[ccp4bb] Looking for PDB His-tag statistics

[Snijder, Arjan]

Dear all,



Best wishes for the new year!



I am looking for some statistics about the fraction of protein structures in 
the pdb that are actually crystallized with a His tag, and from those 
crystallized with a His tag whether these construct were engineered with a 
cleavable tag or not. Has anybody done such an analysis and would want to share 
the outcome?

In the mean time I will mentally prepare myself for re-freshing my perl 
knowledge to write a script to do such an analysis. Any suggestions on how to 
quickly analyse this would be appreciated.



cheers



Arjan



Arjan (Harm Jan) Snijder

Associate Principal Scientist

Astra Zeneca R&D, Mölndal

Discovery Enabling Sciences and Capabilities, DECS

SE-431 83 Mölndal Sweden

tel: ++46 31 7763691

arjan.snij...@astrazeneca.com




--
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notify us and remove it from your system and note that you must not copy, 
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Re: [ccp4bb] Looking for PDB His-tag statistics

[Bosch, Juergen]

I vaguely remember a paper by Aled Edwards, where they did the statistics for 
their targets at SGC. As far as I recall they engineer with cleavable tag 
purify and cyrstallize it first with tag and only if they don't get crystals or 
a structure they cleave off the tag and rerun the screens. I think the numbers 
where 30% of the proteins have been cleaved.

Jürgen

-
Jürgen Bosch
Johns Hopkins Bloomberg School of Public Health
Department of Biochemistry & Molecular Biology
Johns Hopkins Malaria Research Institute
615 North Wolfe Street, W8708
Baltimore, MD 21205
Phone: +1-410-614-4742
Lab:  +1-410-614-4894
Fax:  +1-410-955-3655
http://web.mac.com/bosch_lab/

On Jan 7, 2011, at 9:40 AM, Snijder, Arjan wrote:


Dear all,

Best wishes for the new year!

I am looking for some statistics about the fraction of protein structures in 
the pdb that are actually crystallized with a His tag, and from those 
crystallized with a His tag whether these construct were engineered with a 
cleavable tag or not. Has anybody done such an analysis and would want to share 
the outcome?
In the mean time I will mentally prepare myself for re-freshing my perl 
knowledge to write a script to do such an analysis. Any suggestions on how to 
quickly analyse this would be appreciated.

cheers

Arjan

Arjan (Harm Jan) Snijder
Associate Principal Scientist
Astra Zeneca R&D, Mölndal
Discovery Enabling Sciences and Capabilities, DECS
SE-431 83 Mölndal Sweden
tel: ++46 31 7763691
arjan.snij...@astrazeneca.com





Confidentiality Notice: This message is private and may contain confidential 
and proprietary information. If you have received this message in error, please 
notify us and remove it from your system and note that you must not copy, 
distribute or take any action in reliance on it. Any unauthorized use or 
disclosure of the contents of this message is not permitted and may be unlawful.

Re: [ccp4bb] selenomethinine-labeled protein

[Liu, Deqian]

It is normal for me, expressed a few proteins before and mass conformed all MET 
is SE-met. DQ

From: CCP4 bulletin board [ccp...@jiscmail.ac.uk] On Behalf Of Yibin Lin 
[yyb...@gmail.com]
Sent: Thursday, January 06, 2011 3:06 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] selenomethinine-labeled protein

Dear all,

I try to express selenomethinine-labeled protein in E.coli methinoine 
auxotrophic strain B834 in M9 with 17 amino acid and 50 mg/ml se-met for 18 
hours at 37 degree. I don't why the colour of medium vary from colorless to 
yellow.  Could anybody tell me it is normal or not?

Thanks a lot!

Lin

Re: [ccp4bb] Do carbon scattering include hydrogen

[Andrew T. Torelli]

Hi Kenneth,

There are recognized advantages to including riding hydrogens during 
refinement, even at low resolution.  Others have written on this better than I 
could (the following link is a post to the CCP4bb from the archives):

http://www.ysbl.york.ac.uk/ccp4bb/2002/msg01215.html

-Andy Torelli

-Original Message-
From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of Kenneth 
A. Satyshur
Sent: Thursday, January 06, 2011 11:16 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] Do carbon scattering include hydrogen

Persons of interest:

I understood that refinements in refmac of low res structures without hydrogens 
includes a component of the Hydrogen atom attached to the carbon in the carbon 
scattering factors. Or is this just if the Hydrogen atoms
 are in the riding position. If so, why bother to add hydrogens in the riding 
position. 

Thanks
kas


--
Kenneth A. Satyshur, M.S.,Ph.D.
Associate Scientist
University of Wisconsin
Madison, Wisconsin 53706
608-215-5207

Re: [ccp4bb] Looking for PDB His-tag statistics

[Peter Rose]

The RCSB PDB site (www.pdb.org) provides as sequence motif search as an 
advanced search option. For example to search for a N-terminal (His)6 tag, use 
the following regular expression:

 

^HH

 

or HH to find any sequence with a (His)6 pattern.

 

Click on this link to retrieve all sequences with a (His)6 pattern:

http://www.pdb.org/pdb/search/smart.do?smartSearchSubtype_0=MotifQuery&motif_0=HH

 

For more details about the sequence motif search are available at:

http://www.pdb.org/pdb/staticHelp.do?p=help/advancedsearch/sequenceMotif.html

 

Best regards,

 

Peter Rose

RCSB PDB

 

 

From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of Snijder, 
Arjan
Sent: Friday, January 07, 2011 6:41 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] Looking for PDB His-tag statistics

 

Dear all,

 

Best wishes for the new year!

 

I am looking for some statistics about the fraction of protein structures in 
the pdb that are actually crystallized with a His tag, and from those 
crystallized with a His tag whether these construct were engineered with a 
cleavable tag or not. Has anybody done such an analysis and would want to share 
the outcome?

In the mean time I will mentally prepare myself for re-freshing my perl 
knowledge to write a script to do such an analysis. Any suggestions on how to 
quickly analyse this would be appreciated.

 

cheers

 

Arjan

 

Arjan (Harm Jan) Snijder

Associate Principal Scientist

Astra Zeneca R&D, Mölndal

Discovery Enabling Sciences and Capabilities, DECS

SE-431 83 Mölndal Sweden

tel: ++46 31 7763691

arjan.snij...@astrazeneca.com

 

  _  

Confidentiality Notice: This message is private and may contain confidential 
and proprietary information. If you have received this message in error, please 
notify us and remove it from your system and note that you must not copy, 
distribute or take any action in reliance on it. Any unauthorized use or 
disclosure of the contents of this message is not permitted and may be unlawful.

 

Re: [ccp4bb] Do carbon scattering include hydrogen

[James Holton]

Obviously, this depends a bit on which refinement program you use, and I 
am not familiar with all of them.  However, I have had some 
conversations with Garib "refmac" Murshudov and Pavel "phenix.refine" 
Afonine about this, and the shocking answer appears to be "no".  Carbon 
has six electrons, even if it is a methyl carbon with three unmodeled 
hydrogens stuck to it.  Yes, this does introduce a 33% error in the 
number of electrons at every CH3!  I am also suspicious that free ions 
like "Cl" are often modeled as free elemental Cl, and not the Cl- ion, 
which has 1 more electron.  But this is only a 3% error.


Personally, I think that "something should be done" about disordered 
aliphatic hydrogens (particularly since I don't have to code it).  I 
suggest that simply adding "3" to the "constant" coefficient of the "C" 
form factor to make a "CH3 form factor" might just do the trick.  Not 
only are the electrons brought in by the H atoms generally delocalized 
into the carbon's electron cloud anyway, but at most MX resolutions, the 
whole CH3 group is just one big fat lump of 9 electrons.  
Mathematically, this is equivalent to saying that the B-factor Gaussian 
(1.5 A FWHM when B=30) dominates the 5 much narrower Gaussians of the 
atomic form factor.


That said, I should also add that our illustrious refinement program 
authors have good reasons for doing things the way they do.  Mostly 
because users can say some very unkind things if the new version makes 
their R factors go up.  It is true that riding hydrogens "count" as one 
electron each, but it is not always clear where to put them, and in 
situations like methyl group hydrogens they could be anywhere in the 
"doughnut of uncertainty" traced out by rotating the X-CH3 about its X-C 
bond.  It is tempting to say that this doughnut should be modeled in 
somehow, but an important caveat to remember is that putting an atom in 
the wrong place is twice as bad as leaving it out.  That is, you get one 
difference feature if the atom is missing, but two (one positive, one 
negative) if it is wrong.  So, a "doughnut atom" is expected to at best 
cancel itself out.  One could call this a corollary of of the general 
principles of model building:  "When it doubt, don't".


But what about MolProbity? and all those neat inter-digitating 
hydrogens?  Should it not be possible for the refinement program to be 
"smarter" about where it puts riding hydrogens?  Well, that is always 
possible, but I don't think MolProbity is exactly built into refmac.  
You can, however, run MolProbity before you put your model into refinement!


The problem, of course, is if you have a 4.5 A structure with all the 
hydrogens built in you will get hateful comments containing the word 
"parameters" from undereducated reviewers.  I say "undereducated" 
because getting the electron count right is actually MORE important for 
low-angle structure factors than it is for high-angle ones (the extreme 
case of this being F000 itself).  Yes, explicit hydrogen atoms do slow 
down the refinement, and clutter the graphics, but they do not really 
add much by way of "free parameters".  Not if the geometric restraints 
are sensible.  In fact, the central lesson of MolProbity is that 
hydrogens do introduce (at least potentially) better geometric 
restraints.  Sort of like what an elastic network model can do (ahem).


-James Holton
MAD Scientist

On 1/6/2011 8:15 PM, Kenneth A. Satyshur wrote:

Persons of interest:

I understood that refinements in refmac of low res structures without
hydrogens includes a component of the Hydrogen atom attached to the
carbon in the carbon scattering factors. Or is this just if the Hydrogen atoms
  are in the riding position. If so, why bother to add hydrogens in the riding
position.

Thanks
kas


--
Kenneth A. Satyshur, M.S.,Ph.D.
Associate Scientist
University of Wisconsin
Madison, Wisconsin 53706
608-215-5207

[ccp4bb] The density shown in Pymol and Coot is different.

[Zhihong Yu]

Hi,all

I refined a protein-ligand complex structure using Refmac5 and got the map
coefficient "A.mtz" file. I want to represent the electron density in Pymol,
so I transformed "A.mtz" into "A.map" ccp4-format map file using FFT in
ccp4i (parameters are: generate "simple" map in "CCP4" format to cover
"asymmetric unit", all others are default). When I load "A.map" inot Pymol
and show eletron density around the ligand, I found the electron density was
somewhat different comparing to that shown in coot using "A.mtz", density in
Pymol (using "A.map") was much stronger than that in Coot (using "A.mtz")
when showing at 1.0 sigma level. Where does the differenc come from? Should
I set any other parameters when I transform mtz to map using FFT program?

Thanks in advance!

Zhihong

Re: [ccp4bb] The density shown in Pymol and Coot is different.

[Zhihong Yu]

Hi, Nian,

Thanks for your reply. I figured out the problem just now. The difference
indeed came from my FFT transformation. I just used all default parameters
when I transform "A.mtz" to "A.map", so in the "Run FFT" dialog, F1= F,
Sigma=SIGF, PHI=PHIC, Weight=Unaasigned, and I got "A.map" under these
conditions. Even when I load "A.map" into Coot, the density is different
with "A.mtz". But when I assigned parameters as following: F1= FWT,
Sigma=Unaasigned, PHI=PHWT, Weight=Unaasigned, and ran the job, I got
another "A1.map", the density is exactly same as "A.mtz" this time whatever
in Pymol or Coot. I'm a rookie in this field, so herein I have another
question about this: Which map should I use for showing the final density,
"A.map" or "A1.map"? What's the difference of these two maps, it looks like
"A.map" is much stronger than "A1.map".

Really thanks for response!

Zhihong



2011/1/7 Nian Huang 

> It has been discussed before. Nothing to do with FFT. Both pymol and
> coot will rescale your map but differently. You have to really look
> into how the programmer wrote their program to know what is going on.
> For now, I suggest you just use coot map which is more realistic to
> me. Also new version of pymol seems to work better.
>
> On Fri, Jan 7, 2011 at 1:42 PM, Zhihong Yu  wrote:
> > Hi,all
> >
> > I refined a protein-ligand complex structure using Refmac5 and got the
> map
> > coefficient "A.mtz" file. I want to represent the electron density in
> Pymol,
> > so I transformed "A.mtz" into "A.map" ccp4-format map file using FFT in
> > ccp4i (parameters are: generate "simple" map in "CCP4" format to cover
> > "asymmetric unit", all others are default). When I load "A.map" inot
> Pymol
> > and show eletron density around the ligand, I found the electron density
> was
> > somewhat different comparing to that shown in coot using "A.mtz", density
> in
> > Pymol (using "A.map") was much stronger than that in Coot (using "A.mtz")
> > when showing at 1.0 sigma level. Where does the differenc come from?
> Should
> > I set any other parameters when I transform mtz to map using FFT program?
> >
> > Thanks in advance!
> >
> > Zhihong
>

Re: [ccp4bb] Do carbon scattering include hydrogen

[Nat Echols]

On Fri, Jan 7, 2011 at 10:49 AM, James Holton  wrote:

> But what about MolProbity? and all those neat inter-digitating hydrogens?
>  Should it not be possible for the refinement program to be "smarter" about
> where it puts riding hydrogens?  Well, that is always possible, but I don't
> think MolProbity is exactly built into refmac.  You can, however, run
> MolProbity before you put your model into refinement!
>

Actually, if I understand you correctly, Phenix already does something very
much like this using Reduce (the component of MolProbity that adds and
optimizes hydrogens), although it currently needs to be run as a separate
step for most functions.

-Nat

Re: [ccp4bb] The density shown in Pymol and Coot is different.

[Matthew Franklin]

Hi Zhihong -

 

You should use 'FWT' and 'PHWT'.  These are the sigma-A weighted 2Fo-Fc map
coefficients (likewise, 'DELFWT' and 'PHDELWT' are the sigma-A weighted
Fo-Fc map coefficients).  The sigma-A weighted 2Fo-Fc map (also known as the
mFo-DFc map) has become the standard electron density map to use for model
building and for paper figures (unless you're using CNS or Phenix, where you
might use a composite-omit version of the same map).  Using the 'F' and
'PHIC' coefficients will produce a simple Fo map phased by the model phases;
this is generally considered to be more model biased than the sigma-A
weighted maps.

 

However, I'm not sure if you're doing the right thing by leaving SIGF
unassigned.  I believe this should be assigned to the experimental error,
which is usually called 'SIGF' in your mtz file.  I don't know if FFT
actually uses this for anything when it's calculating your map file.  I'm
also not sure what you should use for the weight parameter - perhaps 'FOM'?

 

I should also point out that you can ask Refmac to output the sigma-A
weighted 2Fo-Fc and Fo-Fc maps automatically at the end of a refinement run.
If you're using the ccp4i GUI, expand the "Monitoring and output options"
section of the Refmac window, and check the box marked "Generate weighted
difference maps..."  Then you don't need to mess around with FFT at all.

 

Hope that helps (and I hope I didn't make any egregious mistakes there!),

 

Matt

 

 

-- 

Matthew Franklin, Ph. D.

Senior Research Scientist

New York Structural Biology Center

89 Convent Avenue, New York, NY 10027

(646) 275-7165

  _  

From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of
Zhihong Yu
Sent: Friday, January 07, 2011 4:31 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] The density shown in Pymol and Coot is different.

 

Hi, Nian,

 

Thanks for your reply. I figured out the problem just now. The difference
indeed came from my FFT transformation. I just used all default parameters
when I transform "A.mtz" to "A.map", so in the "Run FFT" dialog, F1= F,
Sigma=SIGF, PHI=PHIC, Weight=Unaasigned, and I got "A.map" under these
conditions. Even when I load "A.map" into Coot, the density is different
with "A.mtz". But when I assigned parameters as following: F1= FWT,
Sigma=Unaasigned, PHI=PHWT, Weight=Unaasigned, and ran the job, I got
another "A1.map", the density is exactly same as "A.mtz" this time whatever
in Pymol or Coot. I'm a rookie in this field, so herein I have another
question about this: Which map should I use for showing the final density,
"A.map" or "A1.map"? What's the difference of these two maps, it looks like
"A.map" is much stronger than "A1.map".

 

Really thanks for response!

 

Zhihong

 

 

 

2011/1/7 Nian Huang 

It has been discussed before. Nothing to do with FFT. Both pymol and
coot will rescale your map but differently. You have to really look
into how the programmer wrote their program to know what is going on.
For now, I suggest you just use coot map which is more realistic to
me. Also new version of pymol seems to work better.


On Fri, Jan 7, 2011 at 1:42 PM, Zhihong Yu  wrote:
> Hi,all
>
> I refined a protein-ligand complex structure using Refmac5 and got the map
> coefficient "A.mtz" file. I want to represent the electron density in
Pymol,
> so I transformed "A.mtz" into "A.map" ccp4-format map file using FFT in
> ccp4i (parameters are: generate "simple" map in "CCP4" format to cover
> "asymmetric unit", all others are default). When I load "A.map" inot Pymol
> and show eletron density around the ligand, I found the electron density
was
> somewhat different comparing to that shown in coot using "A.mtz", density
in
> Pymol (using "A.map") was much stronger than that in Coot (using "A.mtz")
> when showing at 1.0 sigma level. Where does the differenc come from?
Should
> I set any other parameters when I transform mtz to map using FFT program?
>
> Thanks in advance!
>
> Zhihong

 

Re: [ccp4bb] The density shown in Pymol and Coot is different.

[Nat Echols]

On Fri, Jan 7, 2011 at 2:34 PM, Matthew Franklin wrote:

>  However, I'm not sure if you're doing the right thing by leaving SIGF
> unassigned.  I believe this should be assigned to the experimental error,
> which is usually called 'SIGF' in your mtz file.  I don't know if FFT
> actually uses this for anything when it's calculating your map file.  I'm
> also not sure what you should use for the weight parameter - perhaps 'FOM'?
>

FWT,PHWT has the weights already applied to the amplitudes*, so no FOM is
necessary.  According to the FFT documentation, the sigmas can be used to
exclude reflections (subject to an additional keyword), but I assume by
default it will just be ignored.

-Nat

(* FWT = "2mFo-DFc", where "m" is the FOM.)

Re: [ccp4bb] The density shown in Pymol and Coot is different.

[Zhihong Yu]

Matthew & Nat,

Thanks very much, your answers made me much clear about the map
manipulating, as well as usage of Refmac and FFT. Just from my results, I
think I agree with Nat that "Sigma" and "Weight" is unnecessary when running
FFT based on a Refmac-resulted mtz file, since my "A1.map" is exactly same
as FWT of "A.mtz" in Coot.

Again thanks for your help!

Zhihong

2011/1/7 Nat Echols 

> On Fri, Jan 7, 2011 at 2:34 PM, Matthew Franklin wrote:
>
>>  However, I'm not sure if you're doing the right thing by leaving SIGF
>> unassigned.  I believe this should be assigned to the experimental error,
>> which is usually called 'SIGF' in your mtz file.  I don't know if FFT
>> actually uses this for anything when it's calculating your map file.  I'm
>> also not sure what you should use for the weight parameter - perhaps 'FOM'?
>>
>
> FWT,PHWT has the weights already applied to the amplitudes*, so no FOM is
> necessary.  According to the FFT documentation, the sigmas can be used to
> exclude reflections (subject to an additional keyword), but I assume by
> default it will just be ignored.
>
> -Nat
>
> (* FWT = "2mFo-DFc", where "m" is the FOM.)
>

Re: [ccp4bb] Do carbon scattering include hydrogen

[George M. Sheldrick]

Dear James,

I did once write a refinement program so I should try to answer your 
question. All hydrogen atoms attached to C and N in standard proteins and 
almost all in DNSs and RNAs can be calculated geometrically with no 
ambiguities. This has been standard practice in SHELX for about 35 years
and I believe that Refmac and Phenix_refine do so too. The bond lengths to 
hydrogen are set to values about 0.1 shorter than the neutron diffraction
values to best fit the electron density. Methyl groups attached to sp3 
atoms are staggered. For other methyls SHELX has a facility to calculate
the electron density around a circle and does 3-fold averaging to find
the best torsion angle. H on O can either be found in this way or the
torsion angle chosen that makes the best H-bond. For macromolecular 
refinements I recommend including hydrogens at any resolution but leaving
out H on O unless (at very high resolution) they can be seen in the 
difference map. Adding the electrons to the C, N or O atoms is not a good 
idea. 

Best wishes, George 

Prof. George M. Sheldrick FRS
Dept. Structural Chemistry,
University of Goettingen,
Tammannstr. 4,
D37077 Goettingen, Germany
Tel. +49-551-39-3021 or -3068
Fax. +49-551-39-22582


On Fri, 7 Jan 2011, James Holton wrote:

> Obviously, this depends a bit on which refinement program you use, and I am
> not familiar with all of them.  However, I have had some conversations with
> Garib "refmac" Murshudov and Pavel "phenix.refine" Afonine about this, and the
> shocking answer appears to be "no".  Carbon has six electrons, even if it is a
> methyl carbon with three unmodeled hydrogens stuck to it.  Yes, this does
> introduce a 33% error in the number of electrons at every CH3!  I am also
> suspicious that free ions like "Cl" are often modeled as free elemental Cl,
> and not the Cl- ion, which has 1 more electron.  But this is only a 3% error.
> 
> Personally, I think that "something should be done" about disordered aliphatic
> hydrogens (particularly since I don't have to code it).  I suggest that simply
> adding "3" to the "constant" coefficient of the "C" form factor to make a "CH3
> form factor" might just do the trick.  Not only are the electrons brought in
> by the H atoms generally delocalized into the carbon's electron cloud anyway,
> but at most MX resolutions, the whole CH3 group is just one big fat lump of 9
> electrons.  Mathematically, this is equivalent to saying that the B-factor
> Gaussian (1.5 A FWHM when B=30) dominates the 5 much narrower Gaussians of the
> atomic form factor.
> 
> That said, I should also add that our illustrious refinement program authors
> have good reasons for doing things the way they do.  Mostly because users can
> say some very unkind things if the new version makes their R factors go up.
> It is true that riding hydrogens "count" as one electron each, but it is not
> always clear where to put them, and in situations like methyl group hydrogens
> they could be anywhere in the "doughnut of uncertainty" traced out by rotating
> the X-CH3 about its X-C bond.  It is tempting to say that this doughnut should
> be modeled in somehow, but an important caveat to remember is that putting an
> atom in the wrong place is twice as bad as leaving it out.  That is, you get
> one difference feature if the atom is missing, but two (one positive, one
> negative) if it is wrong.  So, a "doughnut atom" is expected to at best cancel
> itself out.  One could call this a corollary of of the general principles of
> model building:  "When it doubt, don't".
> 
> But what about MolProbity? and all those neat inter-digitating hydrogens?
> Should it not be possible for the refinement program to be "smarter" about
> where it puts riding hydrogens?  Well, that is always possible, but I don't
> think MolProbity is exactly built into refmac.  You can, however, run
> MolProbity before you put your model into refinement!
> 
> The problem, of course, is if you have a 4.5 A structure with all the
> hydrogens built in you will get hateful comments containing the word
> "parameters" from undereducated reviewers.  I say "undereducated" because
> getting the electron count right is actually MORE important for low-angle
> structure factors than it is for high-angle ones (the extreme case of this
> being F000 itself).  Yes, explicit hydrogen atoms do slow down the refinement,
> and clutter the graphics, but they do not really add much by way of "free
> parameters".  Not if the geometric restraints are sensible.  In fact, the
> central lesson of MolProbity is that hydrogens do introduce (at least
> potentially) better geometric restraints.  Sort of like what an elastic
> network model can do (ahem).
> 
> -James Holton
> MAD Scientist
> 
> On 1/6/2011 8:15 PM, Kenneth A. Satyshur wrote:
> > Persons of interest:
> >
> > I understood that refinements in refmac of low res structures without
> > hydrogens includes a component of the Hydrogen atom attached to the
> > carbon in the carbo