  probed	  with	  α-­‐Hcp	  to	  generate	  the	  upper	  panel.	  The	  α-­‐Hcp	  
was	  then	  stripped	  away	  and	  the	  same	  membrane	  was	  probed	  with	  α-­‐GFP	  to	  produce	  the	  
lower	  panel.	  Positions	  of	  Hcp,	  VipA-­‐PA-­‐GFP,	  and	  VipA-­‐GFP	  with	  their	  calculated	  molecular	  
mass,	  and	  molecular	  markers	  are	  indicated.	   	  
Nature Methods: doi:10.1038/nmeth.2961
	   6	  
Supplementary	  Figure	  5	  |	  Comparison	  of	  fluorescent	  images	  from	  room	  temperature	  
FLM,	  cryo-­‐FLM,	  and	  cryo-­‐PALM.	  
	  
	   	  
	  
(a)	  M.	  xanthus	  SA4137	  (∆vipA	  strain	  containing	  VipA-­‐GFP)	  cells	  on	  a	  coverslip	  imaged	  with	  
a	  100x,	  NA	  1.4	  oil	  immersion	  objective	  lens	  at	  room	  temperature.	  (b)	  M.	  xanthus	  SA4137	  
cells	  frozen	  on	  an	  EM	  grid	  imaged	  with	  a	  60x,	  NA	  0.7	  air	  objective	  lens	  at	  80	  K.	  (c)	  Cryo-­‐
PALM	  images	  of	  M.	  xanthus	  SA5718	  (∆vipA	  strain	  containing	  VipA-­‐PA-­‐GFP)	  cells	  frozen	  on	  
an	  EM	  grid	  at	  80	  K.	  Upper	  panel:	  (σ	  x,y)max	  =	  220	  nm,	  M	  image	  =	  395;	  middle	  panel:	  (σ	  x,y)max	  =	  
180	  nm,	  M	  image	  =	  1148;	  lower	  panel:	  (σ	  x,y)max	  =	  170	  nm,	  M	  image	  =	  308.	  Phase	  contrast	  
images	  revealing	  cell	  outlines	  are	  superposed	  in	  grayscale	  for	  all	  images.	  GFP	  signals	  in	  a	  
and	  b	  are	  shown	  in	  green.	  PA-­‐GFP	  locations	  in	  c	  are	  shown	  by	  color	  gradient,	  yellow	  (high	  
precision)	  to	  red	  (low	  precision).	  Scale	  bar	  1	  μm.	  
	   	  
Nature Methods: doi:10.1038/nmeth.2961
	   7	  
Supplementary	  Figure	  6	  |	  Reducing	  stage	  vibrations	  through	  enhanced	  cryo-­‐FLM	  stage	  
insulation.	  
	  
	  
	  
(a)	  Original	  design	  of	  the	  FEI	  CryoStage2.	  The	  room-­‐temperature	  condenser	  aperture	  needs	  
to	  be	  inserted	  into	  the	  cryo-­‐stage	  through	  a	  “condenser	  opening”	  hole	  in	  the	  insulating	  lid	  
(yellow).	  (b)	  Modified	  cryo-­‐stage.	  A	  1-­‐cm	  thick	  poly(methyl	  methacrylate)	  plate	  was	  
introduced	  to	  reduce	  heat	  loss	  through	  the	  hole	  but	  preserve	  transparency	  for	  light	  
traveling	  through	  the	  condenser	  aperture	  into	  the	  cryo-­‐stage.	  A	  continuous	  air	  stream	  was	  
directed	  onto	  the	  poly(methyl	  methacrylate)	  plate	  in	  order	  to	  prevent	  water	  condensation.	  
	   	  
Nature Methods: doi:10.1038/nmeth.2961
	   8	  
Supplementary	  Figure	  7	  |	  Cryo-­‐PALM-­‐CET	  alignment	  precision.	  
	  
Superposed	  cryo-­‐PALM	  (foreground	  with	  yellow	  to	  red	  color	  gradient)	  and	  EM	  (grayscale	  
background)	  images	  of	  the	  fluorescent	  beads	  located	  in	  the	  (a)	  upper-­‐left,	  (b)	  upper-­‐right,	  
(c)	  lower-­‐left,	  and	  (d)	  lower-­‐right	  corners	  of	  Supplementary	  Movie	  1.	  Black	  and	  red	  cross	  
symbols	  indicate	  bead	  centers	  in	  EM	  and	  cryo-­‐PALM	  images,	  respectively.	  The	  distances	  
between	  the	  centers	  of	  the	  same	  bead	  in	  the	  two	  images	  are	  9	  ±	  2	  nm.	  Scale	  bar	  100	  nm.	  
	   	  
Nature Methods: doi:10.1038/nmeth.2961
	   9	  
Supplementary	  Table	  1	  |	  Parameters	  of	  merit	  for	  the	  acquisition	  and	  analysis	  of	  the	  cryo-­‐
PALM	  images.	  
	  
	   τ	  frame	  
(ms)	   F	  total	  
Image	  size	  
(μm2)	   M	  total	  
(σ	  x,y)max	  
(nm)	   M	  image	  
Figure	  1a	   100	   400	   1.05	  x	  1.05	   386	   160	   294	  
Figure	  1d	   100	   500	   1.05	  x	  1.05	   525	   160	   345	  
Figure	  2a,	  
Supplementary	  
Movie	  2	  
100	   500	   1.05	  x	  1.05	   830	   160	   617	  
Figure	  2e	   100	   500	   1.05	  x	  1.05	   473	   180	   327	  
Supplementary	  
Movie	  1	   100	   500	   1.05	  x	  1.05	   557	   200	   370	  
	  
τ	  frame	  =	  acquisition	  time	  for	  each	  frame	  of	  the	  image	  stack;	  F	  total	  =	  total	  number	  of	  acquired	  
frames	  in	  the	  image	  stack;	  M	  total	  =	  total	  number	  of	  molecules	  localized	  in	  the	  image	  stack;	  
(σ	  x,y)max	  =	  maximum	  acceptable	  position	  error	  for	  inclusion	  in	  the	  final	  image;	  M	  image	  =	  
number	  of	  localized	  molecules	  comprising	  the	  final	  image.	  	  
	   	  
Nature Methods: doi:10.1038/nmeth.2961
	   10	  
Supplementary	  Table	  2	  |	  M.	  xanthus	  strains	  used	  in	  this	  work.	  
	  
Strain	  name	   Genotype	   Reference	  
DK1622	   wild-­‐type	   22	  
SA5707	   ΔT6SS	  (ΔMXAN4800-­‐MXAN4813)	   This	  work	  
SA5716	   ΔvipA	  (ΔMXAN4807)	   This	  work	  
SA5718	   ΔvipA/PpilA-­‐vipA-­‐PA-­‐GFP	  (attB::pSlo5)	   This	  work	  
SA4137	   ΔvipA/PpilA-­‐vipA-­‐GFP	  (attB::pMAT36)	   This	  work	  
	  
	  
Nature Methods: doi:10.1038/nmeth.2961
	   11	  
Supplementary	  Table	  3	  |	  Primers	  used	  in	  this	  work.	  
	  
Primer	   Sequencea	  
oVipA1	   CTAGTCTAGAAGCAAAGAGAGTTCCGTCGCC	  
oVipA3	   CGCGGATCCCTTGGTCTCTTCGGTCAGACC	  
oGFP1	   GCGCGGATCCGCGGCGGCCGGGGGCGGGATGGCCAAGGGCGAGGAGCTG	  
oGFP2	   GCGCGGTACCTTACTTGTACAGCTCGTCCA	  
oPAGFP1	   CGCGGATCCGCGGCGGCCGGGGGCGGGGTGAGCAAGGGCGAGGAG	  
oPAGFP2	   CGGGGTACCTTACTTGTACAGCTCGTCCATGCC	  
mxan4807A	   ATCGCAAGCTTAGACGCTCATCTCCACCTTGGGAAGC	  
mxan4807B	   ACTCGGGATCCGGTAGGGGCGACGGAACTCTCTTTGC	  
mxan4807C	   ACTCGGGATCCGAACTGGGTCTGACCGAAGAGACC	  
mxan4807D	   TAGCGTCTAGAAAGCTCTCCTCGCCGAACACTTCC	  
mxan4800A	   ATCGCAAGCTTCGAACATCTCCAGCGACG	  
mxan4800B	   ATCGCGAATTCCTCTCCGGCGGTGAAGGG	  
mxan4813C	   ATCGCGAATTCCTGCATGTGGCCCTGACC	  
mxan4813D	   TAGCGTCTAGAGGCCTGCGCCTTGCTGG	  
	  
aPrimer	  sequences	  that	  are	  not	  complementary	  to	  the	  template	  are	  indicated	  in	  bold.	  
	  
	  
Nature Methods: doi:10.1038/nmeth.2961


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Correlated cryogenic photoactivated localization microscopy and cryo-electron tomography

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